Characterization of active members in C and N cycles in the subsurface environment of the Witwatersrand Basin

M. Lindsay1, C.Y.M. Lau1, G. Tetteh1, L. Snyder2, T.L. Kieft2, B. Sherwood Lollar3, L. Li3, S. Maphanga4, E. van Heerden5 and T.C. Onstott1

1Princeton University - Department of Geosciences, 2New Mexico Tech, Department of Biology, 3University of Toronto - Department of Geology, 4Gold Fields Ltd., 5University of Free State - Department of Biotechnology

AbstractFracture 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 18O and 2H 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 H2, hydrocarbons from water-rock reaction and 18O and 2H 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 O2 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% CH4 and 20% N2. 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. 2011 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 N2 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 CH4 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 (alphaThiobacillus 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 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 2011. 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 2011. 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 N2.

• 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-0948659 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. 2HH2O and 18OH2O 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 2011.

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)

Me tha no micro bia 30%

Bacter ia 5%

Uncu ltu r ed eu rya rch aeo te

19%

Un iden tif ied a rch aeo n 4 6 %

BE326 Archaeal 16S rDNA library ( J uly 2011)

Methanococci 6%

Methanomicrobia 41%

Bacteria 13%

Unidentified archaeon

40%

BE326 Archaeal 16S rRNA library ( J uly 2011)

beta-proteobacteria

17% gamma-pr oteobactera

6%

uncultured bacteria 6% alpha-

proteobacteria 71%

BE326 Bacterial 16S rRNA library ( J uly 2011)

Beta-Proteobacteria

69%

Gamma-Proteobacteria

1%

Alpha-Proteobacteria

20%

Bacteroidetes 1%

candidate division OP11 or OD1

1% Chlorof lexi 3%

Firmicutes 2% 1%

Proteobacteria 2%

BE326 Bacterial rDNA library (J uly 2011)

beta- proteobacteria 50%

gamm a-Proteobacte ria 4% Unclassified

1%

alph a-Proteobacte ria 3%

Acidobacter ia 1%

Chlorof lexi 2%

F irmicutes 39%

BE326 Bacterial 16S rDNA library (J anuary 2011)

Ge ne Fr equ en cy D escr ipti on

m cr A ge ne 1 6 Un cu ltu red arch ae on clone OL-K R 40 _3 48 -351m _m cr A _7 8

m cr A ge ne 2 1 Un cu ltu red arch ae on clone OL-K R 40 _3 48 -351m _m cr A _7 7

n ifH gen e 1 6 Un cu ltu red bacte r ium clo ne 200 13- 5

n ifH gen e 2 1 Un cu ltu red nitr oge n- f ix ing ba cte r iu m iso la te

n ifH gen e 3 2 Un cu ltu red bacte r ium clo ne AB 0 2

n ifH gen e 4 1 C andi da tu s Desu lfor u dis au daxv ia tor M P1 04C

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.

Methylococcus sp.

10%

Methylocystis sp.

35%

Methylomicrobium

D. audaxviator

33%

Uncertain 20%

BE326 p moA DNA

Figure 9. Composition of pmoA gene clone library of DNA from Beatrix Mine, sample collected in July 2011. 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 measurem

ent

Dissolved

inorganics

ppmDissolved organics

ppm

Trace

metals

ppm Gas %

Temp (°C) 38.1 DN 0.66 DOC 0.34 Mn 0.31 He 3.16

pH 8.55 DIC 2.7 Formate 0.02 Fe0.035

3H2

0.00078

Measured Eh (mV)

-227 SO42- 59.8 Acetate<0.04

Mo0.002

1O2 9.68

Conductivity (mS/cm)

7.15 PO43-0.02

8Lactate 0.09 Cr

0.0044

N2 42.91

Chemet O2 (ppm)

4 NO2-<0.0

4Propanoa

te<0.02

Co0.003

5CO

0.0068

Chemet Soluble Fe

(ppm)0.3 NO3- 0.37 Ni <d.l. CH4 56.19

Chemet Total Fe (ppm)

0 Mg2+0.62

4Cu

0.0036

CO2 0.034

Chemet H2S (ppm)

0.1 Sr2+ 6.43 Zn 0.019NMH

C0.21

Chemet H2O2 (ppm)

0.5 Ba2+ 1.38 W0.045

4

Chemet PO4 (ppm)

0.0075

Al3+ 0.29 As 0.004

Gas Flow Rate

(mL/min)50 Si4+ 11.1 U <d.l.

Water Flow Rate (mL/min)

4,000

S 15.8

Figure 2. Methodology of molecular analyses

(A)

(B)

(C)

N=106

N=105

N=103

Target group Primer name Primer sequence (5’ – 3’) Expected amplicon size (bp)

Archaea ARC-109F ACK GCT CAG TAA CAC GT ~800

ARC-915R GTG CTC CCC CGC CAA TTC CT

Bacteria BAC-S-8Fa AGR GTT YGA TYM TGG CTC AG ~900

BAC-S-926R CCG TCA ATT CMT TTR AGT

Nitrogen-fixers nifH_PolF TGC GAY CCS AAR GCB GAC TC ~340

nifH-PolR ATS GCC ATC ATY TCR CCG GA

Methanotrophs A189mA682m_a

GGN GAY TGG GAC TTY TGGGAA YSC NGA RAA GAA CGM

~500

Methanogens MlasmcrA-rev

GGT GGT GTM GGD TTC ACM CAR TACGT 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)

Methanococci 6%

Methanomicrobia 41%

Bacteria 13%

Unidentified archaeon 40%

BE326 Archaeal 16S rRNA library ( J uly 2011)

Methanococci Methanomicrobia Bacteria

Uncultured euryarchaeote Unidentified archaeon

Sequencing and data-analysis

Ligation and cloning(pGEM-T Easy Kit, Promega

PCR amplification using multiple primer sets(Primer sequences in Table 2)