Molecular Biogeochemistry Lecture
4
• Hopanoids
– Different structures and known bacterial sources,
•
•
•
•
•
C30
C35
Composite
Unsaturated
Methylated
– Biosynthesis
• Squallene hopene cycllase
• Beyond shc
• Genes and taxonomic distribution of hopanoids
– Function
•
•
•
•
Localization
Membrane permeability
Stress responses
Novel functions
1
Hopanoids
• First recognized as a class of C30
pentacyclic triterpenes found in ferns,
ferns mosses and dammar resins
• ‘Hopane’ named after the Dipterocarp plant genus Hopea, itself after botanist John Hope
• Biosynthetic kinship to sterols, tetrahymanol & oleanoids,
oleanoids via squalene recognised in 60’s
© Gaines, Eglinton & Rullkötter
2
Hopanoid Structures: C30 Hopanoids
diplotene
• Diploptene found in all hopanoid producing bacteria
p p detected in most hop
panoid • Diplopterol
producers
• Biosynthetic intermediates only?
diplopterol
3
Side Chain Variations
Composite
Z
NH2
31
O
X
OH
Y
30
OH
HO
NH2
H
N
TETRA: X=OH, NH2, composite; Y = Z = H
NH2
O
OH
O
PENTA: X = OH, NH2, composite; Y = OH, Z = H
X = OH, Y = H, Z = OH
HEXA: X = NH2; Y = Z = OH
4
OH
OH
O
OH
COOH
Analysis Of Biohopanoids
• Hi
Highly
hl functionali
ti
lized,
d amphiphillic
hi hilli
• Not amenable to conventional GC‐MS
• Side chain cleavage (Rohmer et al., 1984)
– Periodic acid/sodium borohydride
– Product structure directly related to number and position of functional groups in side chain
• Specific nature of functional groups lost
5
Periodic Acid Oxidation
OH
TETRA
O
OH O
O
OH
32
OH
H5IO6/NaBH4
OH
O
32 hopanol
32‐hopanol
H
OH
PENTA
OH
31
OH
OH
OH
H5IO6/NaBH4
OH
31‐hopanol
OH
HEXA
OH
OH
30
OH
OH
OH
OH
H5IO6/NaBH4
30‐hopanol
NH2
Topic #1: How was analysis of functionalized (i.e. C35) hopanoids improved? (Helen Talbot papers)
6
Hopanoid Structures: C35 Hopanoids
HO
bacteriohopanetetrol
HO
OH
•
•
OH
Thought to be most common hopanoid produced
Found in most but not all hopanoid producers:
• Cyanobacteria
• Gram‐positive heterotrophs
• Gram
Gram‐negative
negative heterotrophs
• Obligte/Faculative methylotrophs
• Purple nonsulfur bacteria
• Sulfate reducing bacteria
7
Hopanoid Structures: C35 Hopanoids
bacteriohopanepentols
HO
HO
HO
OH
OH
HO
OH
•
•
C35 hopanoids
h
d with
h 5 hydroxyl
h d
l groups ((pentafunctionalized
f
l d)
Only observed in cyanobacteria
8
HO
OH
OH
Hopanoid Structures: C35 Hopanoids
Aminohopanoids
HO
HO
OH
HO
OH
HO
HO
OH
aminobacteriohop
panetriol
NH2
HO
OH
aminobacteriohopanetetrol
NH2
HO
OH
aminobacteriohopanepentol
NH2
9
Hopanoid Structures: C35 Hopanoids
Aminohopanoids
HO
HO
OH
•
aminobacteriohopanetriol
p
NH2
3 h
hydroxyl
d
l groups with
i h amino
i group at C
C‐35
35
• Type I/II methylotrophs
• Nitrogen fixing bacteria
• Beijerinckkia
• Purple nonsulfur bacteria
• Rhodopseudomonas
• Actinobacteria
• Streptomyces
10
Hopanoid Structures: C35 Hopanoids
Aminohopanoids
HO
OH
•
•
HO
OH
aminobacteriohop
panetetrol
NH2
4 hydroxyl groups with amino group at C‐35
Type I, X, II methanotrophs
• Methylosinus
• Type II facultative
• Methylococcus
• Type X obligate
obligate
• Methylomonas
• Type I facultative
11
Hopanoid Structures: C35 Hopanoids
Aminohopanoids
HO
HO
OH
•
•
HO
OH
aminobacteriohop
panep
pentol
NH2
5 hydroxyl groups with amino group at C‐35
Type X methanotrophs
• Methylococcus
• Type X obligate
• Methylocaldum
• Type I/X obligate
obligate
12
Methylotroph vs Methanotroph
• Type I, X, II methylotrophs
• Methylotrophs: aerobic bacteria use C‐1
C 1 compounds as
carbon and energy source
• Methanotrophs: subset of methylotrophs; can use methane
as carbon and energy source
• obligate and/or facultative
13
Type distinction made primarily on how they assimilate formaldehyde:
•
Type II
• Serine pathway
• All α‐Proteobacteria
•
Type I
• ribulose
b l
monophosphate
h h
(
(RuMP)
)
pathway
• β‐Proteobacteria (no CH4 oxidation)
• γ‐Proteobacteria
Proteobacteria (CH4 oxidation)
oxidation)
•
Type X
• subset of Type I
• RuMP + some serine pathway enzymes
• Can grow at higher temperatures
• Usually have a higher G + C content
• γ‐Proteobacteria
Hanson and Hanson, Microbiological Reviews, 1996: p. 439‐471
14
Aminohopanoids as proxies for aerobic methanotrophy?
