The deep phreatic thermal explorer (DEPTHX) is an autonomous underwater vehicle designed to navigate an unexplored environment, generate high-resolution three-dimensional (3-D) maps, collect biological samples based on an autonomous sampling decision, and return to its origin. In the spring of 2007, DEPTHX was deployed in Zacatón, a deep (∼318 m), limestone, phreatic sinkhole (cenote) in northeastern Mexico. As DEPTHX descended, it generated a 3-D map based on the processing of range data from 54 onboard sonars. The vehicle collected water column samples and wall biomat samples throughout the depth profile of the cenote. Post-expedition sample analysis via comparative analysis of 16S rRNA gene sequences revealed a wealth of microbial diversity. Traditional Sanger gene sequencing combined with a barcoded-amplicon pyrosequencing approach revealed novel, phylum-level lineages from the domains Bacteria and Archaea; in addition, several novel subphylum lineages were also identified. Overall, DEPTHX successfully navigated and mapped Zacatón, and collected biological samples based on an autonomous decision, which revealed novel microbial diversity in a previously unexplored environment. Key Words: Autonomous—Robotics—Microbial diversity—16S rRNA. Astrobiology 10, 201–213.
Get full access to this article
View all access options for this article.
References
1.
AcinasS.G., MarcelinoL.A., Klepac-CerajV., PolzM.F.2004. Divergence and redundancy of 16S rRNA sequences in genomes with multiple rrn operons. J. Bacteriol, 186:2629–2635.
2.
AmannR., BinderB.J., OlsonR.J., ChisholmS.W., DevereuxR., StahlD.A.1990. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol., 56:1919–1925.
3.
AmendJ.P., RogersK.L., Meyer-DombardD.R. 2004>Microbially mediated sulfur-redox: energetics in marine hydrothermal vent systems. Sulfur Biogeochemistry Past and Future, GSA Special Paper 379. AmendJ.P., EdwardsK.J., LyonsT.W. Geological Society of America: Boulder, CO, 17–34.
4.
AshelfordK.E., ChuzhanovaN.A., FryJ.C., JonesA.J., WeightmanA.J.2006. New screening software shows that most recent large 16S rRNA gene clone libraries contain chimeras. Appl. Environ. Microbiol., 72:5734–5741.
5.
BakermansC., MadsenE.L.2002. Diversity of 16S rDNA and naphthalene dioxygenase genes from coal-tar-waste-contaminated. Microb. Ecol., 44:95–106.
6.
BeattyJ.T., OvermannJ., LinceM.T., ManskeA.K., LangA.S., BlankenshipR.E., Van DoverC.L., MartinsonT.A., PlumleyF.G.2005. An obligately photosynthetic bacterial anaerobe from a deep-sea hydrothermal vent. Proc. Natl. Acad. Sci. U.S.A., 102:9306–9310.
7.
BergsteinT., CavariB.Z.1983. Sulfide utilization by the photosynthetic bacterium Chlorobium phaeobacteroides. Hydrobiologia, 106:241–246.
8.
BreitbartM., HoareA., NittiA., SiefertJ., HaynesM., DinsdaleE.A., EdwardsR.A., SouzaV., RohwerF., HollanderD.2008. Metagenomic and stable isotopic analyses of modern freshwater microbialites in Cuatro Cienegas, Mexico. Environ. Microbiol., 11:16–34.
9.
CampbellB.J., EngelA.S., PorterM.L., TakaiK.2006. The versatile e-proteobacteria: key players in sulphidic habitats. Nat. Rev. Microbiol., 4:458–468.
10.
CannoneJ.J., SubramanianS.2002. The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs. BMC Bioinformatics, 3:2.
11.
CasamayorE.O., SchaferH., BanerasL., Pedros-AlioC., MuyzerG.2000. Identification of and spatio-temporal differences between microbial assemblages from two neighboring sulfurous lakes: comparison by microscopy and denaturing gradient gel electrophoresis. Appl. Environ. Microbiol., 66:499–508.
12.
ChaoA.1987. Estimating the population size for capture-recapture data with unequal catchability. Biometrics, 43:783–791.
13.
