Characterization of active members in C and N cycles in the subsurface environment of the Witwatersrand Basin M. Lindsay 1, C.Y.M. Lau 1, G. Tetteh 1, L. Snyder 2, T.L. Kieft 2, B. Sherwood Lollar 3, L. Li 3, S. Maphanga 4, E. van Heerden 5 and T.C. Onstott 1 1 Princeton University - Department of Geosciences, 2 New Mexico Tech, Department of Biology, 3 University of Toronto - Department of Geology, 4 Gold Fields Ltd., 5 University of Free State - Department of Biotechnology Abstract Fracture fluid from various depths and locations in Beatrix gold mine (Gold Fields Ltd.), located in the Welkom region on the 2.9 Ga Witwatersrand Basin of South Africa has been previously studied. Research has shown differential geochemistry data and distinctive community structure which varies from the dominance of different Proteobacterial classes in waters with paleometeoric 18 O and 2 H signatures including methanotrophs to one dominated by Firmicutes including Candidatus Desulforudis audaxviator-like taxa, which are associated with more saline waters with high concentrations of dissolved H 2, hydrocarbons from water-rock reaction and 18 O and 2 H signatures above the Global Meteoric Water Line. Archaea seem to be a minority and all are euryarchaeota including methanogenic genera. The question is:Which of them are actively driving the subsurface C and N cycles? At shaft 3 on level 26, 1.3 kmbls, fracture water from 42 m behind the tunnel wall located in the Main quartzite formation was collected and analyzed. The temperature, pH, Eh, dissolved O 2 and salinity of this hydrocarbon-containing fracture water ranged from 35 to 38°C, 8.2 to 8.8, -30 to -100 mV, 0.3 to 30  M and 4.2 to 4.3 ppt, respectively. Gas comprised 60% CH 4 and 20% N 2. The same fracture formerly yielded Halicephalobus mephisto, the first reported subsurface nematode. Microorganisms were captured on filters in two field seasons. Defined by 16S rDNA, 2011 January sample contains  - Proteobacteria (50%), Firmicutes (39%) and  - and  -Proteobacteria (7%). Of the Firmicutes, 90% were represented by Ca. D. audaxviator. All archaea detected are closestly related to sequences also reported from South African gold mines, with Crenarchaeota accounting for 77% of the clones. Prospective methane-oxidation and production were assessed by amplifying genes encoding for particulate methane monooxygenase alpha subunit (pmoA) and methyl-coenzyme M reductase alpha subunit (mcrA). PmoA genes of Type II methanotrophs were found three times more than Type I methanotrophs. A pmoA gene sequence represents 42% of the library matches only and is identical to a putative protein sequence annotated on Ca. D. audaxviator genome, but further analysis is required to validate its candidature of methanotrophy. The cluster of mcrA gene sequences is related to a novel group of anaerobic methanotrophs (ANME) defined by environmental sequences July samples from the same borehole revealed an absence of Firmicutes. Two  - Proteobacterial sequences dominated the bacterial 16S rDNA clone library, accounting for 54% and 25%. The first 16S rRNA clone library for the region confirmed a complete lack of Firmicutes and active Proteobacteria (71%  -, 17%  - and 6%  -Proteobacteria). Only 3% of the active community is confidently inferred as methylotrophs while 22% belongs to N 2 fixer Rhizobium sp. which has been demonstrated to stimulate methanotrophic growth and 28% is related to Polymorphum gilvum, which is known for n-alkane degradation. Active members responsible for CH 4 metabolism will be supported by presenting the results of archaeal 16S rRNA, pmoA, mcrA and nitrogenase gene diversities. The lack of Firmicutes in July samples could be attributed to collection methods: different filter membrane, faster flowrate but shorter sampling duration, and less total volume of water filtered. Figure 2. Sample collection at borehole 2 in Beatrix Gold Mine shaft #3, level 26. A sterlized S.S. manifold was attached that allowed the simultaneous collection of different samples at different flow rates. Bacteria Clone Libraries Archaea Clone Libraries DNA RNA Agrobacterium (alpha) Polymorphum gilvum (alpha Thiobacillus sp. (beta) A bacterium from Beatrix Mine A beta-Proteobacterium A bacterium of Rhodospirillaceae (alpha) 39 OTUs 12 OTUs DNA RNA Unid. archaeon pMC2A35 hydrothermal vent archaeon Methanobacterium (SA-12) hot spring archaeon 9 OTU ’ s Figure 4. Bacterial compositions of BE326 borehole water in (A) DNA sample collected in January 2011, (B) DNA and (C) RNA samples collected in July Beta-Proteobacteria and Firmicutes (90% was Ca. Desulforudis audaxviator) together accounted for 90% of the DNA community in Jan 2011 sample. Beta- Proteobacteria remained as the most dominant group in the clone library of bacterial 16S rDNA generated from the DNA of Jul 2011 sample, where as the Firmicutes population became one of the minorities. The diversity obtained from the RNA extracted from the same sample affirmed the prevalence of Proteobacteria in the borehole, yet alpha-Proteobacteria were more active