• Biogenic methane from methanogenesis is severely 13C depleted; reflected in biomass from methanotrophs
• Aminohop
panoid identification coup
pled with stable isotop
pic analyysis good indicator of methanotrophy in given modern environment
HO
OH
HO
OH
NH2
HO
HO
OH
HO
OH
NH2
aminobacteriohopanetetrol
aminobacteriohopanepentol
15
Unexpected occurrence of hopanoids at gas seeps in
the Black Sea
Volker Thiel a,*, Martin Blumenberg b, Thomas Pape ,
Richard Seifertb, Walter Michaelis b,*
b
a Geowissenschaftliches Zentrum der Universität Göttingen, Goldschmidtstr. 3, 37022 Göttingen, Germany
b Institut für biogeochemie und Meereschemie, Universität Hamburg, Bundesstr. 55, 20146 Hamburg, Germany
Received 12 July 2002; accepted 1 October 2002
(returned to author for revision 20 August 2002)
Occurrence of unusual steroids and hopanoids derived from
aerobic methanotrophs at an active marine mud volcano
Marcus Elvert a,*, Helge Niemann b,c
aOrganic Geochemistry Group, Department of Geosciences, University of Bermen, Leobener Strasse, D-28359 Bremen, Germany
b Max Planck Institute for MArine Microbiology, Celsiusstrasse 1, D-28359 Bremen, Germany
c Alfred Wegener Institute for Polar and Marine research, D-27515 Bremerhaven, Germany
Received 26 July 2007; received in revised form 5 November 2007; accepted 15 November 2007
Available online 22 November 2007
Aerobic methanotrophy in the oxic-anoxic transition zone
of the Black Sea water column
Martin blumenberg*, Richard Seifert, Walter Michaelis
Institute of Biogeochemistry and Marine Chemistry, University of Hamburg, Bundesstrasse 55, 20146 Hamberg, Germany
Received 8 February 2006; received in revised form 17 AAugust 2006; accepted 30 August 2006
Available online 30 October 2006
16
Hopanoid Structures: Composite Hopanoids
• Hydroxyl or amino group at C‐35 linked to diverse complex moieties guanidine substituted bacteriohopanetetrol cyclitol ether
bacteriohopanetetrol cyclitol ether
•
•
•
•
Cyanob
bacteria
α‐proteobacteria
• Acetic acid bacteria
• Type II methylotrophs
β‐proteobacteria
• Burkholderia
γ‐proteobacteria
• Azotobacter
•
17
Methylobacterium organophilum
• α‐proteobacteria
• Type II methylotroph
Hopanoid Structures: Composite Hopanoids
• Hydroxyl or amino group at C‐35 linked to diverse complex
moieties •
•
•
bacteriohopanetetrol glycoside
18
Cyanobacteria
α‐proteobacteria
proteobacteria
• Acetic acid bacteria
• Type II methylotrophs
• Zymomonas mobilis
β‐proteobacteria
• Burkholderia
Ring Variations
11 known ring systems
Δ11
2
3
Δ6
19
Hopanoid Structures: Unsaturated hopanoids
• Acetic acid bacteria most abundant producers
• Also produce Δ6Δ11 double unsaturation
• Recentlyy discovered in Burkholderia
• Other unsaturated BHPs found in small amounts in
• Cyanobacteria
• Methylosinus
Unsaturated bacteriohopanetetrol cyclitol ethers
• Methylocaldum
20
Hopanoid Structures: Methylated hopanoids
•
• Methylation at C‐2
•
HO
HO
OH
OH
H3C
HO
HO
OH
OH
OH
H3C
21
Cyanobacteria
• Produce all of these structures
α‐Proteobacteria
• Only methylate some of these structures
• Varies between bacterial classes
• Rhodopseudomonas
• Bradyrhizobium
