Research Article Distinct Habitats Select Particular

Research ArticleDistinct Habitats Select Particular Bacterial Communities inMangrove Sediments

Lidianne L Rocha Geoacutergia B Colares Vanessa L R NogueiraFernanda A Paes and Vacircnia M M Melo

Laboratorio de Ecologia Microbiana e Biotecnologia (LEMBiotech) Departamento de Biologia Centro de CienciasUniversidade Federal do Ceara Campus do Pici Bloco 909 Avenida Mister Hull sn 60455-970 Fortaleza CE Brazil

Correspondence should be addressed to Vania M M Melo vmmmelogmailcom

Received 11 September 2015 Revised 9 January 2016 Accepted 27 January 2016

Academic Editor Johann Peter Gogarten

Copyright copy 2016 Lidianne L Rocha et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

We investigated the relationship among environmental variables composition and structure of bacterial communities in differenthabitats in a mangrove located nearby to an oil exploitation area aiming to retrieve the natural pattern of bacterial communities inthis ecosystemThe T-RFLP analysis showed a high diversity of bacterial populations and an increase in the bacterial richness fromhabitats closer to the sea and without vegetation (S1) to habitats covered byAvicennia schaueriana (S2) and Rhizophora mangle (S3)Environmental variables in S1 and S2 were more similar than in S3 however when comparing the bacterial compositions S2 andS3 shared more OTUs between them suggesting that the presence of vegetation is an important factor in shaping these bacterialcommunities In silico analyses of the fragments revealed a high diversity of the class Gammaproteobacteria in the 3 sites althoughin general they presented quite different bacterial composition which is probably shaped by the specificities of each habitat Thisstudy shows that microhabitats inside of a mangrove ecosystem harbor diverse and distinct microbiota reinforcing the need toconserve these ecosystems as a whole

1 Introduction

Mangroves are coastal ecosystems that have been seriouslythreatened by anthropogenic activities Worldwide man-grove areas have been used for urban development tourismoil exploitation agriculture and shrimp farming Between1980 and 2005 about 36 million hectares of mangrove waslost [1] Competition for land is themajor cause of devastationand losses over time Brazil the second largest mangrove areain the world has lost approximately 50000 ha of mangrovesin the last 25 years [2]

Although these ecosystems are well known for theirtypical flora and associated fauna comparatively only a fewstudies deal with their microbial diversity [3ndash8] On the otherhand studies on cultivable microorganisms have advancedin the isolation and identification of organisms capable ofdegrading xenobiotics including oil hydrocarbons [3 5 9ndash11]

In the environment microorganisms fulfill various nichesand are fundamental for the functioning of mangroves being

particularly important in controlling the geochemistry ofthese habitats [12 13] Recently using metagenomics andpyrosequencing Andreote et al [14] Nogueira et al [15] andAlzubaidy et al [16] retrieved a large volume of informationon the microbial composition and function in tropical man-groves Although these studies represent a valuable contri-bution to our understanding of microbial life more studiesare necessary to access the microbial ecology of mangrovesediments from distinct zones inside the mangrove as wellas those submitted to different anthropogenic threats [17]

Genetic fingerprinting techniques provide a pattern orprofile of the genetic diversity in a microbial communitywhich are important in distinguishing PCR products thathave different nucleotide sequences Terminal-RestrictionFragment Length Polymorphism (T-RFLP) has proven to bea valuable tool to study bacterial community structure incomplex environments such as sediments and soils [3 18 19]Also tools for web-based phylogenetic alignment exist thatallow the retrieval of hypothetical microbial diversity These

Hindawi Publishing CorporationInternational Journal of MicrobiologyVolume 2016 Article ID 3435809 6 pageshttpdxdoiorg10115520163435809

2 International Journal of Microbiology

500250 0 500

(m)

4∘419984000998400998400S

4∘4199840030998400998400S

4∘429984000998400998400S

4∘4299840030998400998400S

4∘419984000998400998400S

4∘4199840030998400998400S

4∘429984000998400998400S

4∘4299840030998400998400S

37∘21

9984000998400998400W 37

∘20

99840030

998400998400W37∘21

99840030

998400998400W37∘22

9984000998400998400W

37∘21

9984000998400998400W 37

∘20

99840030

998400998400W37∘21

99840030

998400998400W37∘22

9984000998400998400W

N

S

S1S2

S3

BrazilAtlantic Ocean

Icapuiacute

Cearaacute

Figure 1 Sampling sites in Barra Grande mangrove Icapuı CearaBrazil

in silico methods such as the resources available on theMiCA3 (Microbial Community Analysis III) website makethe identification of specific organisms in a community basedon the length of Terminal-Restriction Fragments (T-RFs)possible as they predict T-RFs from known and depositedsequences in databases that can be compared with the sub-mitted T-RFs [20 21]