ChaoA., LeeS.M.1992. Estimating the number of classes via sample coverage. J. Am. Stat. Assoc., 87:210–217.
14.
ColeJ.R., WangQ., CardenasE., FishJ., ChaiB., FarrisR.J., Kulam-Syed-MohideenA.S., McGarrellD.M., MarshT., GarrityG.M., TiedjeJ.M.2009. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res., 37:D141–D145.
15.
ColwellR.K.2005. EstimateS: Statistical estimation of species richness and shared species from samples, Version 8. User's guide and application. http://purl.oclc.org/estimates.
16.
DeLongE.F., PaceN.R.2001. Environmental diversity of Bacteria and Archaea. Syst. Biol., 50:470–478.
17.
DeSantisT.Z., HugenholtzP., KellerK., BrodieE.L., LarsenN., PicenoY.M., PhanR., AndersenG.L.2006a. NAST: a multiple sequence alignment server for comparative analysis of 16S rRNA genes. Nucleic Acids Res., 34. 10.1093/nar/gkl244.
18.
DeSantisT.Z., HugenholtzP., LarsenN., RojasM., BrodieE.L., KellerK., HuberT., DaleviD., HuP., AndersenG.L.2006b. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl. Environ. Microbiol., 72:5069–5072.
19.
DeWeerdK.A., MandelcoL., TannerR.S., WoeseC., SuflitaJ.M.1990. Desulfomonile tiedjei gen. nov. and sp. nov., a novel anaerobic, dehalogenating, sulfate-reducing bacterium. Arch. Microbiol., 154:23–30.
20.
Diaz-CalderonA., BackesP.G., BonitzR.G.2007. An autonomous robotic scooping approach for planetary sample acquisition. IEEE/RSJ International Conference on Intelligent Robots and Systems (IROS), 10. 10.1109/IROS.2007.4398966.
FairfieldN., KantorG.A., WettergreenD.2006. Towards particle filter SLAM with three dimensional evidence grids in a flooded subterranean environment. IEEE Internal Conference on Robotics and Automation (ICRA).
23.
FairfieldN., KantorG.A., WettergreenD.2007. Real-time SLAM with octree evidence grids for exploration in underwater tunnels. Journal of Field Robotics, 24:3–21.
24.
FrankD.N.2008. XplorSeq: a software environment for integrated management and phylogenetic analysis of metagenomic sequence data. BMC Bioinformatics, 9. 10.1186/1471-2105-9-420.
25.
GaryM.O., SharpJ.M.Jr.2006. Volcanogenic karstification of Sistema Zacatón, Mexico. Perspectives on Karst Geomorphology, Hydrology, and Geochemistry—A Tribute Volume to Derek C. Ford and William B. White. HarmonR.S., WicksC.M.GSA Special Paper 404,Geological Society of America: Boulder, CO, 79–89.
26.
GaryM.O., FairfieldN., StoneW.C., WettergreenD., KantorG.A., SharpJ.M.Jr.2008. 3D mapping and characterization of Sistema Zacatón from DEPTHX (DEep Phreatic THermal eXplorer)Proceedings of KARST08: 11th Sinkhole conference ASCE, 1–11.
27.
GihringT.M., MoserD.P., LinL.-H., DavidsonM., OnstottT.C., MorganL., MillesonM., KieftT.L., TrimarcoE., BalkwillD.L., DollhopfM.E.2006. The distribution of microbial taxa in the subsurface water of the Kalahari Shield, South Africa. Geomicrobiol. J., 23:415–430.
28.
HamadyM., WalkerJ.J., HarrisJ.K., GoldN.J., KnightR.2008. Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nat. Methods, 5:235–237.
29.
HandK.P., CarlsonR.W., ChybaC.F.2007. Energy, chemical disequilibrium, and geological constraints on Europa. Astrobiology, 7:1006–1022.
HoH.T.K., LipmanL.J.A., WostenM.S.M., van AstenA.J.A.M.2008. Arcobacter spp. possess two very short flagellins of which FlaA is essential for motility. FEMS Immunol. Med. Microbiol., 53:85–95.
32.
HoehlerT.M., AmendJ.P., ShockE.L.2007. A “follow the energy” approach for astrobiology. Astrobiology, 7:819–823.
