Supplementary information
Thermodynamic prediction of stoichiometry
Calculations for Ph oxidation coupled to partial denitrification followed Rittmann
and McCarty (2001). ΔG values were obtained from Helgeson et al. (1998) with the
5
exception of Ph and Pr, which were determined using the group contribution
method as described in Mavrovouniotis (1991).
10
15
ΔGpc = energy of formation of pyruvate from substrate
ΔGpc = 35.09 - ΔGsubstrate,
n = -1 if ΔGpyruvate synthesis < 0, n = +1 if ΔGpyruvate synthesis > 0
ΔGp = energy for the formation of cellular material from pyruvate
ΔGp = 18.8 kJ/e- eq with NH4+ as a N source
(5)
ε: efficiency of energy transfer, 0.4 – 0.7 for anaerobic heterotrophs
ΔGcell synth = (ΔGpyruvate synth/ εn) + (ΔGpyruvate synth/ εn)
ΔGR = reaction free energy of the coupled electron acceptor and donor
ΔGR = ΔGdenitrification – ΔGPh oxidation
A = # eq of the electron donor oxidized per eq of cell synthesis
A = [(ΔGpyruvate synth/ εn) + (ΔGcell synth/ εn)]/ε ΔGR
fS0 = e- eq cell mass /e- eq donor fe0 = e- eq cell mass/ e- eq acceptor
fS0 = 1/(1 +A)
fe0 = 1 - fS0
(1)
(2)
(3)
(4)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
20
25
30
NO3- + 2H+ + 2e-  NO2- + H2O
6(-CH3) + 10(–CH2) + 4(–CH)  C20H42
C20H42 + 60H2O  20HCO3- + 142H+ + 122e6(-CH3) + 9(-CH2) + 4(-CH)  C19H40
C19H40 +57H2O  19HCO3- + 135H+ + 116e-
ΔG = -81.52 kJ/e- eq
ΔG = 107.76 kJ/mol
ΔG = 19.57 kJ/e- eq
ΔG = 99.97 kJ/e- eq
ΔG = 16.62 kJ/e- eq
For Ph with ε = 0.7:
ΔGpyruvate synth = (35.09 kJ/e- eq) – (107.76 kJ/e- eq) = -72.63 kJ/e- eq
ΔGcell synth = (-72.63 kJ/e- eq /0.7) + (18.8 kJ/e- eq /0.7) = -76.9 kJ/e- eq
ΔGR = (-81.52 kJ/e- eq) – (19.57 kJ/e- eq) = -101.09 kJ/e- eq
A = [(-72.63 kJ/e- eq) /0.7 + (18.8 kJ/e- eq) /0.7] /[ 0.7*-101.09 kJ/e- eq] = 1.09
fS0 = 1/(1 + 1.09) = 0.48
fe0 = 1 – 0.48 = 0.52
For Ph with ε = 0.4:
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
35
40
ΔGcell synth = (-72.63 kJ/e- eq /0.4) + (18.8 kJ/e- eq /0.4) = -134.58 kJ/e- eq
A = [(-72.63 kJ/e- eq) /0.4 + (18.8 kJ/e- eq) /0.4] /[ 0.4*-81.52 kJ/e- eq] = 3.33
fS0 = 1/(1 + 2.62) = 0.23
fe0 = 1 – 0.46 = 0.77
Electron acceptor
0.5NO3- + H+ + e-  0.5NO2- + 0.5H2O
Electron donor
0.01C20H42 + 0.6H2O  0.2HCO3- + 1.42H+ + eCell synthesis
0.25HCO3- + 0.05NH4+ + 1.2H+ + e-  0.05C5H7O2N + 0.65H2O
(24)
(25)
(26)
(27)
(28)
(29)
45
For Ph with ε = 0.7:
0.01C20H42 + 0.26NO3- + 0.024NH4+ + 0.028H2O  0.08HCO3- + 0.024C5H7O2N +
0.26NO2- + 0.32H+
(28)
NO3-: HCO3- = 3.25
(30)
50
For Ph with ε = 0.4:
0.01C20H42 + 0.39NO3- + 0.012NH4+ + 0.07H2O  0.14HCO3- + 0.012C5H7O2N +
0.39NO2- + 0.37H+
(31)
NO3-: HCO3- = 2.70
(32)
55
60
65
70
For Pr with ε = 0.7:
ΔGpyruvate synth = (35.09 kJ/e- eq) – (99.97 kJ/e- eq) = -64.88 kJ/e- eq
ΔGcell synth = (-64.88 kJ/e- eq /0.7) + (18.8 kJ/e- eq /0.7) = -65.83 kJ/e- eq