In this context we hypothesized that the zonation ofman-grove species as well as the daily fluctuations imposed bythe tidal regimes shapes the microbial communities thatare present in these habitats making them unique to eachmangrove area In order to test our hypothesis we employedT-RFLP to access the composition and structure of bacterialcommunities of sediments in vegetated and nonvegetatedareas in a mangrove located in Northeastern Brazil and itsrelation to the biotic and abiotic variables in a region knownfor petroleum exploitation which is considered a risk area foroil contamination

2 Materials and Methods

21 Study Area and Sediment Sampling Barra Grande man-grove is located in Icapuı on the extreme east coast of thestate of Ceara Northeastern Brazil (37∘201015840W 4∘401015840S) in aregion comprised of an extensive tidal flat covering anarea of 126031 ha (Figure 1) Due to the rather flat profileof the studied area the sampling sites remain uncoveredat low tide (01m) and are subsequently flooded by thetide Sediments from three different sites at depths between0 and 10 cm were collected following the shoreline in aperpendicular transect Site 1 (S1) was the closest to thesea in an area without vegetation the second site (S2) waslocated in an area of Avicennia schaueriana forest and the

third site (S3) was located in a region of a robust forest ofRhizophora mangle The sites were 150m apart from eachother At each site five sediment samples (0ndash10 cm depth)were randomly collected using a cylindrical sampler (30 cmlong and 10 cm in diameter) and transferred to sterile jarsThe samples were kept in an ice-cooled box for about 2hours before being transported to the laboratory In thelaboratory the five replicate samples from each site werehomogenized in order to obtain composed and representativesamples of each habitat and a portion was stored at minus20∘Cfor DNA extraction and the remaining fraction was usedfor sediment analyses Granulometry was performed by drysieving [22] and organic matter was determined by weightloss on ignition described in Schulte and Hopkins [23] Theenvironmental variables pH salinity and temperature of thesedimentsrsquo percolated water were measured directly in thefield using a multiparameter probe (Multiparameter DisplaySystem Model 650 YSI Yellow Springs OH USA)

22 Study of Bacterial Community Structure Bacterial com-munities were analyzed by T-RFLP following the protocoldescribed by Marsh [24] The extraction of total DNA fromsediment samples was performed using the PowerSoil DNAIsolation Kit (Mo Bio Laboratories Carlsbad CA USA)following the manufacturersrsquo protocol DNA samples wereamplified by PCR using the primers 63F labeled with thefluorophore 6-carboxyfluorescein (6-FAM) at the 51015840 end and1389R [25] PCRs were performed according to the followingprogram initial denaturation at 94∘C for 3min 25 cycles of94∘C for 1min 55∘C for 1min 72∘C for 2min and a finalextension at 72∘C for 10min PCR products were purifiedusing the commercial kit QIAquick PCR Purification (QIA-GENValencia CAUSA) Afterwards samples were digestedseparately with restriction enzymesHhaI andMspI followingthe manufacturersrsquo recommendations (New England Bio-labs Beverly MA USA) Digestion products were dried at40∘C and sent to the Research Technology Support FacilityDepartment of Plant Biology Michigan State University(MSU East Lansing MI USA) Facility where T-RF profileswere generated The analysis was performed using 2 120583L ofthe digestion with 8 120583L of a solution containing the internalstandard MapMakertrade 1000 (BioVentures Inc MurfreesboroTN USA) labeled with ROX (6-carboxy-X-rhodamine) andthe running buffer (deionized formamide) DNA fragmentswere detected by capillary electrophoresis on an ABI Prism3100 Genetic Analyzer (Applied Biosystems Foster CityCA USA) automatic sequencer The T-RFs were visualizedusing the GeneScan Analysis Software (Applied Biosys-tems) exported to Excel and analyzed with the Ibest tool(httpmicaibestuidahoedu) using the height of 50 unitsof fluorescence as an initial point for the electropherogramanalysis andnormalized by calculating the relative abundanceof each T-RF from the fluorescence intensity area T-RFs inthe range of 50ndash990 bp were used for the analysis T-RFs thatdiffered by less than 1 bp were considered identical The fileswere exported from Ibest and analyzed by the T-Align tool(httpinismorucdiesimtalignindexhtml) Each individualT-RFwas considered anOTU (Operational TaxonomicUnit)

International Journal of Microbiology 3

Table 1 Environmental variables of sites S1 S2 and S3 from BarraGrande mangrove soils state of Ceara Northeastern Brazil

