environmental signals from ‘biochemical dustbins’ · environmental signals from ‘biochemical...
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Environmental Signals from ‘Biochemical Dustbins’
Richard EvershedOrganic Geochemistry Unit
Bristol Biogeochemistry Research CentreSchool of Chemistry
Environmental ‘Dustbins’ for Biochemical Products
Plant matterAnimal matterGaseous products
Mineral products
Atmosphere Water
Lake sedimentsOcean sediments
Soil
Microbial matter
Soils and peatsArchaeological remains
RecyclingGeological deposits
Return to the living world
Chemical and
microbialprocessing
(decay)
Living world D
ead or ganic matter
ANALYTICAL CHEMISTRY
Molecular and stable isotopic compositions
ORGANIC GEOCHEMISTRY
Reconstructing past climate and
environments (peat bogs and lakes)
BIOGEOCHEMISTRY
Role of microbes and invertebrates in soil
organic matter cycling
BIOMOLECULAR PALAEONTOLOGY
Fossilisation processes and
palaeoenvironmentalinformation
BIOMOLECULAR ARCHAEOLOGY
Reconstructing human activity in the past
Analytical chemistry of molecules past and present
Biomarker compounds
CO2H
CH3
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH3
CH2
CH2
CH2
CH C
H2
CH
CH
CH
CH2
CH2
CH
CH3
CH3CH3
CH3 CO2H
Tree resin biomarker
Plant leaf wax biomarker
Bacterial fatty acid biomarker
Molecules that can be linked to biological sources due to their characteristic structures
O
OHCH3
CH C
H2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
O
OH
CH3
n-alkane
Abieticacid
C29
Analytical Chemistry: ‘Needle in a Haystack’ Methods
Need to determine either one or a few compoundsfrom materials containing many thousands of substances
Analytical Chemistry: ‘Needle from a Haystack’
Biochemically complex samples100,000s of compounds
Lipid extract1,000s of compounds
Fraction10s of compounds
Separation
Separation
Solvent extraction
Gas chromatographyDo we have biomarkers? How much do we have?
Separation
GC/mass spectrometryWhat are the structures of the biomarkers?
GC/isotope ratio/MSWhat are the stable isotope values of the biomarkers?
State-of-the-art instrumentation vital for this type of work
Gas chromatography based analytical methods
Inte
nsity
Retention time
Gas chromatography
Separates compounds according to differences in boiling point and polarity
O2/Cu/Pt 850oC
13CO2 + 12CO2
Isotope ratio mass spectrometry
D/H + H2
Isotope ratio mass spectrometry
1400oC
Organic mass spectrometry
δD values
Mass spectra
δ13C valuesStructures
Palaeoclimate reconstruction‘Using the past to predict the future’
‘Using the past to predict the future’
Ocean sediments
Ice cores
Tree rings
Using the past to predict the future:Peat bogs as archives of past climate
Ombrotrophic mire formation
Atmosphere the only source of nutrients
Plants growing on surface highly
sensitive to climate change
Mineral substratum
Lake sediment
Fen peat
Bog peat
Peat bogs as archives of past climate
Bolton Fell Moss,
Cumbria
C29
C23
Sedge biomarker
Sphagnum moss biomarker
Identification of biomarkers by analysis
of modern plants
(Prefer drier conditions)
(Prefer wetter conditions)
Sedge fossil
Sphagnumfossil
GC analysis of peat n-alkanes
C23
Retention time
Sphagnum mossbiomarker
Sedge biomarker
Bacterial b io m
a rk er
C31
n-Alkanes recording the changes in plants forming peat bogs in the past
0.01
1770
1820
1870
1920
1970
0.1 1 10
Calendar years (AD)
Calendar years (A
D)
0 100
Sphagnum (%)Peat core
n-C23
Cha
ngin
g v e
geta
tion
Log [C23/31]
Increasing bog surface wetness
Present day
Past 210Pb/14C
Effect of temperature on 2H/1H ratio (δD)2H/1H content at higher latitudes (>30o) varies seasonally
with a maximum in the summer and a minimum in winterThe
Summer: high δD
Winter: low δDHence, δD of plant biochemical should reflect differences
in temperature during growing season
1770
1820
1870
1920
1970
15.0 15.2 15.4 15.6
Years (AD)
-180 -160 -140
Cha
ngin
g t e
mpe
ratu
re
Temperature Increasing δD
n-Alkane δD values correlate with recorded temperature for the past 200 years
Cal
enda
r yea
rs (A
D)
Peat core Present
day
Past
The ACCROTELM Project
Generating calibrated records of rainfall and temperature change from peat bogs across Europe using biomarkers and a range of other indicators
No need to lie!No need to lie!
