Characterization of active members in C and N cycles in the
subsurface environment of the Witwatersrand Basin
Poster Number B43G-0499
M.
1
Lindsay ,
1Princeton
C.Y.M.
1
Lau ,
G.
1
Tetteh ,
L.
2
Snyder ,
T.L.
2
Kieft ,
B. Sherwood
3
Lollar ,
L.
3
Li ,
S.
4
Maphanga ,
5
Heerden
E. van
and T.C.
1
Onstott
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
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 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.
Bacteria Clone Libraries
(A)
Archaea Clone Libraries
BE3 2 6 Ba ct e r ia l 1 6 S r D N A libr a r y ( Ja n u a r y 2 0 1 1 )
(A)
BE3 2 6 Ar ch a e a l 1 6 S r D N A libr a r y ( Ju ly 2 0 1 1 )
N=47
N=106
Firmicutes
39%
Unidentified
archaeon 46%
beta-proteobacteria
50%
Bacteria 5%
Chloroflexi
2%
Acidobacteria
1%alpha-Proteobacteria
gamma-Proteobacteria
3%
Unclassified 4%
1%
Uncultured
euryarchaeote
19%
BE3 2 6 Ba ct e r ia l r D N A libr a r y ( Ju ly 2 0 1 1 )
(B)
candidate division
OP11 or OD1
1%
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.
Bacteroidetes
1%
Firmicutes
2% Proteobacteria
Chloroflexi 1%
2%
3%
(B)
BE3 2 6 Ar ch a e a l 1 6 S r RN A libr a r y ( Ju ly 2 0 1 1 )
BE3 2 6 Ar cha e a l 1 6 S r RN A libr a r y ( July 2 0 1 1 )
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.
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.
Water Flow
Rate
(mL/min)
4,000
S
ppm
0.66
2.7
Dissolved
Trace
ppm
ppm
organics
metals
DOC
0.34 Mn
0.31
Formate 0.02 Fe 0.0353
59.8
Acetate
0.028
Lactate
<0.04 Mo
0.09
Cr
<0.04 Propanoate <0.02 Co
Gas
%
He
H2
3.16
0.00078
0.0021
O2
9.68
0.0044
N2
42.91
0.0035
CO
AlphaProteobacteria
20%
GammaProteobacteria
1%
N=105
Methanococci 6%
Archaea
Primer name
0.37
0.624
Ni
Cu
<d.l.
CH4
0.0036
CO2
41%
BetaProteobacteria
69%
BE3 2 6 p m oA D N A
BE3 2 6 Ba ct e r ia l 1 6 S r RN A libr a r y ( Ju ly 2 0 1 1 )
56.19
betaproteobacteria
17%
0.034
Zn
0.019 NMHC 0.21
1.38
W
0.0454
0.29
As
0.004
11.1
U
<d.l.
alphaproteobacteria
71%
Uncertain
20%
Methanococci
Methanomicrobia
Bacteria
Uncultured euryarchaeote
Unidentified archaeon
N=103
gammaproteobactera
6%
uncultured bacteria
6%
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)
Expected amplicon
size (bp)
~800
ARC-915R
GTG CTC CCC CGC CAA TTC CT
BAC-S-8Fa
AGR GTT YGA TYM TGG CTC AG
BAC-S-926R
CCG TCA ATT CMT TTR AGT
nifH_PolF
TGC GAY CCS AAR GCB GAC TC
nifH-PolR
ATS GCC ATC ATY TCR CCG GA
Methanotrophs
A189m
A682m_a
GGN GAY TGG GAC TTY TGG
GAA YSC NGA RAA GAA CGM
~500
Methanogens
Mlas
mcrA-rev
GGT GGT GTM GGD TTC ACM CAR TA
CGT TCA TBG CGT AGT TVG GRT AGT
~500
~900
~340
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. BetaProteobacteria 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.
DNA
Note: Primers were adopted or modified from literature
39 OTUs
DNA
9 OTU’s
RNA
Unid. archaeon pMC2A35
hydrothermal vent archaeon
Methanobacterium (SA-12)
hot spring archaeon
9 OTU’s
RNA
Agrobacterium (alpha)
Polymorphum gilvum (alpha
Thiobacillus sp. (beta)
A bacterium from Beatrix Mine
A beta-Proteobacterium
A bacterium of Rhodospirillaceae (alpha)
12 OTUs
Methylocystis
sp.
35%
Methylomicrobiu
m
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.
Functional gene BLAST results
Ge ne
m cr A ge ne 1
m cr A ge ne 2
n ifH ge n e 1
n ifH ge n e 2
Frequ en cy
6
1
6
1
D escr ipti on
Un cu ltu red
Un cu ltu red
Un cu ltu red
Un cu ltu red
arch ae on clo ne OL-K R 40 _3 48 -35 1m _m cr A _7 8
arch ae on clo ne OL-K R 40 _3 48 -35 1m _m cr A _7 7
ba cte rium clo ne 200 13 -5
nitr oge n-f ixing ba cte riu m iso late
2 Un cu ltu red ba cte rium clo ne AB 0 2
1 C an dida tu s Desu lf oru dis au da xvia to r 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.
Conclusions and Further Work
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 -orDNA sequences.
• 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.
PCR amplification using multiple primer sets
(Primer sequences in Table 2)
Acknowledgements
Ligation and cloning
(pGEM-T Easy Kit, Promega
Sequencing and data-analysis
Figure 2. Methodology of molecular analyses
D.
audaxviator
33%
n ifH ge n e 3
n ifH ge n e 4
15.8
Primer sequence (5’ – 3’)
Methylococcus
sp.
10%
Bacteria
13%
Bacteria
13%
0.0068
6.43
ACK GCT CAG TAA CAC GT
Nitrogen-fixers
Methanomicrobia
41%
Methanomicrobia
(C)
ARC-109F
Bacteria
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.
Unidentified
archaeon 40%
Unidentified
archaeon
40%
Table 2. Primer pairs used in this study
Target group
N=43
Methanococci
6%
Table 1. Water chemistry of borehole waterBE326 as of July 2012
Field
Dissolved
measurement
inorganics
DN
Temp (°C) 38.1
pH
8.55
DIC
Measured Eh
-227 SO42(mV)
Conductivity
7.15 PO43(mS/cm)
Chemet O2
4
NO2(ppm)
Chemet
Soluble Fe
0.3
NO3(ppm)
Chemet Total
0
Mg2+
Fe (ppm)
Chemet H2S
0.1
Sr2+
(ppm)
Chemet H2O2
0.5
Ba2+
(ppm)
Chemet PO4
0.0075 Al3+
(ppm)
Gas Flow Rate
50
Si4+
(mL/min)
Methanomicrobia
30%
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 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.
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.