Variable S1 S2 S3Temperature 3118 plusmn 04 3101 plusmn 06 346 plusmn 03pH 924 plusmn 025 1045 plusmn 02 88 plusmn 015Salinity 537 plusmn 05 465 plusmn 03 46 plusmn 04Sand () 920 plusmn 02 930 plusmn 015 673 plusmn 02Silt + clay () 80 plusmn 02 70 plusmn 015 327 plusmn 02Organic matter () 24 plusmn 034 27 plusmn 025 84 plusmn 06

23 Diversity Indices The relative abundance of OTUs wasused to calculate diversity indices for each sampleThe Shan-non index (1198671015840) by log

2 the Simpson diversity (120582) and the

Pielou equitability (1198691015840) [26] were calculated using the pro-gram Primer 6 (Primer E Ivybridge United Kingdom)

24 Assignment of T-RFs to Bacterial Taxa The web-basedanalysis tool (PAT+) provided by MiCA3 (httpmicaibestuidahoedupatphp) was used to identify OTUs for T-RFpeaks based on the RDP (Ribosomal Database Project)Release 960 16S rRNA gene database [21]

3 Results

31 Characterization of Mangrove Sediments Habitats S1 andS2 were relatively similar in most environmental factorsexcept for the presence of vegetation in S2 and salinity wasthe only analyzed variable shared between S2 and S3 apartfrom the presence of vegetation Sediments were classified asfine sand at S1 and S2 and coarse silt at S3 the latter presentinga higher silt + clay and organic matter contentThemeasuredenvironmental variables temperature pH salinity organicmatter content and sediment particle size from the threehabitats of the Barra Grande mangrove are shown in Table 1

32 Bacterial Community Structure and Composition Threedifferent community structures with a higher similaritybetween S2 and S3 were observed both inside forested areasThe digestion with HhaI generated a larger number of OTUs(120 T-RFs) thanMspI (87 T-RFs)This means thatHhaI bestresolved the constituent community in the analyzed samplesThus results from the digestion with HhaI were selected forfurther analyses

The relative abundance of OTUs (Figure 2) revealed thatS1 had a lower richness (34 T-RFs) and a great abundanceof three OTUs S3 showed the highest number of T-RFs(73) but a lower relative abundance and was considered themost diverse site in terms of bacterial OTUs S2 showedintermediate characteristics when compared with the othersites showing some abundant T-RFs and also an intermediatenumber of OTUs (43) Only two T-RFs were identical amongthe sites as shown in Figure 3 In addition S1 shares onlythree OTUs with S2 and S3 whereas S2 and S3 share 20OTUsTherefore S1 showed the highest percentage of uniqueOTUs (765) followed by S3 (6575) and S2 (4186) The

S3S2S1

Relat

ive a

bund

ance

()

53137 3935232533 250 22043200 16669 1612815961 15459 1428911815 11531 1141410148 10022 97559358 8689 76587524 7441 72186454 5902 567756 5484 5374

0

10

20

30

40

50

60

70

80

90

100

Others gt 2

Figure 2 T-RFs and their abundance for bacterial communities ofthe Brazilian mangrove soils (S1 S2 and S3) derived from HhaIdigestion The fragment represented as ldquoothersrdquo refers to the sum ofall fragments with a relative abundance less than 2

26

4818

S1 (34)

S2 (43) S3 (73)

23 3

20

Figure 3 Venn diagrams showing the potential number and sharedT-RFs for bacterial communities from Brazilian mangrove soils (S1S2 and S3) derived from HhaI digestion

comparison of diversity indices (Table 2) showed an increasein terms of evenness richness and diversity from S1 to S3

Using PAT+ inMiCAwe predicted the potential bacterialgroups based on digestion pattern of the fragments obtainedby T-RFLP T-RFs 54 and 65 which were shared by the threesites were mainly represented by uncultured bacteria of theBacteroidetes and Proteobacteria phyla Among the possible


Table 2 Diversity indices generated by T-RFLP profiles of the stud-ied mangrove sites

Sample OTUs 119869

1015840a119867

1015840(log 119890)b 120582

c

S1 34 05247 185 0239S2 43 07519 2828 00961S3 73 08096 3474 00555aPieloursquos equitabilitybShannon-Weaverrsquos diversitycSimpsonrsquos diversity

species that can be attributed to T-RF 54 are Flavobacteriumsp Capnocytophaga sp Vibrio sp and Photobacterium spwhereas many species of Bacteroidetes from the Flavobacte-riaceae family and some Alphaproteobacteria were assignedto fragment 65

