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

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br15

:0

a15:

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w9

br17

:0

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a17:

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18:1

w11

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w7c 19

:1

19:1

Methanotroph PLFAs

0

2000

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br15

:0

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n18:

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18:1

w11

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:1

19:1

Grazed

Total soil bacterial PLFAs

Bronydd Mawr NCaPK

0

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br15

:0

a15:

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n18:

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w11

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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