ΔGR = (-81.52 kJ/e- eq) – (16.62 kJ/e- eq) = -98.14 kJ/e- eq
A = [(-64.88 kJ/e- eq) /0.7 + (18.8 kJ/e- eq) /0.7] /[ 0.7*-98.14 kJ/e- eq] = 0.96
fS0 = 1/(1 + 0.96) = 0.49
fe0 = 1 – 0.49 = 0.51
(33)
(34)
(35)
(36)
(37)
For Pr with ε = 0.4:
ΔGcell synth = (-64.88 kJ/e- eq /0.4) + (18.8 kJ/e- eq /0.4) = -115.2 kJ/e- eq
A = [(-64.88 kJ/e- eq) /0.4 + (18.8 kJ/e- eq) /0.4] /[ 0.4*-98.14 kJ/e- eq] = 2.93
fS0 = 1/(1 + 2.93) = 0.25
fe0 = 1 – 0.25 = 0.75
(38)
(39)
(40)
Electron acceptor
(1) 0.5NO3- + H+ + e-  0.5NO2- + 0.5H2O
Electron donor
(2) 0.01C19H40 + 0.57H2O  0.19HCO3- + 1.35H+ + eCell synthesis
(3) 0.25HCO3- + 0.05NH4+ + 1.2H+ + e-  0.05C5H7O2N + 0.65H2O
(41)
(42)
(43)
75
For Pr with ε = 0.7:
0.01C19H40 + 0.25NO3- + 0.026NH4+  0.063HCO3- + 0.026C5H7O2N + 0.25NO2- +
0.25H+ + 0.007H2O
(44)
NO3 : HCO3 = 3.92
(45)
80
For Pr with ε = 0.4:
0.01C19H40 + 0.38NO3- + 0.013NH4+ + 0.33H2O  0.13HCO3- + 0.013C5H7O2N +
0.38NO2- + 0.3H+
(46)
NO3 : HCO3 = 2.94
(47)
85
Ph and Pr degradation rate constant calculations
Pr degradation rate constants were calculated from the loss of Pr over 40
days. Pr concentration at any time point was determined by using the stoichiometry
determined for ε = 0.7 or ε = 0.4 and the moles of DIC measured. The ratio of DIC to
Pr was 6.25 for ε = 0.7, and 12.75 for ε = 0.4. We applied zero, first and second-order
90
models to explain the kinetics of Pr degradation. Based up the R2 values of linear
fits to the Pr degradation models and for comparison with described hydrocarbon
biodegradation rate constants (Grossi et al., 2000; Larter et al., 2003), we report the
first order rate constant. For Pr, k = 1.1 yr-1 (r2 0.76, n=8) to 2.5 yr-1 (r2 0.77, n = 8).
Values used in determining the linear fit (time = k*ln[Pr] +b) are reported in Table
95
S-2. Ph degradation rate constants were calculated as described for Pr. The ratio of
DIC to Ph was 8.0 for ε = 0.7, and 14.25 for ε = 0.4. For Ph, k = 0.10 yr-1 (r2 0.83,
n=15) to 0.20 yr-1 (r2 0.83, n = 15). Values used for determining the rate constant are
reported in Table S-3.
100
Analysis of labeled Ph purity and yield
Growth of 28 l Haloferax sulfurifontis SD1 resulted in 33.4 g lyophilized
biomass. A modified Bligh-Dyer extraction and acid methanolysis of the biomass
yielded 60.9 mg saponified material. After derivatization with BSTFA, analysis
with GC-MS revealed the saponified material to be archaeol (S-1). Assignment of
105
archaeol followed MS fragmentation patterns of Teixidor and Grimalt (1992) and
Steihl et al. (2005). Major MS fragments (m/z) of the archaeol-TMS derivative
include: the M+. (724 m/z); M – C20H41 (445 m/z); M – C20H41OH (426 m/z); M –
C20H41OSi(CH3)3 (369 m/z); C20H41 (278 m/z); and CH2CHCH2OSi(CH3)3 (130 m/z).