Considering the dominant fragments at each site S1showed three different fragments T-RF 100 associated withuncultured halophilic bacteria T-RF 325 represented byuncultured bacteria including species of Gammaproteobac-teria and the cultured bacterium Vibrio parahaemolyticusand T-RF 394 corresponding to an uncultured bacteriumAt S2 four dominant fragments were detected T-RF 57including Alphaproteobacteria such as uncultured Azospiril-lum sp and the Bacteroidetes Mariniflexile fucanivorans andcultured and unculturedCytophaga spp T-RF 56 comprisingAlphaproteobacteria as Sneathiella sp uncultured Mesorhi-zobium sp various species ofThalassospira members of Rho-dospirillaceae some uncultured Gammaproteobacteria fromPiscirickettsiaceae and groups of the phylum Bacteroidetessuch as Fluviicola sp Aequorivita sp A antarctica A sub-lithincola Subsaxibacter sp Persicivirga sp Salinimicrobiumsp Mariniflexile gromovii Myroides sp M odoratimimusM profundi M pelagicus Gelidibacter sp G algens and anuncultured Sphingobacterium T-RF 77 which could not beidentified by the web-based tool and T-RF 94 which wasidentified as Escherichia coli

S3 showed three dominant fragments T-RF 55 repre-sented by uncultured bacteria and Capnocytophaga sp T-RF 72 represented by uncultured members of the order Bac-teroidales T-RF 167 which consisted of undetermined uncul-tured bacteria and several uncultured Gammaproteobacte-ria and cultured representatives such as Pseudomonas spPseudoalteromonas sp Shewanella sp Salicola sp S salis Smarasensis and Halovibrio denitrificans

4 Discussion

In this study we observed differences in the bacterial commu-nity structure and composition that could be attributed to thespecific characteristics of each sampledmangrove habitat It iswell known thatmangroves are under the influence ofmarineand terrestrial environments which generate gradients inthe texture of sediments and organic matter content and insalinity as a result of sea and freshwater inputs [27 28] Takingthis into consideration it is expected that the fluctuatingenvironmental conditions shape the microbial communitiesin mangroves

Peixoto et al 2011 [8] have shown that mangrove micro-bial communities are heterogeneously distributed withinmangroves and between different mangroves The authorsexplain these differences based on the sharp environmentalgradients over short spatial scales that include pollutantsreductive-oxidative balance (redox state) pH and nutrientdistribution The aerobicanaerobic interface is a criticalboundary that characterizes soil community structures

Also microbial populations seem to be influenced by thepresence and type of mangrove species Gomes et al 2010[29] demonstrated that even under the fluctuating condi-tions found in mangroves the rhizosphere effect which iswell described for terrestrial plants was also evidenced in thisecosystem Alzubaidy et al 2016 [16] found a predominanceof Bacteroidetes in the rhizosphere of Avicennia while apredominance of Actinobacteria was evidenced in nonvege-tated sediments Ramırez-Elıas et al [30] studying culturablepopulations showed that the species in the Lagunculariarhizosphere harbored the highest microbial population whencompared to other mangrove species

Sites S2 and S3 which are habitats covered by A schaue-riana and R mangle respectively shared more similarities interms of microbial composition with each other than with S1the site without vegetationThe numbers of OTUs detected inS1 S2 and S3 were 34 43 and 73 respectively This increasein the richness from S1 to S3 was observed which suggeststhat besides local abiotic variables the nature of exudatesand nutrients provided by each plant species select specificcommunities [31]

The observed differences in community structure werereflected in the relative abundance as well as in T-RF compo-sition Regarding the percentage of unique T-RFs it is notablethat this mangrove harbors quite different bacterial commu-nities as confirmed by the high value of exclusivity especiallyat S1 which was 765 and the low level of similarity amongthe sites considering that only two T-RFs were shared bythem Thus these data confirm that local differences wereresponsible for distinguishing bacterial populations

We detected an increase in the evenness of bacterial com-munities over the three sites that is S1 showed lower evennessand the presence of some dominant OTUs Due to the prox-imity of the sea the microbiota in S1 is under the influenceof tidal hydrodynamics which probably led to the selectionof several species that are more adapted to marine environ-ments At S3 awider distribution ofOTUswas observedwitha lower occurrence of dominant OTUs demonstrating thatthe environmental conditions have not favored any particularOTU Intermediate characteristics were shown at S2 whichcan be explained by its location in an area inside the vegeta-tion (A schaueriana) like S3 but with many abiotic variablessimilar to those found at S1 due to its proximity to the sea

Putative community compositions were determinedusing a phylogenetic assignment tool (PAT) developed byKent et al [20] using MiCA3 in which the sizes of T-RFpeaks in mangrove soils were matched with T-RF sizesderived in silico from the 16S rRNA gene sequences ofphylotypes in the RDP database The results from PATwere used to examine the bacterial community compositionat different levels of phylogenetic resolution At the three


studied habitats (S1 S2 and S3) there was prevalence ofuncultured bacteria which shows the wide gap in extantdata on microbial diversity considering the large number ofunknown organisms [32]