than others. Figure 5. Venn diagram of OTU (operational taxonomic unit) distribution between bacterial community in DNA and RNA samples collected in July Using aligned sequences, similarities were calculated using Mothur (Schloss et al. 2011). Seven OTUs were common to both communities, whereas DNA community contained a higher number of unique OTUs than RNA community. Figure 7. Shared OTU ’ s (operational taxonomic unit) between archaea cDNA and DNA samples. Using aligned sequences, similarities were calculated using Mothur (Schloss et al. 2011) and 4 OTU ’ s had sequences from both the DNA and cDNA data sets. 18 OTU ’ s had only cDNA -or- DNA sequences. Poster Number B43G-0499 Figure 1. Sampling location. Beatrix Gold mine (28°14'24.06"S, 26°47'45.25"E) is located near Welkom in the Free State province of South Africa, 240 km southwest of Johannesburg on the southern rim of the Witwatersrand Basin. Conclusions and Further Work Some differences between cDNA and DNA results for both Archaea and Bacteria demonstrated the composition of active community differs from the total DNA community The active community specifically had Methanobacterium, uncultured archaeon and bacteria, unidentified archaeon and bacteria, uncultured euryarchaeote, beta-proteobacteria, gamma-proteobacteria, and a large number of alpha-proteobacteria (mostly rhizobia) Shared OTU ’ s between cDNA and DNA further confirmed that presence of active members D. audaxviator found in both DNA results and cDNA nifH results - it is active in the subsurface fixing N 2. No sulphate-reducing bacteria (dsrAB gene) was detected, however, after a few attempts of PCR amplifications. Positive amplification of the pmoA gene from cDNA library was achieved, yet it was problematic to get them transformed and sequenced. Further results are pending. Acknowledgements I would like to thank the Princeton University EEB department and the Office of the Dean of the College for their support and funding. Additional support was provided by NSF grant #EAR to T.C. Onstott. A very special thanks to Dr. Maggie Lau and Dr. TC Onstott for their constant guidance and feedback on this project. Functional gene BLAST results Figure 3.  2 H H2O and  18 O H2O for fissure water for numerous sampling sites, including Beatrix (squares) (Ward et al. 2004). The global meteoric waterline is indicated. Water from Beatrix for both January 2011 and July 2011 fall on trend with the GMWL. Figure 6. Archaeal compositions of BE326 borehole water in (A) DNA and (B) RNA samples collected in July Both communities reveals prevalence Euryarchaeota of Methanobacterium (SA-12) and unidentified/ uncultured archaeon while the DNA shows less Methanobacterium than the cDNA. Frequency of SA specific archaea within the DNA was 3 Methanobacterium, 3 uncultured archaea SAGMA-F, and 1 other uncultured archaeon. For the cDNA, the only South Africa specific result was in the Methanobacterium, all SA-12. South African methanogens (e.g. Methanobacterium sp. SA-12, OTU belonging to SAGMA-F cluster) Table 3. BLAST results from PCR product of selected metabolic genes, mcrA and nifH. There were only 2 different results for the mcrA gene, both from the same set of clones. The nifH gene revealed the presence of D. audaxviator as well as other nitrogen-fixers. Figure 9. Composition of pmoA gene clone library of DNA from Beatrix Mine, sample collected in July Prevalence of D. audaxviator and Methyocystis sp. Figure 8. (Ward, 2004) Phylogenetic tree for mcrA genes. The blue sequences represent the dominant strains found in BE326 samples, and are part of Novel group 2. They are also related to the ANME (anaerobic methane oxidation) group, which could indicate that the mcrA gene for these organisms is reversed. Table 1. Water chemistry of borehole waterBE326 as of July 2012 Field measurement Dissolved inorganics ppm Dissolved organics ppm Trace metals ppmGas% Temp (°C)38.1DN0.66DOC0.34Mn0.31He3.16 pH8.55DIC2.7Formate0.02Fe0.0353H Measured Eh (mV) -227SO Acetate<0.04Mo0.0021O29.68 Conductivity (mS/cm) 7.15PO Lactate0.09Cr0.0044N Chemet O2 (ppm) 4NO2-<0.04Propanoate<0.02Co0.0035CO Chemet Soluble Fe (ppm) 0.3NO3-0.37Ni<d.l.CH Chemet Total Fe (ppm) 0Mg Cu0.0036CO Chemet H2S (ppm) 0.1Sr2+6.43Zn0.019NMHC0.21 Chemet H2O2 (ppm) 0.5Ba2+1.38W Chemet PO4 (ppm) Al3+0.29As0.004 Gas Flow Rate (mL/min) 50Si4+11.1U<d.l. Water Flow Rate (mL/min) 4,000S15.8 Figure 2. Methodology of molecular analyses (A) (B) (C) N=106 N=105 N=103 Target groupPrimer namePrimer sequence (5’ – 3’)Expected amplicon size (bp) ArchaeaARC-109FACK GCT CAG TAA CAC GT~800 ARC-915RGTG CTC CCC CGC CAA TTC CT BacteriaBAC-S-8FaAGR GTT YGA TYM TGG CTC AG~900 BAC-S-926RCCG TCA ATT CMT TTR AGT Nitrogen-fixersnifH_PolFTGC GAY CCS AAR GCB GAC TC~340 nifH-PolRATS GCC ATC ATY TCR CCG GA MethanotrophsA189m A682m_a GGN GAY TGG GAC TTY TGG GAA YSC NGA RAA GAA CGM ~500 MethanogensMlas mcrA-rev GGT GGT GTM GGD TTC ACM CAR TA CGT TCA TBG CGT AGT TVG GRT AGT ~500 Note: Primers were adopted or modified from literature Table 2. Primer pairs used in this study N=47 N=43 (A) (B)