• Methylobacterium
• Beijerinckia
Hopanoid Structures: Methylated hopanoids
• Methylation at C‐3
•
•
Acetic acid bacteria
Type I and
d Type X meth
hanotroph
hs
Methylhopane biomarker hydrocarbons in Hamersley Province sediments provide
evidence for Neoarchean aerobiosis
Jennifer L. Eigenbrodea,b,* , Katherine H. Freeman a, Roger E. Summons c
a
Department of Geosciences and Penn State astrobiology Research Center, The Pennysylvania State University, University Park, PA, 16802, United States
bGeophysical Laboratory, Carnegie institution of Washington, Washington, DC 20015, United States
cDepartment of Earth, Atmospheric, and planetary Sciences, Massachusetts Institute of Technology, Cambridge MA 02139, United States
Late Archean molecular fossils from the Transvaal Supergroup record the
antiquity of microbial diversity and aerobiosis
Jacob R. Waldbauer a, Laura S. Sherman b,1, Dawn Y. Sumner c, Roger E. Summons b,*
a Joint Program in Chemical Oceanography, Massachusetts Institute of Technology and Woods Hole Oceanographic Institution, Cambridge, MA 02139, United States
b Department of Earth, Atmospheric, and Planetary Sciences, Massachusetts Institute of Technology, Cambridge MA 02139, United States
c Department of Geology, University of California, Davis, CA 95616, United States
22
Signature of 3‐Methylhopanoids
Isotopic
Distribution and C‐isotopic
fractionation in hopanoids of M.
capsullatus as functiion off growth
h stage
23
Summons
et al., GCA, 1994
Tetrahymanol
• Not a hopanoid
• Discovered in ciliated protozoan Tetrahymena pyriformis
• Also found in
• Other ciliates
• An anaerobic rumen fungus: Piromonas communis
• A fern: Oleandra wallicii
• Two α
α‐Proteobacteria
Proteobacteria::
• Rhodopseudomonas palustris
• Bradyrhizobium japonicum
• Proposed to function as sterol surrogates; particularly in anaerobic unicellular eukaryotes
24
Hopanoid and Sterol Biosynthesis
squalene
Enz‐AH+
squalene
l
h
hopene cycllase Enz‐AH+
oxidosqualene cyclase hopene lanosterol
?
bacteriohopanetetrol cholesterol
25
Hopanoid Biosynthesis
squalene hopene cyclase
(Shc)
26
Squalene hopene cyclase
• Most well studied hopanoid
biosynthesis protein
• Purified from 7 organisms
• Crystall structure from A.
acidocaldarius
• Catalyzes one of the most complex
enzymatic one
one‐step
step reaction
• Shc can generate several minor
hydrocarbons in vitro
• Diplopt
Diplopterol
erol (1) viewed as a side
product
• Tetrahymanol (2) catalyzed
from squalene not by Shc but
Stc
• Loose substrate specificity (Table 2)
• More so than the
idosquallene cycllase
oxid
27
Loose substrate specificity of Shc
28
Phylogenetic analysis of Shc
•
•
•
•
•
Welander PV, et al. PNAS, 107: p. 8537‐8542
29
Phylogenetic tree of bacterial species
Blue bar = presence of Shc
Before
Bef
ore genome analysis of Shc, it was thought about 50% of bacteria made hopanoids
• Lipid surveys of approx. 90 strains
that
BLAST analysis
l i off Sh
Shc shows
h
th t is
i onlly about 10%
Shc found in
• Firmicutes
• Actinobacteria
• Thermotogae
• Cyanobacteria
• Planctomycetes
• Acidobactria
• Proteobacteria
α, β,
• δ,
δ α
β γ
• Not ε
Evolutionary link between Shc and Osc?