Taking into account the possible groups associated withT-RFs it can be observed that besides the uncultured bacte-ria the main common OTUs were phylogenetically affiliatedwith the Bacteroidetes with a large number belonging to theFlavobacteriaceae In Brazilian mangrove sediments somebacteria affiliated with the Bacteroidetes were also observedin clone libraries but at a low number compared to thephylum Proteobacteria which appears to be dominant inthese environments [8 33 34] Considering the dominantgroups at each habitat in the studied mangrove an overallabundance of Proteobacteria andBacteroideteswas observed

At site S1 which lies in a region close to the sea withoutvegetation there was prevalence of uncultured halophilicbacteria uncultured Gammaproteobacteria andVibrio para-haemolyticus At S2 located in the root zones of A schaue-riana several uncultured bacteria were found with a pre-dominance of Alphaproteobacteria some of them knownnitrogen fixers such as an uncultured Azospirillum whichwas previously reported in roots of A marina and in therhizosphere of Suaeda monoecious in a mangrove fromIndia [35] Several bacterial strains affiliated with the genusThalassospira were detected which were previously isolatedfromoil-contaminated seawater It was found that these coulddegrade several polycyclic aromatic hydrocarbons (PAHs)including naphthalene dibenzothiophene phenanthreneand fluorene [36] These strains might play important rolesin the bioremediation of marine oil spills and considering thelocation of the studied mangrove in a risk area for oil con-tamination the presence of possible oil degraders indicatesthe potential of this environment to respond effectively topossible contamination by petroleum hydrocarbons

Among the OTUs found at S3 some were identified asbelonging to the phylum Proteobacteria such as Vibrio spand Photobacterium (Proteobacteria) and Rhodovulum sul-fidophilum a marine photosynthetic bacterium (Alphapro-teobacteria) Some members of the genus Pseudomonaswhich are able to metabolize petroleum hydrocarbons [37]were also detected

Altogether the data showed distinct bacterial communi-ties among the three mangrove habitats due to the presenceand type of vegetation and the divergence environmentalvariables in which these habitats are submitted In generalall the potential bacteria corresponding to the identified T-RFs are typical of marine environments and play importantroles in maintaining the dynamic balance of the ecosystemThis study highlights the importance of preservingmangroveecosystems as a whole due to the uniqueness of each habitat

5 Conclusion

The main contribution of this study was to demonstrate thatmangrove soils hold highly diverse bacterial populations withincreasing richness from the sea to forested areas selectedby the local differences The existence of unique bacterialcommunities to each mangrove habitat covered by distinct

plant species clearly demonstrates the importance of the con-servation of this ecosystem as a whole

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to thank Conselho Nacional de Des-envolvimento Cientıfico e Tecnologico (CNPq) for the Lidi-anne L Rocha doctoral scholarship and for the financial sup-port They also thank Coordenacao de Aperfeicoamento dePessoal de Nıvel Superior (CAPES) for Georgia B Colarespostdoctoral fellowship

References

[1] Food and Agriculture Organization of the United NationsmdashFAO ldquoThe worldrsquos mangroves 1980ndash2005rdquo 2007 ftpftpfaoorgdocrepfao010a1427ea1427e00pdf

[2] M Spalding M Kainuma and L Collins World Atlas ofMangroves UNEP-WCMC Cambridge UK 2010

[3] E M S Brito R Guyoneaud M Goni-Urriza et al ldquoCharac-terization of hydrocarbonoclastic bacterial communities frommangrove sediments in Guanabara Bay Brazilrdquo Research inMicrobiology vol 157 no 8 pp 752ndash762 2006

[4] E M S Brito R Duran R Guyoneaud et al ldquoA case study ofin situ oil contamination in a mangrove swamp (Rio De JaneiroBrazil)rdquo Marine Pollution Bulletin vol 58 no 3 pp 418ndash4232009

[5] N C M Gomes L R Borges R Paranhos F N Pinto L CS Mendonca-Hagler and K Smalla ldquoExploring the diversity ofbacterial communities in sediments of urbanmangrove forestsrdquoFEMS Microbiology Ecology vol 66 no 1 pp 96ndash109 2008

[6] B Gonzalez-Acosta Y Bashan N Y Hernandez-Saavedra FAscencio and G De La Cruz-Aguero ldquoSeasonal seawater tem-perature as the major determinant for populations of culturablebacteria in the sediments of an intact mangrove in an aridregionrdquo FEMS Microbiology Ecology vol 55 no 2 pp 311ndash3212006

[7] J-B Liang Y-Q Chen C-Y Lan N F Y Tam Q-J Zanand L-N Huang ldquoRecovery of novel bacterial diversity frommangrove sedimentrdquo Marine Biology vol 150 no 5 pp 739ndash747 2007

