GOVPH
MENU

 

Prokaryotic Community Analysis of a Hyperalkaline Spring
in the Philippines Using 16S rRNA Gene Clone Library Construction

 

Ronan Q. Baculi1,3, Nacita B. Lantican1*,Francis L. de los Reyes III2 and Asuncion K. Raymundo1


1Microbiology Division, University of the Philippines Los Baños,
College, Laguna, Philippines
2Department of Civil, Construction, and Environmental Engineering,
North Carolina State University
3Current Address : Department of Biology, College of Science,
University of the Philippines Baguio City, Philippines


corresponding author: This email address is being protected from spambots. You need JavaScript enabled to view it.; This email address is being protected from spambots. You need JavaScript enabled to view it.

ABSTRACT

The prokaryotic diversity associated with serpentinization-driven Manleluag Hyperalkaline (pH 11) Spring in Pangasinan, Philippines was investigated. DNA extracted directly from the sediment samples was used to construct clone libraries based on bacterial and archaeal 16S rRNA gene sequences. Phylogenetic analysis of 16S rRNA gene sequences from the clone library revealed that the clones were grouped into Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Cyanobacteria, Bacteroidetes, and Firmicutes. Analysis of the archaeal 16S rRNA clones revealed the presence of sequences associated with members of Euryarchaeota and Thaumarchaeota. Most of the sequences from Euryarchaeota were related to Methanobacteria and Methanomicrobia. Some clones show little affiliation with known taxa and may represent novel sequences of organisms adapted to the hyperalkaline conditions. The populations found suggest the type of metabolisms that drive this specific environment, which include ammonia oxidation, and hydrogen-based and methanogenic metabolisms. This study represents the first analysis of prokaryotic diversity from community DNA of a hyperalkaline environment in the Philippines.

 

INTRODUCTION

Natural nonsaline alkaline environments are not common while saline alkaline soda lakes and soda deserts have been systematically studied. Nonsaline alkaline environments are much rarer and their microbial populations have not been well-documented. The genesis of nonsaline alkaline environments is related to a geochemical process known as serpentinization (Tiago et al. 2004). This process is exothermic and large quantities of hydrogen gas, methane and low-molecular weight organic compounds can emanate from these serpentinizing regions (Brazelton et al. 2011). Serpentinization is therefore a potential source of reducing power and organic carbon for organisms inhabiting the ultramafic subsurface (Brazelton et al. 2012). . . . read more