HongS.-H., BungeJ., JeonS.-O., EpsteinS.S.2006. Predicting microbial species richness. Proc. Natl. Acad. Sci. U.S.A., 103:117–122.
35.
HuberJ.A., MorrisonH.G., HuseS.M., NealP.R., SoginM.L., WelchD.B.M.2009. Effect of PCR amplicon size on assessments of clone library microbial diversity and community structure. Environ. Microbiol., 11:1292–1302.
36.
HugenholtzP., KyrpidesN.C.2009. A changing of the guard. Environ. Microbiol., 11:551–553.
37.
JeonS.-O., BungeJ., StoeckT., BargerK.J.-A., HongS.-H., EpsteinS.S.2006. Synthetic statistical approach reveals a high degree of richness of microbial eukaryotes in an anoxic water column. Appl. Environ. Microbiol., 72:6578–6583.
38.
JeonS.-O., AhnT.-S., HongS.-H.2008. A novel archaeal group in the phylum Crenarchaeota found unexpectedly in an eukaryotic survey in the Cariaco basin. J. Microbiol., 46:34–39.
39.
KlappenbachJ.A., SaxmanP.R., ColeJ.R., SchmidtT.M.2001. rrndb: the ribosomal RNA operon copy number database. Nucleic Acids Res., 29:181–184.
40.
KodamaY., WatanabeK.2004. Sulfuricurvum kujiense gen. nov., sp. nov., a facultatively anaerobic, chemolithoautotrophic, sulfur-oxidizing bacterium isolated from an underground crude-oil storage cavity. Int. J. Syst. Evol. Microbiol., 54:2297–2300.
41.
KrajickK.2007. Robot seeks new life—and new funding—in the abyss of Zacatón. Science, 315:322–324.
42.
KuninV., CopelandA.C., LapidusA., MavromatisK., HugenholtzP.2008. A bioinformatics guide to metagenomics. Microbiol. Mol. Biol. Rev., 72:557–578.
LuthyL., FritzM., BachofenR.2000. In situ determination of sulfide turnover rates in a meromictic alpine lake. Appl. Environ. Microbiol., 66:712–717.
45.
MacaladyJ.L., DattaguptaS., SchaperdothI., JonesD.S., DruschelG.K., EastmanD.2008. Niche differentiation among sulfur-oxidizing bacterial populations in cave waters. ISME J., 2:590–601.
46.
MadridV.M., TaylorG.T., ScrantonM.I., ChistoserdovA.Y.2001. Phylogenetic diversity of bacterial and archaeal communities in the anoxic zone of the Cariaco Basin. Appl. Environ. Microbiol., 67:1663–1674.
47.
Medina-SanchezJ.M., Villar-ArgaizM.2006. Solar radiation–nutrient interaction enhances the resource and predation algal control on bacterioplankton: a short-term experimental study. Limnol. Oceanogr., 51:913–924.
48.
MillerW.G., ParkerC.T., RubenfieldM., MendzG.L., WostenM.M., UsseryD.W., StolzJ.F., BinnewiesT.T., HallinP.F., WangG., MalekJ.A., RogosinA., StankerL.H., MandrellR.E.2007. The complete genome sequence and analysis of the epsilonproteobacterium Arcobacter butzleri. PLoS ONE, 26:e1358.
49.
MoralesS.E., CosartT.F., JohnsonJ.V., HolbenW.E.2009. Extensive phylogenetic analysis of a soil bacterial community illustrates extreme taxon evenness and the effects of amplicon length, degree of coverage, and DNA fractionation on classification and ecological parameters. Appl. Environ. Microbiol., 75:668–675.
NunouraT., HirayamaH., TakamiH., OidaH., NishiS., ShimamuraS., SuzukiY., InagakiF., TakaiK., NealsonK.H., HorikoshiK.2005. Genetic and functional properties of uncultivated thermophilic crenarchaeotes from a subsurface gold mine as revealed by analysis of genome fragments. Environ. Microbiol., 7:1967–1984.
52.
PaceN.R.1997. A molecular view of microbial diversity and the biosphere. Science, 276:734–740.
53.