[8] R Peixoto G M Chaer F L Carmo et al ldquoBacterial commu-nities reflect the spatial variation in pollutant levels in Brazilianmangrove sedimentrdquo Antonie van Leeuwenhoek vol 99 no 2pp 341ndash354 2011

[9] H F Santos F L Carmo J E S Paes A S Rosado and R SPeixoto ldquoBioremediation of mangroves impacted by petro-leumrdquo Water Air amp Soil Pollution vol 216 no 1 pp 329ndash3502011

[10] A C F Dias F D Andreote F Dini-Andreote et al ldquoDiversityand biotechnological potential of culturable bacteria fromBrazilian mangrove sedimentrdquo World Journal of Microbiologyand Biotechnology vol 25 no 7 pp 1305ndash1311 2009

[11] L L Rocha G B Colares A L Angelim T B Grangeiro andV M M Melo ldquoCulturable populations of Acinetobacter can


promptly respond to contamination by alkanes in mangrovesedimentsrdquo Marine Pollution Bulletin vol 76 no 1-2 pp 214ndash219 2013

[12] G Holguin P Vazquez and Y Bashan ldquoThe role of sedimentmicroorganisms in the productivity conservation and reha-bilitation of mangrove ecosystems an overviewrdquo Biology andFertility of Soils vol 33 no 4 pp 265ndash278 2001

[13] K Kathiresan and B L Bingham ldquoBiology of mangroves andmangrove ecosystemsrdquo Advances in Marine Biology vol 40 pp81ndash251 2001

[14] F D Andreote D J Jimenez D Chaves et al ldquoThemicrobiomeof Brazilianmangrove sediments as revealed bymetagenomicsrdquoPLoS ONE vol 7 no 6 Article ID e38600 2012

[15] V L R Nogueira L L Rocha G B Colares et al ldquoMicrobiomesand potential metabolic pathways of pristine and anthropizedBrazilian mangrovesrdquo Regional Studies inMarine Science vol 2pp 56ndash64 2015

[16] H Alzubaidy M Essack T B Malas et al ldquoRhizospheremicro-biome metagenomics of gray mangroves (Avicennia marina) inthe Red Seardquo Gene vol 576 no 2 part 1 pp 626ndash636 2016

[17] A Strangmann Y Bashan and L Giani ldquoMethane in pristineand impaired mangrove soils and its possible effect on estab-lishment of mangrove seedlingsrdquo Biology and Fertility of Soilsvol 44 no 3 pp 511ndash519 2008

[18] W-T Liu T L Marsh H Cheng and L J Forney ldquoChar-acterization of microbial diversity by determining terminalrestriction fragment length polymorphisms of genes encoding16S rRNArdquoApplied and EnvironmentalMicrobiology vol 63 no11 pp 4516ndash4522 1997

[19] B K Singh L Nazaries S Munro I C Anderson and CD Campbell ldquoUse of multiplex terminal restriction fragmentlength polymorphism for rapid and simultaneous analysis ofdifferent components of the soil microbial communityrdquoAppliedand Environmental Microbiology vol 72 no 11 pp 7278ndash72852006

[20] A D Kent D J Smith B J Benson and E W Triplett ldquoWeb-based phylogenetic assignment tool for analysis of terminalrestriction fragment length polymorphism profiles of microbialcommunitiesrdquoApplied and EnvironmentalMicrobiology vol 69no 11 pp 6768ndash6776 2003

[21] C Shyu T Soule S J Bent J A Foster and L J Forney ldquoMiCAa web-based tool for the analysis of microbial communitiesbased on terminal-restriction fragment length polymorphismsof 16S and 18S rRNA genesrdquoMicrobial Ecology vol 53 no 4 pp562ndash570 2007

[22] K Suguio Introducao a Sedimentologia Edgard BlucherEDUSP Sao Paulo Brazil 1973

[23] E E Schulte and B G Hopkins ldquoEstimation of soil organicmatter by weight loss-on-ignitionrdquo in Soil Organic MatterAnalysis and Interpretation F R Magdoff M A Tabatabai andE A Hanlon Jr Eds pp 21ndash31 Soil Science Society of AmericaMadison Wis USA 1996

[24] T L Marsh ldquoCulture-independent microbial community anal-ysis with terminal restriction fragment length polymorphismrdquoMethods in Enzymology vol 397 pp 308ndash329 2005

[25] AMOsborn E R BMoore andKN Timmis ldquoAn evaluationof terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structureand dynamicsrdquoEnvironmentalMicrobiology vol 2 no 1 pp 39ndash50 2000