REFERENCES
ABRAJANO TA, STURCHIO NC, BOHLKE JK, LYON GL, POREDA RJ,STEVENS CM. 1988. Methane-hydrogen gas seeps, Zambales Ophiolite, Philippines: Deep or shallow origin? Chemical Geology 71: 211-222.
ABRAJANO TA, STURCHIO NC, KENNEDY BM, MUELENBACHS K, LYON GL, BOHLKE JK. 1990. Geochemistry of reduced gas related to serpentinization of the Zambales Ophioliye, Philippines. Appl Geochem  5:625-630.
ARCILLA C, PAGUICAN E, FERRER C, ALEXANDER W, MCKINLEY I, MIYOSHI S. 2007. IPHAP: Information on the Philippine Bentonites, Hyperalkaline Waters and Potential Analogue Sites.Retrieved from http://www.andra.fr/lille2007/abstract_lille2007/donnees/pdf/275_276_P_AP_13.pdf on 31 May 2013.
BATH  AH, CHRISTOFI N, NEAL C, PHILIP JC,CAVE MR, MCKINLEY IG,  BERNER U. 1987. Trace element and microbiological studies of alkaline groundwaters in Oman, Arabian Gulf: a natural analogue for cement pore-waters. Rep Fluid Processes Research Group Brit Geol Surv FLPU 87–92.
BLANK JG, GREEN SJ, BLAKE D, VALLEY JW, KITA NT, TREIMAN A, DOBSON PF. 2009. An alkaline spring system within the Del Puerto Ophiolite (California, USA): a Mars analog site. Planetary and Space Science 57: 533– 540.
BOONE DR, WHITMAN WB, ROUVIERE P. 1993. Diversity and taxonomy of methanogens. In: Perry JG. (ed). Methanogenesis: Ecology, Physiology, Biochemistry and Genetics. Chapman and Hall, USA: Inc.p.35-80.
BRAZELTON WJ, MEHTA MP, KELLEY DS, BAROSS JA. 2011. Physiological differentiation within a single-species biofilm fueled by serpentinization. mBio 4(2): e00127-11.
BRAZELTON WJ, NELSON B, SCHRENK MO. 2012. Metagenomic evidence for H2 oxidation and H2 production by serpentinite-hosted subsurface microbial communities. Frontiers in Microbiology 2, doi: 10.3389/ fmicb.2011.00268
BUCKLEY DH, GRABER JR, SCHMIDT TM. 1998. Phylogenetic analysis of nonthermophilic members of the kingdom Crenarchaeota and their diversity and abundance in soils. Appl Environ Microbiol 64: 4333–4339.
CEJA-NAVARRO JA, RIVERA FN,PATINO L, VILA A, CROSSA  J, GOVAERTS B, DENDOOVEN  L. 2010. Phylogenetic and multivariate analysis to determine the effects of different tillage and residue management practices on soil bacterial communities. Appl Environ Microbiol 76(11): 3685-3691.
DELONG EF. 1992. Archaea in coastal marine environments. P Natl Acad Sci USA 89: 5685–5689.
ELSAIED HE, MARUYAMA A. 2008. Biodiversity of archaea in Manzala lake, Egypt, based on 16S rRNA gene. Egypt. J Genet Cytol 37, 57-72.
FEINSTEIN LM, JUN SUL W, BLACKWOOD CB. 2009. Assessment of bias associated with incomplete extraction of microbial DNA from soil. Appl Environ Microbiol 75(16):5428-5433.
GARCIA JL, OLLIVIER B, WHITMAN WB. 2006. The order methanomicrobiales. In: Dworkin M. et al. (ed.) The prokaryotes Archaea. Bacteria: Firmicutes, Actinomycetes, New York: Springer. p.208-30.
GRAY ND, HEAD IM. 2001. Linking genetic identity and function in comminuties of uncultured bacteria. Environ Microbiol 3:481-492.
HORIKOSHI K. 2011. Extremophiles handbook. New York: Springer. p.19-26.
JANSSEN  PH. 2006. Identifying the dominant soil bacterial taxa in libraries of 16S rRNA and 16S rRNA genes. Appl Environ Microbiol 72(3): 1719-1728.
KASAI Y, TAKAHATA Y, HOAKI T, WATANABE K. 2005. Physiological and molecular characterization of a microbial community established in unsaturated petroleum- contaminated soil. Environ Microbiol 7(6), 806-818.
KELLEY DS, KARSON JA, FRUH-GREEN GL, YOERGER DR, SHANK TM, BUTTERFIELD DA, HAYES JM, SCHRENK MO, OLSON EJ, PROSKUROWSKI G, JAKUBA M, BRADLEY A, LARSON B, LUDWIG K, GLICKSON  D, BUCKMAN K, BRADLEY AS, BRAZELTON WJ, ROE K, ELEND MJ, DELACOUR A, BERNASCONI SM, LILLEY MD, BAROSS JA, SUMMONS RE, SYLVA SP. 2005. A serpentinite-hosted ecosystem: the Lost City hydrothermal field. Science 307  1428–1434.