After treatment of archaeol with HI, extraction with benzene recovered 50.0
110
mg phytyl iodide. Initially, the phytyl iodide extract had a distinct purple color, but
became faintly yellow following a second extraction with benzene and 5% sodium
thiosulfate. Reductive dehalogenation of phytyl iodide with Zn and CH3CO2H
produced 39.8 mg Ph. GC-MS showed Ph to be the dominant peak, but also showed
the presence of several additional peaks (Fig. S-2). Purification with Supelclean LC-
115
Si SPE tubes (6 ml, Supelco, Bellefonte, PA) resulted in the elution of 22.2 mg Ph.
GC-MS showed a single peak, assigned as phytane from comparison with the NIST
08 mass spectra library (Fig. S-2).
120
Fig. S-1. Total ion chromatogram and mass spectrum of saponified 13C-labeled
diphytanyl glycerol diether from Haloferax sulfurifontis SD1.
Fig. S-2. Total ion chromatogram and mass spectra of 13C-labeled Ph before and
125
after SPE purification.
Fig. S-3. Quantitative FISH counts of isoprenoid degrading enrichment over the
course of seven transfers, showing the fraction of DAPI stained cells hybridizing to a
130
Pseudomonas stutzeri probe (Pseu15, triangles), a Commamonas probe (Cte659,
squares) and a Bacteriodales probe (CFB719b, diamonds). Error bars represent ±1
standard deviation based upon the counting of a minimum of 1000 cells.
135
Fig. S-4. (A) Keeling plot of δ13C for DIC produced during incubations on 13Clabeled Ph vs 1/[DIC]. Data points represent average of 3 replicate incubations and
error bars are ± 1 standard deviation. (B) Contribution of background carbon (δ13C 40‰) and 13C-labeled Ph (δ13C +325‰) to DIC production in a two component
mixture. Background carbon is the major source of DIC produced in the initial 0-20
140
days, while 13C-labeled Ph is the source of DIC throughout the remainder of the
incubation.
Table S-1
Oligonucleotide probes used.
Formamide
Probe
Target group
Sequence (5’ 3’)
Target site
Reference
(%)
(Amann et al.,
EUB338a
Most Bacteria
GCTGCCTCCCGTAGGAGT
0 – 50
16S (338-355)
1995)
EUB338-
(Daims et al.,
Planctomycetales
IIa
GCAGCCACCCGTAGGTGT
0 – 50
16S (338-355)
1999)
EUB338-
(Daims et al.,
Verrucomicrobiales
IIIa
GCTGCCACCCGTAGGTGT
(Simplicispira sp.)
16S (338-355)
1999)
(Schleifer et al.,
Comamonadaceae
CTE659
0 – 50
TTCCATCCCCCTCTGCCG
40
16S (659-676)
1992)
Pseudomonas
Pseu15
GCAAGCTCCACTCATCCGCT
stutzeri
CFB719b
Bacteriodales
a Combined
145
(Demaneche et
AGCTGCCTTCGCTATCGG
in equimolar amounts to make EUBMIX.
35
16S (64-83)
al., 2008)
30
16S (719-737)
This study
Table S-2
Values used for Pr first-order rate constant determination.
ε = 0.7
n=8
150
ε = 0.4
Time
(days)
HCO3(µM)
Pr (µM)
ln[Pr]
Pr (µM)
ln[Pr]
0
0
729.0
6.59
729.0
6.59
7
508.9
647.6
6.47
689.1
6.54
20
1152.6
544.6
6.30
638.6
6.46
39
1078.6
556.5
6.32
644.4
6.47
0
0
729.0
6.59
729.0
6.59
7
565.3
638.6
6.46
684.7
6.53
20
1040.5
562.5
6.33
647.4
6.47
39
1154.7
544.3
6.30
638.5
6.46
Table S-3
Values used for Ph degradation first-order rate constant determination.