Biogeochemistry of the methane cycle:
‘Stable isotope fishing for methane eating bacteria’
Methane (CH4) and global warming
• CH4 contributes (mole for mole) 26 times more to climate change than CO2
• During the past century CH4 methane has contributed 15-25% to global warming
Etheridge et al . (1998)
• Global CH4 concentrations have been increasing for the past 300 years reaching a global mean of 2 ppmv
• Sources of CH4– Wetlands– Agriculture– Landfill Sites– Energy production and use – Natural gas venting– Biomass burning– Domestic Sewage
• Sinks of CH4
– Chemical reactions in the atmosphere with OH
.
Soils via methane oxidising bacteria (methanotrophic bacteria:methane eating bacteria)
Sources and sinks of methane
Source
Sink
2 ppmv
• An enhanced understanding of the methanotrophicbacteria would help in managing methane emissions
e.g. forest management, fertilizer effects, etc.
–
Methane oxidising bacteria – methanotrophic bacteria
• Two main groups:
Low affinity methanotrophs oxidise methane at high concentrations and have been isolated and characterised
CH4 CH3OH HCHO HCO2H CO2
Divided into three main groups– Type I: C16 fatty acids– Type II: C18 fatty acids– Type X: mixture of characteristics
High affinity methanotrophs oxidise methane at low concentrations but cannot be isolated and cultured
•
• Major problem - these are the soil methane sink!
Culture independent methods:Phospholipid fatty acid (PLFA) analysis of soil microbes
• Phospholipids are major components of cell walls
O
OH
OH
PO
O
O
O
O
O
O
O
OHO
OH
• Phospholipids short-lived upon cell death• PLFAs represent living microbes
Phospholipid
Chemical release_
Phospholipid fatty acids (PLFAs)
GC profiling of PLFAs of soil microbes
• Wide range of microbial groups represented but impossible to say which PLFAs come from methanotrophs
‘Stable isotope fishing for methane eating bacteria’Selective 13C-labelling of subset of microbial population
13CH4
Incubate
Highly complex soil microbial community
Only bacteria consuming 13CH4
become 13C-labelled
• Challenge: detection of very low concentrations of 13C-labelled PLFA amongst 10,000 x the concentration of unlabelled PLFAs
Synthetic air + 13CH4
Soil incubationsFlow through chamber
Soils
Chamber flushed every 24 hSynt
hetic
air
+ 13
CH
4
Maxfield et al. Environ. Microbiol. (in press)
Exposure of soils to different concentrations of 13CH4 : high and low affinity methanotrophs
• Analyse PLFAs by GC-IRMSto locate 13C-label
• Only the PLFAs from the methanotrophic bacteriatake up 13CH4
• Dominance of 18:1 PLFAindicates Type II methanotrophic bacteria oxidising methane at both high and low concentrations
• Concentrations of 13C-labelledPLFAs indicates methanotrophsare ca. 0.01% of all soil bacteria
δ13 C
val
ues
13C
-PLF
A c
once
ntra
tion
PLFA Crossman et al. Org. Geochem. (2005)
• Run by IGER (Institute of Grassland & Environmental Research)
• Long term study: 6 different soil treatments over the past 14 years
• Wider investigation the impact of grazing and other agricultural practices on uplands
Effects of low input farming in the Brecon Beacons, South Wales
What is the impact of the various treatments
on methanotrophicbacteria?
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
br15
:0
a15:
0
n16:
0
16:1
w9
br17
:0
i17:
0
a17:
0
n17:
0
n18:
0
18:1
w11
18:1
w7c 19
:1
19:1
N Ca P K fertilizer
0
2
4
6
8
10
12
br15
:0
a15:
0
n16:
0
16:1
w9
br17
:0
i17:
0
a17:
0
n17:
0
n18:
0
18:1
w11
18:1
w7c 19
:1
19:1
Methanotroph PLFAs
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
br15
:0
a15:
0
n16:
0
16:1
w9
br17
:0
i17:
0
a17:
0
n17:
0
n18:
0
18:1
w11
18:1
w7c 19
:1
19:1
Grazed
Total soil bacterial PLFAs
Bronydd Mawr NCaPK
0
2
4
6
8
10
12
br15
:0
a15:
0
n16:
0
16:1
w9
br17
:0
i17:
0
a17:
0
n17:
0
n18:
0
18:1
w11
18:1
w7c 19
:1
19:1
N Ca P K fertilizerDecreased
methanotrophicbacteria
Grazed
PLFA
μg
g-1
13C
-labe
lled
PLFA
μg
g-1
Phospholipid fatty acid (PLFA)
Effect of a common fertilizer on soil methanotrophs
Increased soil bacteria
Reconstructing human activity in the past
‘Molecules that time forgot’
Archaeological BiomarkersAncient biomolecules that carry information concerning
human activity in the pastCan be any class of compound preserved in organic residues at
archaeological sites, e.g. DNA, protein, lipids, etc.