PorterM.L., EngelA.S.2008. Diversity of uncultured epsilonproteobacteria from terrestrial sulfidic caves and springs. Appl. Environ. Microbiol., 74:4973–4977.
54.
PriceM.N., DehalP.S., ArkinA.P.2009. FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Mol. Biol. Evol., 26:1641–1650.
55.
RauéH.A., KlootwijkJ., MustersW.1988. Evolutionary conservation of structure and function of high molecular weight ribosomal RNA. Prog. Biophys. Mol. Biol., 51:77–129.
56.
ReysenbachA.-L., PaceN.R.1995. Protocol 16. Reliable amplification of hyperthermophilic archaeal 16S rRNA genes by the polymerase chain reaction. Thermophiles. Archaea: A Laboratory Manual 3. Cold Spring Harbor Laboratory Press: New York, 101–107.
57.
RobertsonC.E., HarrisJ., SpearJ.R., PaceN.R.2005. Phylogenetic diversity and ecology of environmental archaea. Curr. Opin. Microbiol., 8:638–642.
58.
RogersK.L., AmendJ.P.2005. Archaeal diversity and geochemical energy yields in a geothermal well on Vulcano Island, Italy. Geobiology, 3:319–332.
59.
SchlossP.D., HandelsmanJ.2005. Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl. Environ. Microbiol., 71:1501–1506.
60.
ShimizuS., AkiyamaM., IshijimaY., HamaK., KunimaruT., NaganumaT.2006. Molecular characterization of microbial communities in fault-bordered aquifers in the Miocene formation of northernmost Japan. Geobiology, 4:203–213.
61.
SoginM.L., MorrisonH.G., HuberJ.A., WelchD.M., HuseS.M., NealP.R., ArrietaJ.M., HerndlG.J.2006. Microbial diversity in the deep sea and the underexplored “rare biosphere.”Proc. Natl. Acad. Sci. U.S.A., 103:12115–12120.
62.
SpearJ.R., WalkerJ.J., McCollomT.M., PaceN.R.2005. Hydrogen and bioenergetics in the Yellowstone geothermal ecosystem. Proc. Natl. Acad. Sci. U.S.A., 102:2555–2560.
63.
SpearmanC.E.1904. The proof and measurement of association between two things. Am. J. Psychol., 15:72–101.
64.
SquyresS.W., ReynoldsR.T., CassenP.M., PealeS.J.1983. Liquid water and active resurfacing on Europa. Nature, 301:225–226.
65.
StamatakisA.2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics, 22:2688–2690.
66.
StamatakisA., HooverP., RougemontJ.2008. A rapid bootstrap algorithm for the RAxML web servers. Syst. Biol., 57:758–771.
67.
StoneW.C.2007. Design and deployment of a 3D autonomous subterranean submarine exploration vehicle [Proceedings #UUST07]Conference on Unmanned, Untethered Submersable Technology, Durham, NH, August 20–22.
68.
Van de PeerY., ChappelleS., de WachterR.1996. A quantitative map of nucleotide substitution rates in bacterial rRNA. Nucleic Acids Res., 24:3381–3391.
69.
van den EndeF.P., van GemerdenH.2003. Sulfide oxidation under oxygen limitation by Thiobacillus thioparus isolated from a marine microbial mat. FEMS Microbiol. Ecol., 13:69–77.
70.
WagnerM., RogerA.J., FlaxJ.L., BrusseauG.A., StahlD.A.1998. Phylogeny of dissimilatory sulfite reductases supports an early origin of sulfate respiration. J. Bacteriol., 180:2975–2982.
71.
WallrabensteinC., HauschildE., SchinkB.1995. Syntrophobacter pfennigii sp. nov., new syntrophically propionate-oxidizing anaerobe growing in pure culture with propionate and sulfate. Arch. Microbiol., 5:346–352.
72.
WoeseC., MagrumL.J., GuptaR., SiegelR.B., StahlD.A., KopJ., CrawfordN., BrosiusJ., GutellR., HoganJ.J., NollerH.F. 1980. Secondary structure model for bacterial 16S ribosomal RNA: phylogenetic, enzymatic and chemical evidence. Nucleic Acids Res., 8:2275–2293.