ε = 0.7
n = 18
155
ε = 0.4
Time
(days)
HCO3(µM)
Ph (µM)
ln[Ph]
Ph (µM)
ln[Ph]
0
0
699.8
6.55
699.8
6.55
7
13.7
697.7
6.55
698.6
6.55
20
35.0
695.5
6.54
697.3
6.55
47
58.5
692.5
6.54
695.7
6.54
82
118.8
686.9
6.53
692.5
6.54
120
341.4
660.0
6.49
677.4
6.52
0
0
699.8
6.55
699.8
6.55
7
11.4
698.3
6.55
699.0
6.55
20
43.8
694.3
6.54
696.7
6.55
47
72.0
690.8
6.54
694.7
6.54
82
177.6
677.5
6.52
687.3
6.53
120
387.6
651.3
6.48
672.5
6.51
0
0
699.8
6.55
699.8
6.55
7
13.7
698.0
6.55
698.8
6.55
20
26.7
696.4
6.55
697.9
6.55
47
45.2
694.1
6.54
696.6
6.55
82
76.2
690.2
6.54
694.4
6.54
120
318.3
660.0
6.49
677.4
6.52
References
160
Amann, R.I., Ludwig, W., Schleifer, K.H., 1995. Phylogenetic identification and in
situ detection of individual microbial cells without cultivation. Microbiology Reviews
59, 143-169.
Daims, H., Bruhl, A., Amann, R., Schleifer, K.-H., Wagner, M., 1999. The domainspecific probe EUB338 is insufficient for the detection of all Bacteria: development
and evaluation of a more comprehensive probe set. Systematic and Applied
Microbiology 22, 434-444.
165
170
175
Demaneche, S., Sanguin, H., Pote, J., Navarro, E., Bernillon, D., Mavingui, P.,
Wildi, W., Vogel, T.M., Simonet, P., 2008. Antibiotic-resistant soil bacteria in
transgenic plant fields. Proceedings of the National Academy of Sciences USA 105,
3957-3962.
Grossi, V., Raphel, D., Hirschler-Rea, A., Gilewicz, M., Mouzdahir, A., Bertrand, J.C., Rontani, J.-F., 2000. Anaerobic biodegradation of pristane by a marine
sedimentary bacterial and/or archaeal community. Organic Geochemistry 31, 769.
Helgeson, H.C., Owens, C.E., Knox, A.M., Richard, L., 1998. Calculation of the
standard molal thermodynamic properties of crystalline, liquid, and gas organic
molecules at high temperatures and pressures. Geochimica et cosmochimica acta
62, 985-1081.
Larter, S., Wilhelms, A., Head, I., Koopmans, M., Aplin, A., Di Primio, R., Zwach,
C., Erdmann, M., Telnaes, N., 2003. The controls on the composition of biodegraded
oils in the deep subsurface—part 1: biodegradation rates in petroleum reservoirs.
Organic Geochemistry 34, 601-613.
180
Mavrovouniotis, M.L., 1991. Estimation of standard Gibbs energy changes of
biotransformations. The Journal of Biological Chemistry 266, 14440-14445.
Rittmann, B.E., McCarty, P.L., 2001. Environmental Biotechnology: Principles and
Applications. McGraw-Hill.
185
190
Schleifer, K.H., Amann, R., Ludwig, W., Rothemund, C., Springer, N., Dorn, S.,
1992. Nucleic acid probes for the identification and in situ detection of
pseudomonads. In: Galli, E., Silver, S., Withold, B. (Eds.), Pseudomonas: Molecular
Biology and Biotechnology. American Society for Microbiology, Washington, pp. 127134.
Stiehl, T., Rullkötter, J., Nissenbaum, A., 2005. Molecular and isotopic
characterization of lipids in cultured halophilic microorganisms from the Dead Sea
and comparison with the sediment record of this hypersaline lake. Organic
Geochemistry 36, 1242-1251.
195
Teixidor, P., Grimalt, J.O., 1992. Gas chromatographic determination of isoprenoid
alkylglycerol diethers in archaebacterial cultures and environmental samples.
Journal of Chromatography A 607, 253-259.