30
Hopanoid Biosynthesis: Beyond Shc
31
HpnH: Generating the Adenosyl hopane Intermediate
32
HpnH: Generating the Adenosyl hopane Intermediate
• The hpnH gene identified in
Methylobacterium extorquens
• Hp
pnH annotated as a radical SAM
protein
• Transfers adenosine ribose to
diploptene to from adenosyl hopane
• Mechanism not experimentally
verified
• BLAST analysis of HpnH shows that
all Shc containing genomes contain
this protein
• Leads to idea that all hopanoid
producers can make functionalized
hopanoids (i.e., C35 hopanoids)
• Questions the use of adenosyl
hopane as a biomarker for soil
bacteria – just an intermediate
produced by all hopanoid producing
bacteria
33
HpnG: Removal of adenine
34
HpnG: Removal of adenine
• The hpnG gene also first identified in
Methylobacterium extorquens
• HpnG annotated as a nucleoside hydrolase
• Removes adenine nucleotide to form
ribosyl hopane
• Mechanism not experimentally
verified
• BLAST analysis of HpnG inconclusive
• High similarity to other nucleosides
not involved in hopanoid biosynthesis
• Presumably all Shc and HpnH
containing genomes would have this
protein as well
35
Conversion
of ribosyl
hopane
to formyl hopane
• Equilibrium between open and closed form of ribose
• Hypothesis is that no enzyme needed to catalyze this step
36
HpnO: Addition of amino group
37
HpnO:
of amino group
Addition
• The hpnO gene identified in Rhodopseudomonas palustris
• HpnO annotated as an aminotransferase
• Presumably adds amine group to formyl hopane
• Mechanism not experimentally verified
• BLAST analysis of HpnO shows limited numb
ber off aminohopanoid
i h
id producers
d
• Confirms presence in strains known to make aminohoapnoids
• Demonstrates potentially new aminohopanoids producers
38
HpnO: Phylogeny
39
Unknown
biosynthetic steps
•
•
Gene specifically
p
y needed to produce
p
bacteriohopanetetrol not discovered yet
No composite hopanoid biosynthesis genes known
40
Methylation at C‐2: R. palustris Experiments
eutB
hpnN2
hpnP
eutC
4266 hpnC
4265
hpnE
shc
4258 4257 hpnH ispH
hpnG
hpnD
4259
squalene
Enz‐AH
Enz
AH+
hopene methionine
2‐methylhopene Welander PV, et al. PNAS, 107: p. 8537‐8542
41
hpnN1
hpnQ
hpnO
4252 4251
hpnP encodes for the C‐2 methylase
Wild type
diploptene
diplopterol
2‐methyldiplop
y p pterol
2‐methyldiploptene
25.00
24.00
ΔhpnP
24.00
27.00
26.00
diploptene
25.00
28.00
29.00
28.00
29.00
diplopterol
27.00
26.00
Welander PV, et al. PNAS, 107: p. 8537‐8542
Time (min)
42
HpnP: Methylation at C‐2
eutB
hpnN2
hpnP
eutC
4266 hpnC
4265
hpnE
shc
4258 4257 hpnH ispH
hpnG
hpnD
4259
•
hpnQ
hpnO
4252 4251
HpnP annotated as a B‐12 binding radical SAM
• Uses S‐adenosylmethionine radical to add CH3 to C
C‐2
2
• Mechanism not experimentally verified
squalene
Enz‐AH
Enz
AH+
hopene hpnN1
methionine
2‐methylhopene Welander PV, et al. PNAS, 107: p. 8537‐8542
43
Phylogenetic analysis of the HpnP methylase
0.2
sto
cp
un
cti
othe
c
for
m
eP
e sp
CC
73
PCC
7
10
2
4 25
Cyanobacteria
ac
ob
oe
l
G
No
Cyan
N wino
N h ogradskyi Nb255
amb
u rg
ens
is X1
4
9
s BP1
A
2
Cya
noth
e
4
hanic
um CM
M chlo
romet
OR
S20
60
erium sp 446
Meethylobact
lan
s
Mn
odu
boxi
d
Oligo
trop
h
ce s
p
28
PCC
742
4
31
s
an
er
l
o
ot
di
1
ra
J00
M uli B
p
o
M pextorquens AM1
M
M
ex
t or
qu
en
sP
A1
M4
ens D
icca ATCC9039
Beijerinckia ind
Methy
locella
silves
tris BL
2
ovor
ans O
M5
M
JC
Bra
dyr
hiz
ob
i um
sp
B jap
BT A
oniicu
i1
m US
DA11
0
us
1
torqu
M ex
isB18
stris B
R palu
53
isA
B
is
str
alu
p
78
R
S2
R
O
sp
um
i
b
izo
rh
y
ad
Br
te
e
ac
io l
rv
42
C7
PC
22
PCC78
ce sp
e
h
t
o
Cyan
TIE1
a c ar
stris
R palu
N b 31 1
A
Ha
CG
A0
0
p
acter s
s
stri
tris
Nitrob
alu
alu
s
R palustris BissB5
Rp
Rp
coccuss elongatu
Thermosynecho
other Rhizobiales
Cand Koribacter versatilis Ellin345
Acidobacterium
Methylobacteria
Welander PV, et al. PNAS, 107: p. 8537‐8542
44
Top
pic 4: Are 2‐methyylhop
panes good biomarkers for cyyanobacteria and/or O2‐p
photosyynthesis?
