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Organic Geochemistry xxx (2007) xxx–xxx
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OrganicGeochemistry
RO
OFHydrogen isotope fractionation in freshwater algae:
I. Variations among lipids and species
Zhaohui Zhang *, Julian P. Sachs 1
Department of Earth, Atmospheric and Planetary Sciences, Building E34-166, Massachusetts Institute of Technology,
77 Massachusetts Avenue, Cambridge, MA 02139, USA
Received 16 May 2006; received in revised form 19 November 2006; accepted 3 December 2006
REC
TED
PAbstract
Five species of freshwater green algae, including three strains of Botryococcus braunii (two A Race, one B Race), Eudo-
rina unicocca and Volvox aureus, were cultured under controlled conditions in media containing different concentrations ofdeuterium. The hydrogen isotopic ratios of lipids in the algae, including alkadienes, botryococcenes, heptadecenes, fattyacids, and phytadiene, were measured by gas chromatography–isotope ratio-mass spectrometry (GC–IRMS) and foundto closely track water dD values. While correlation coefficients (R2) in excess of 0.99 for all lipids in all species suggest thatlipid dD values can be used to determine water dD values, hydrogen isotope fractionation was found to vary systematicallybetween lipids and lipid homologues within a single alga, as well as for the same lipid between species of algae. Undersimilar growth conditions, two species of Chlorophyceae (Eudorina unicocca and Volvox aureus) and three species of Tre-bouxiophyceae (Botryococcus braunii) produced palmitic acid (C16 fatty acid) that differed by 90–100& relative to water.Ubiquitous lipids such as palmitic acid, with a multitude of aquatic and terrestrial sources, are therefore not good targetsfor D/H-based paleohydrologic reconstructions. In addition to the use of source-specific biomarkers that derive unambig-uously from a single family or species, paleohydrologic applications of lipid D/H ratios will need to consider the as yetunstudied potential influence that environmental parameters such as nutrients, light and temperature, etc., may have onD/H fractionation during lipid synthesis.� 2006 Elsevier Ltd. All rights reserved.
R30313233
CO1. Introduction
Hydrologic variations are difficult to reconstructfrom the geologic record and are poorly reproduced
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0146-6380/$ - see front matter � 2006 Elsevier Ltd. All rights reserveddoi:10.1016/j.orggeochem.2006.12.004
* Corresponding author. Present address: Department of Geo-sciences, University of Massachusetts, 611 North Pleasant Street,Amherst, MA 01003, USA. Tel.: +1 413 545 1755; fax: +1 413545 1200.
E-mail address: [email protected] (Z. Zhang).1 Present address: University of Washington, School of Ocean-
ography, Box 355351, Seattle, WA 98195, USA.
Please cite this article in press as: Zhang, Z., Sachs, J.P., HydGeochem. (2007), doi:10.1016/j.orggeochem.2006.12.004
by climate models, yet they are essential for under-standing the natural variation of climate, especiallyin the tropics. Hydrogen and oxygen isotope ratiosof lake and ocean water reveal hydrologic variationscaused by the higher vapor pressure of HHO andH2
16O relative to HDO and H218O. However, these
ratios are often not preserved in the geologic recorddue to isotopic exchange or diagenesis or, as withmineral phases, are influenced by non-hydrologicprocesses. The ‘holy grail’ of paleohydrologic recon-structions is therefore a robust recorder of water
.
rogen isotope fractionation in freshwater algae: ..., Org.
T
41424344454647484950515253545556575859606162636465666768697071727374757677787980818283848586878889909192
93949596979899100101102103104105106107108109110111112113114115
116
117
118119120121122123124125126127128129130131132133134135136137138139140141
2 Z. Zhang, J.P. Sachs / Organic Geochemistry xxx (2007) xxx–xxx
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isotopic variations that does not undergo alterationon the timescale of study.
The hydrogen isotopic composition of algal lipidbiomarkers in lake sediments holds great potentialfor water isotopic reconstruction. All hydrogen inalgae derives from water and algae do not transpire,a process that overprints dD values in land plants.Whereas hydrogen atoms covalently bound toorganic nitrogen, sulfur, and oxygen are labile andexchange quickly and reversibly with hydrogenatoms in water (Werstiuk and Ju, 1989), hydrogenatoms in lipids are bound to carbon (Estep andHoering, 1980) and are nonexchangable (Epsteinet al., 1976; Sauer et al., 2001). Compared to bulkorganic matter, individual lipid biomarkers are notsubject to isotopic changes caused by preferentialdegradation of less stable compounds.