KIMURA M. 1980. A simple method for estimating evolutionary of base substitution through comparative studies of nucleotide sequences. J Mol Evol 16: 111–120.
LANE DJ. 1991. 16S/23S rRNA sequencing. In: Nucleic Acid Techniques in Bacterial Systematics (Stackebrandt E, Goodfellow M. eds. Chichester: John Wiley and Sons. pp. 115–147.
LARKIN MA, BLACKSHIELDS G, BROWN NP, CHENNA R, MCGETTIGAN PA,  MCWILLIAM H, VALENTIN F, WALLACE IM, WILM A, LOPEZ R, THOMPSON JD, GIBSON TJ, HIGGINNS DG . 2007. ClustalW and ClustalX version 2.0. Bioinformatics 23:2947–2948.
MCCOLLOM  T,  SEAWALD J. 2001. A reassessment of the potential for reduction of dissolved CO2 to hydrocarbons during serpentinization of olivine. Geochimica et Cosmochimica Acta 65(21): 3769–3778.
McCOLLOM TM, SEEWALD JS. 2013. Serpentinites: serpentinites, hydrogen and life. Elements 9(2):129-134.
MOSER DP, GIHRING TM, BROCKMAN FJ, FREDRICKSON JK, BALKWILL DL,  DOLLHOPF ME, LOLLAR BS, PRATT LM, BOICE E, SOUTHAM G, WANGER G, BAKER BJ, PFIFFNER SM, LIN LH, ONSTOTT TC. 2005. Desulfotomaculumand Methanobacterium spp. dominate 4-to 5-kilometer-deep fault. Appl Environ Microbiol 71: 8773–8783.
NICOLAS A, BOUDIER F, ILDEFONSE B, BALL E. 2000.  Accretion of Oman and United Arab Emirates ophiolite - Discussion of a new structural map. Mar Geophys Res 21(3-4):147-180.
PEDERSEN K, NILSSON E,ARLINGER J, HALLBECK L, O’NEILL A. 2004. Distribution, diversity and activity of microorganisms in the hyper-alkaline spring waters of Maqarin in Jordan. Extremophiles 8: 151-164.
PERNER TJ, FOGHT JM. 2010. Mature fine tailings from oil sands processing harbor diverse methanogenic communities. Cana J Microbiol 56(6): 459-470.
REYSENBACH  AL, GOTZ D, YERNOOL D. 2002. Microbial diversity of marine and terrestrial thermal springs. In: Biodiversity of Microbial Life. Staley JT, Reysenbach AL (ed). New York:Wiley-Liss, Inc. p. 345-421.
SAMBROOK  J, FRITSCH EF, MANIATIS T. 1989. Molecular cloning: a laboratory manual,  2nd ed. New York: Cold Spring Harbor Laboratory Press.
SAITOU N, NEI, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4): 406–425.
SATYANARAYANA T,  CHANDRALATA R, SHIVAJI S. 2005. Extremophilic Microbes: Diversity and perspectives. Curr Sci 89 (1): 10.
SCHRENK  MO, KELLEY DS, BOLTON SA, BAROSS JA. 2004. Low archaeal diversity linked to subseafloor geochemical processes at the Lost City Hydrothermal field, Mid Atlantic Ridge.  Environ Microbiol 6: 1086-1095.
SCHRENK MO,BRAZELTON WJ, LANG SQ. 2013. Serpentinization, carbon and deep life. Reviews in Mineralogy and Geochemistry 75:575-606.
TAMURA K, PETERSON D, PETERSON N, STECHER G, NEI M, KUMAR S. 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28: 2731-2739.
TIAGO I, CHUNG A, VERISSIMO A. 2004. Bacterial Diversity in a Nonsaline Alkaline Environment: Heterotrophic Aerobic Populations. Appl Environ Microbiol 70(12): 7378–7387.
TIAGO I,  VERISSIMO A. 2012. Microbial and functional diversity of a subterranean high pH groundwater associated to serpentinization. Environ Microbiol15(6):1687-706.
VARGAS E, PASCUA C, ARCILLA C, HONRADO ML,  ALEXANDER W,  NAMIKI K,  FUJII N,  YAMAKAWA M, SATO T, MCKINLEY I. 2009. Origin of the Manleluag Hyperalkaline Hot Spring, Philippines. Proceedings Goldschmidt 2009 Conference, 2009 June 22-26. Davos: Geochim Cosmochim Acta, Goldschmidt Conference Abstracts, A1375.
WAGNER, A.,BLACKSTONE N, CARTWRIGHT P, DICK M, MISOF B, SNOW P,  WAGNER GP, BARTEL SJ, MURTHA M, PENDLETON J. 1994. Surveys of gene families using polymerase chain reaction: PCR selection and PCR drift. Syst Biol 43: 250-26.