[26] A E Magurran Measuring Biological Diversity BlackwellOxford UK 2004

[27] M B K Prasad and A L Ramanathan ldquoSedimentary nutrientdynamics in a tropical estuarine mangrove ecosystemrdquo Estuar-ine Coastal and Shelf Science vol 80 no 1 pp 60ndash66 2008

[28] G B Colares and V M M Melo ldquoRelating microbial com-munity structure and environmental variables in mangrovesediments inside Rhizophora mangle L habitatsrdquo Applied SoilEcology vol 64 pp 171ndash177 2013

[29] N C M Gomes D F R Cleary F N Pinto et al ldquoTakingroot enduring effect of rhizosphere bacterial colonization inmangrovesrdquo PLoS ONE vol 5 no 11 Article ID e14065 2010

[30] M A Ramırez-Elıas R Ferrera-Cerrato A Alarcon et alldquoIdentification of culturable microbial functional groups iso-lated from the rhizosphere of four species of mangroves andtheir biotechnological potentialrdquo Applied Soil Ecology vol 82pp 1ndash10 2014

[31] Y Bashan and G Holguin ldquoPlant growth-promoting bacteriaa potential tool for arid mangrove reforestationrdquo Trees vol 16no 2-3 pp 159ndash166 2002

[32] S K Dubey A K Tripathi and S N Upadhyay ldquoExplorationof soil bacterial communities for their potential as bioresourcerdquoBioresource Technology vol 97 no 17 pp 2217ndash2224 2006

[33] A C F Dias F D Andreote J Rigonato M F Fiore I S Meloand W L Araujo ldquoThe bacterial diversity in a Brazilian non-disturbed mangrove sedimentrdquo Antonie van Leeuwenhoek vol98 no 4 pp 541ndash551 2010

[34] R G Taketani N O Franco A S Rosado and J D van ElsasldquoMicrobial community response to a simulated hydrocarbonspill in mangrove sedimentsrdquo The Journal of Microbiology vol48 no 1 pp 7ndash15 2010

[35] S Ravikumar G RamanathanN Suba and L Jeyaseeli ldquoQuan-tification of halophilic Azospirillum from mangrovesrdquo IndianJournal of Marine Sciences vol 31 no 2 pp 157ndash160 2002

[36] Y Kodama L I Stiknowati A Ueki K Ueki and K Watan-abe ldquoThalassospira tepidiphila sp nov a polycyclic aromatichydrocarbon-degrading bacterium isolated from seawaterrdquoInternational Journal of Systematic and Evolutionary Microbiol-ogy vol 58 no 3 pp 711ndash715 2008

[37] J D VanHamme A Singh andO PWard ldquoRecent advances inpetroleum microbiologyrdquo Microbiology and Molecular BiologyReviews vol 67 no 4 pp 503ndash549 2003

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Enzyme Research



Microbiology


500250 0 500

(m)

4∘419984000998400998400S

4∘4199840030998400998400S

4∘429984000998400998400S

4∘4299840030998400998400S

4∘419984000998400998400S

4∘4199840030998400998400S

4∘429984000998400998400S

4∘4299840030998400998400S

37∘21

9984000998400998400W 37

∘20

99840030

998400998400W37∘21

99840030

998400998400W37∘22

9984000998400998400W

37∘21

9984000998400998400W 37

∘20

99840030

998400998400W37∘21

99840030

998400998400W37∘22

9984000998400998400W

N

S

S1S2

S3

BrazilAtlantic Ocean

Icapuiacute

Cearaacute

Figure 1 Sampling sites in Barra Grande mangrove Icapuı CearaBrazil

in silico methods such as the resources available on theMiCA3 (Microbial Community Analysis III) website makethe identification of specific organisms in a community basedon the length of Terminal-Restriction Fragments (T-RFs)possible as they predict T-RFs from known and depositedsequences in databases that can be compared with the sub-mitted T-RFs [20 21]

In this context we hypothesized that the zonation ofman-grove species as well as the daily fluctuations imposed bythe tidal regimes shapes the microbial communities thatare present in these habitats making them unique to eachmangrove area In order to test our hypothesis we employedT-RFLP to access the composition and structure of bacterialcommunities of sediments in vegetated and nonvegetatedareas in a mangrove located in Northeastern Brazil and itsrelation to the biotic and abiotic variables in a region knownfor petroleum exploitation which is considered a risk area foroil contamination