45
Methylation at C‐3: M. capsulatus experiments
I
A
HO
HO
OH
HO
OH
Wild type
II
NH2
III
IV
18 20 22 24 26 28 30 32
I
B
ΔhpnR
methionine
II
18 20 22 24 26 28 30 32
C
III
I
ΔhpnR +
pPVW100
II
IV
18 20 22 24 26 28 30 32
Time (min)
(min)
I and II: desmethyl aminohopanoids
III and IV: C‐3 methylated aminohopanoids
46
Methylation at C‐3
HO
HO
OH
methionine
HO
OH
NH2
• HpnR also annotated as a B‐12 binding radical SAM
• Uses S‐adenosylmethionine radical to add CH3 to C‐3
• Mechanism not experimentally verified
• Very low sequence identity to HpnP
• Although share the B‐12
B 12 binding and radical SAM motifs
• Raises evolutionary questions about the similarity of these two methylations
Topic 5: What is the C‐2 and C‐3 methylation mechanism proposed earlier in the literature? How is it different from the use of radical SAM chemistry?
47
Phylogenetic analysis of the HpnR methylase
• Very few bacteria with HpnR in their genomes have been tested for hopanoid production
• Two have been tested (*) and no 3‐methylhopanoids reported.
• If HpnR is correlated to 3‐
methyylhop
panoid production in other organisms:
• Expands diversity of 3‐
methylhoapnoid producers beyond methanotrophs and acetic acid bacteria
• Actionbacteria
• α, γ, and
d β‐P
Proteob
t bactteriia
• Nitrospirae
• Acidobacteria
U l ified
d organiism
• Unclassifi
48
Functional Role of Hopanoids?
Rohmer & Ourisson, 1976
Rohmer et al., 1979
Kannenberg & Poralla, 1980
Many lines of evidence show an association of hopanoids with cellular membranes
But majority were in vitro studies.
What about in vivo studies?
49
Hopanoid localization in Nostoc punctiforme
•
•
•
Hopanoids localize to the outer membrane; none to the cytoplasmic
• Also observed in M. capsulatus
Akinetes are resting state structures that do not do oxygenic photosynthesis
Functional role not involved in oxygenic photosynthesis
50
Outer membrane versus cytoplasmic membrane
LPS
Outer membrane
Cytoplasmic membrane
• Gram‐negative bacteria have outer membrane in addition to cytoplasmic membrane
• Studies are finding that hopanoids localize to this membrane
• Hopanoid membrane studies were all done in cytoplasmic membrane models
• Do they apply in vivo?