Bronze Age soil
Resins, tars, pitches and
bitumens
Skeletal remains
Bog bodyRoman
cosmetic
Plant remains
Animal mummy
CO2H CO2H
CO2Me CO2Me CO2Me
Abietic acid Pimaric acid
Heating
GC/MS of Wood Tar From King Henry VIII’s Flagship
Archaeological Information Retrievable from Preserved Ancient Biomolecules
• Commodities• Specific uses of artefacts• Ancient technologies• Diet• Agriculture• Hunting
• Origin of peoples• Trade• Cultural differences• Ritual activities• Dating• Decay (taphonomy)
• Abundant
• Durable
• Diagnostic
• Preserve organic residues
Potsherds
Surface residues
Absorbed residues
Organic Residues in Archaeological Pottery
GC/MS analysis of lipids from a medieval cooking vessel
Partial gas chromatogram
Mass spectra
Modern cabbage leaf wax
Late Saxon/Medieval cooking vessel
Rel
ativ
e in
tens
ity
Time (min)
Wild- type cabbage
GC ‘fingerprints’
Animal Fats in Archaeological Pottery
• Degraded animal fats are identified by their characteristic distributionof mono-, di- and triacylglycerols and abundant saturated fatty acids
Uses of Fats (and Oils) in Antiquity
• Essential dietary component• Illuminants (lamp fuel and candles)• Polishes and lubricants• Art materials, e.g. binders• Base for perfumes and ointments• Embalming agents and religious commodities
• Man would have actively recovered fat for these activities
• Often mixed fat with other commodities – pottery vessels would have been used in all these activities
Dudd and EvershedScience (1998)
Classification of animal fats based on compound-specific δ13C values of C16:0 and C18:0 fatty acids
*
Fats from modern animals
δ13C16:0
δ13 C
18:0
Isotopic difference between C 18:0 in ruminant carcass (adipose) and milk fats
Copley et al. PNAS (2003)
Dairying in Antiquity:The traditional evidence
• Pictures
• Perforated vessels Said to be cheese strainers/presses?
• Archaeological bonesAnimals raised for dairy/meat/traction have different herd structures
• Relatively easy to identify domestic animals but much more difficult to establish exactly what they were used for
Southern British sites investigated in milk project Prehistoric Pottery
• 14 sites spanning early Neolithic to Iron Age
• ca. 1000 individual vessels analysed
Importance of Dairying in Prehistoric Britain
Copley et al. Proc. Nat. Acad. Sci. USA (2003)
Dairying established when agriculture introduced into Britain 6000 years ago
Pottery from all sites show the presence of milk fat residues ( = potsherd residue)
Domestication
Secondary ProductsRevolution
Inc. exploitation of animals for meat and
milk products
4,000 BC
7,000 BCNW Europe C and E
EuropeSE Europe Middle and
Near East
Model for the Emergence and Spread of Dairying Across Europe in the Neolithic
Arrival of pottery and agriculture
(with dairying)
Pottery production
Inc. exploitation of animals for meat (and milk?)
Origins of dairying: sites and sherds• >2,200 sherds selected from 23 sites
Animal fats detected in
over 300 sherds
Dairy fats in SE European and Near Eastern Pottery
• Strongest evidence in NW Turkey
• Evidence for processing milk products in pottery at 6500 BC
Non ruminant carcass fatRuminant
carcass fat
Milk fat
Acknowledgements
Archaeological chemistry
Mark CopleyAnna MukherjeeHazel MottramSophie Aillaud
Stephanie DuddStephanie Charters
Vanessa StrakerBas Payne
Andrew SherrattJen Coolidge
Instruments and much more
Ian BullRob BerstanJim Carter
Andy Gledhill
Funding
SERCNERC
English HeritageRoyal Society
Wellcome TrustLeverhulme Trust
HEFC
BiogeochemistryZoe CrossmanPete MaxfieldEd Hornibrook
Phil Ineson
Palaeoclimatereconstruction
Chris NottShuscheng Xie
Richard PancostFrank Chambers
Organic Geochemistry Unit 2005