2 Materials and Methods

21 Study Area and Sediment Sampling Barra Grande man-grove is located in Icapuı on the extreme east coast of thestate of Ceara Northeastern Brazil (37∘201015840W 4∘401015840S) in aregion comprised of an extensive tidal flat covering anarea of 126031 ha (Figure 1) Due to the rather flat profileof the studied area the sampling sites remain uncoveredat low tide (01m) and are subsequently flooded by thetide Sediments from three different sites at depths between0 and 10 cm were collected following the shoreline in aperpendicular transect Site 1 (S1) was the closest to thesea in an area without vegetation the second site (S2) waslocated in an area of Avicennia schaueriana forest and the

third site (S3) was located in a region of a robust forest ofRhizophora mangle The sites were 150m apart from eachother At each site five sediment samples (0ndash10 cm depth)were randomly collected using a cylindrical sampler (30 cmlong and 10 cm in diameter) and transferred to sterile jarsThe samples were kept in an ice-cooled box for about 2hours before being transported to the laboratory In thelaboratory the five replicate samples from each site werehomogenized in order to obtain composed and representativesamples of each habitat and a portion was stored at minus20∘Cfor DNA extraction and the remaining fraction was usedfor sediment analyses Granulometry was performed by drysieving [22] and organic matter was determined by weightloss on ignition described in Schulte and Hopkins [23] Theenvironmental variables pH salinity and temperature of thesedimentsrsquo percolated water were measured directly in thefield using a multiparameter probe (Multiparameter DisplaySystem Model 650 YSI Yellow Springs OH USA)

22 Study of Bacterial Community Structure Bacterial com-munities were analyzed by T-RFLP following the protocoldescribed by Marsh [24] The extraction of total DNA fromsediment samples was performed using the PowerSoil DNAIsolation Kit (Mo Bio Laboratories Carlsbad CA USA)following the manufacturersrsquo protocol DNA samples wereamplified by PCR using the primers 63F labeled with thefluorophore 6-carboxyfluorescein (6-FAM) at the 51015840 end and1389R [25] PCRs were performed according to the followingprogram initial denaturation at 94∘C for 3min 25 cycles of94∘C for 1min 55∘C for 1min 72∘C for 2min and a finalextension at 72∘C for 10min PCR products were purifiedusing the commercial kit QIAquick PCR Purification (QIA-GENValencia CAUSA) Afterwards samples were digestedseparately with restriction enzymesHhaI andMspI followingthe manufacturersrsquo recommendations (New England Bio-labs Beverly MA USA) Digestion products were dried at40∘C and sent to the Research Technology Support FacilityDepartment of Plant Biology Michigan State University(MSU East Lansing MI USA) Facility where T-RF profileswere generated The analysis was performed using 2 120583L ofthe digestion with 8 120583L of a solution containing the internalstandard MapMakertrade 1000 (BioVentures Inc MurfreesboroTN USA) labeled with ROX (6-carboxy-X-rhodamine) andthe running buffer (deionized formamide) DNA fragmentswere detected by capillary electrophoresis on an ABI Prism3100 Genetic Analyzer (Applied Biosystems Foster CityCA USA) automatic sequencer The T-RFs were visualizedusing the GeneScan Analysis Software (Applied Biosys-tems) exported to Excel and analyzed with the Ibest tool(httpmicaibestuidahoedu) using the height of 50 unitsof fluorescence as an initial point for the electropherogramanalysis andnormalized by calculating the relative abundanceof each T-RF from the fluorescence intensity area T-RFs inthe range of 50ndash990 bp were used for the analysis T-RFs thatdiffered by less than 1 bp were considered identical The fileswere exported from Ibest and analyzed by the T-Align tool(httpinismorucdiesimtalignindexhtml) Each individualT-RFwas considered anOTU (Operational TaxonomicUnit)








3 Results




S3S2S1

Relat

ive a

bund

ance

()

53137 3935232533 250 22043200 16669 1612815961 15459 1428911815 11531 1141410148 10022 97559358 8689 76587524 7441 72186454 5902 567756 5484 5374

0

10

20

30

40

50

60

70

80

90

100

Others gt 2


26

4818

S1 (34)

S2 (43) S3 (73)

23 3

20






Sample OTUs 119869

1015840a119867

1015840(log 119890)b 120582

c





4 Discussion














5 Conclusion





Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








3 Results




S3S2S1

Relat

ive a

bund

ance

()

53137 3935232533 250 22043200 16669 1612815961 15459 1428911815 11531 1141410148 10022 97559358 8689 76587524 7441 72186454 5902 567756 5484 5374

0

10

20

30

40

50

60

70

80

90

100

Others gt 2


26

4818

S1 (34)

S2 (43) S3 (73)

23 3

20






Sample OTUs 119869

1015840a119867

1015840(log 119890)b 120582

c





4 Discussion














5 Conclusion





Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Sample OTUs 119869

1015840a119867

1015840(log 119890)b 120582

c





4 Discussion














5 Conclusion





Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







5 Conclusion





Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Distinct Habitats Select Particular