• Currently no in vitro system available to model the outer membrane
51
Other in vivo studies: R. palustris shc mutant
eutB
tB
h N2
hpnN2
h P
hpnP
eutC
h C
4266 hpnC
4265
h E
hpnE
shc
h
i H
4258 4257 hpnH
h H ispH
h G
hpnG
hpnD
4259
squalene
squalene hopene cyclase (Shc) hopene 52
h N1
hpnN1
hpnQ
h O
hpnO
4252 4251
Δshc strain no longer produces hopanoids
tetrahymanol
triglycerides
BHTs and 2‐MeBHTs
Wild type
diploptene
20
Δshc
aminoBHT
25
30
squalene
20
35
40
35
40
triglycerides
25
30
Time (hours)
53
Sensitivity of hopanoid mutant to pH
unbuffered medium
0.4
Wild
TIE
-1 type
Δshc
shc
0.5
0.3
0.4
OD600
OD600
O
MOPS buffered , pH 7 medium
0.6
0.2
0.1
0.3
0.2
Wild
TIE
-1 type
0.1
Δshc
shc
0
0
0
50
100
150
0
200
100
150
200
Time (hours)
Time (hrs)
pH 7.2
2
50
pH 8.2
pH 7.0
54
pH 7.0
Membrane integrity of the Δshc mutant is compromised
Aerobic Growth 30oC 0 5% bile
0.5%
bil salts
lt
0.5
0.5
0.4
0.4
03
0.3
03
0.3
OD600
OD600
Aerobic Growth 30oC No bil
bile sallts
Wild
type
wild
type
0.2
Wild
type
wild
type
0.2
Δshc
Δshc
Δshc
Δshc
0.1
0.1
0
0
0
20
40
60
80
0
100
20
40
60
Time (hours)
Time (hours)
55
80
100
Sensitivity to bile salts is indicative of a permeable OM
OM
Periplasm
CM
Bile salts
LPS
Porin
56
Membrane integrity of other hopanoids mutants is NOT compromised
Does this indicate a novel function for amino and methylated hopanoids?
0.5
0.5
No bile salts
0.5% bile salts
0.3
0.3
OD600
0.4
OD600
0.4
wild type
Δ h
Δshc
0.2
0.2
ΔhpnH
ΔhpnO
0.1
0.1
ΔhpnP
ΔhpnN
0
0
0
20
40
60
80
0
100
20
40
60
80
Time (hours)
ΔhpnH: Only C30 hopanoids produced
ΔhpnO: No amino hopanoid production
ΔhpnP:
h P No
N methylated
th l t d h
hopanoid
id production
d ti
ΔhpnN: No hopanoids in outer membrane
57
100
Functional role for 3‐methylhopanoids in stationary phase survival?
Growth experiments with M. capsulatus hpnR mutant
Growth over time at 37°C
Cell survival assay
0.16
1 0E+10
1.0E+10
0.12
ΔhpnR
1.0E+08
1.0E+06
0.08
1.0E+04
0.04
Colony Forrming Units
Optical Density (600nm)
M. capsulatus
0
0
0.5
1
1.5
2
2.5
0 16
0.16
0.12
1.0E+02
1.0E+00
Day 2
1.0E+10
Day 7
R. palustris
Day 14
ΔhpnP
1.0E+08
1.0E+06
0.08
wild type
0.04
1.0E+04
ΔhpnR
1.0E+02
0
0
25
2.5
5
7.5
7
5
10
12.5
12
5
1.0E+00
15
Day 3
Time (Days)
58
Day 7
Day 14
Functional role for 3‐methylhopanoids in stationary phase survival?
Growth experiments with M. capsulatus hpnR mutant
Cell survival assay
1 0E+10
1.0E+10
M. capsulatus
ΔhpnR
1.0E+08
1.0E+06
Colony Forrming Units
1.0E+04
3 methyls?
3‐methyls?
1.0E+02
1.0E+00
Day 2
1.0E+10
Day 7
R. palustris
Day 14
ΔhpnP
1.0E+08
1.0E+06
1.0E+04
1.0E+02
1.0E+00
Day 3
Methylococcus cyst
59
Day 7
Day 14
Diagnostic Bacteriohopanes?
19
25
2
3
21
22
11
16
10
29
35
32
17
14
1
OH
OH
R2
OH
R1
diagenesis
maturation
R3
4
cyanobacteria
cyanobacteria
δ13C ‐20 to ‐35 ‰
19
25
2
3
4
10
21
22
17
11
14
1
OH
16
29
OH
35
32
O
OH
OH
NH 2
diagenesis
maturation
methanotrophic bacteria
R3
methanotrophs δ13C ‐45 to ‐80 ‰
• Will the physiological and biochemical data reveal that certain hopanes are better proxies for microbial processes rather than a specific bacterial group? 60
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12.158 Molecular Biogeochemistry
Fall 2011
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