Despite the strong potential of algal lipids inD/H reconstructions technical challenges in contin-uous flow-isotope ratio mass spectrometry have lim-ited their application. With these technical problemsall but overcome in recent years (cf. Burgoyne andHayes, 1998; Hilkert et al., 1999; Sessions et al.,1999) the next step in developing compound-specifichydrogen isotopic reconstructions of water D/Hratios is demonstrating that lipid biomarker dD val-ues closely track water dD values. Several recentstudies have addressed this issue (Sessions et al.,1999; Sessions, 2006; Sauer et al., 2001; Huanget al., 2002, 2004; Sachse et al., 2004; Chikaraishiet al., 2004a,b,c, 2005; Chikaraishi, 2006; Englebr-echt and Sachs, 2005; Schouten et al., 2006). How-ever, this is the first to systematically evaluate thefidelity with which dD values in a variety of lipidbiomarkers from cultured freshwater microalgaerecord water dD values.
Estep and Hoering (1980, 1981) pioneered hydro-gen isotopic biogeochemistry by measuring the dD
values of total organic matter in axenic algal cul-tures that were �90& to �110& relative to thewater. They concluded that (1) photosynthesis wasthe primary process causing hydrogen isotopic frac-tionation in plants, (2) some additional fraction-ation occurred in the dark reactions during thesynthesis of malic or pyruvic acids and lipids, and(3) little hydrogen isotope fractionation occurredduring respiration.
Englebrecht and Sachs (2005) cultured the mar-ine microalga Emiliania huxleyi and demonstratedthat alkenones closely tracked water dD values(R2 > 0.999). Huang et al. (2004) showed that dD
values of palmitic acid (C16:0 fatty acid) in core-
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top sediments from lakes in eastern North Americawere well-correlated with both water dD values ofthe lakes and air temperature (R2 = 0.89), andSachse et al. (2004) showed that n-alkanes in coretop sediments from lakes in a meridional transectthrough central Europe co-varied with dD valuesof precipitation.
To better constrain the extent to which algal lip-ids record water dD values and to address the spe-cies-dependence of hydrogen isotope fractionationin lipids we cultured five species of freshwater greenalgae – Eudorina unicocca, Volvox aureus (bothbelong to Chlorophyceae) and three strains of Bot-
ryococcus braunii (Trebouxiophyceae) – under con-trolled conditions in the laboratory, with eachstrain grown in five media containing differentenrichments of deuterium. We measured dD valuesof several lipids from each culture, including alkadi-enes, botryococcenes, heptadecene, fatty acidmethyl esters (FAMEs), phytadiene and free fattyacids. Reported here are the results of hydrogen iso-topic fractionation in those lipids from the fivefreshwater algal species.
2. Methods
2.1. Algal cultures
2.1.1. Algal species
Five species of freshwater green algae were cul-tured: Eudorina unicocca, Volvox aureus and threestrains of Botryococcus braunii. Relative to redand brown algae, green algae are closer in originto land plants (Kirk, 1998).
Botryococcus braunii is a colonial member of Tre-bouxiophyceae (Senousy et al., 2004), characterizedby high production of lipids and widely distributedin freshwater lakes and ponds. Interest in Botryo-
coccus braunii arose in the 1960s when the algaewere discovered to synthesize large amounts ofhydrocarbons (cf. Maxwell et al., 1968; Douglaset al., 1969). Axenic inoculants of three strains ofBotryococcus braunii were provided by Dr. PierreMetzger of the Laboratoire de Chimie Bioorganiqueet Organique Physique in France. Two were fromthe A race, one from Morocco (a small pool inOukaidem, Atlas) and the other from Lake Titicaca(Bolivia). The third was from the B race, from Mar-tinique (French West Indies).
Eudorina unicocca and Volvox aureus both belongto Chlorophyta (green algae), chlorophyceae, volvo-cales and they are the most common single-celled
rogen isotope fractionation in freshwater algae: ..., Org.
142143144145146147148149150151152153154155156157
158159160161162163
165165
166167168169170171172173174175176177178179180181182183184185186187
188189190
191192193194195196197198199200201202203204205206
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green algae in natural water bodies. Each possessesbiflagellate cells held together in a coenobium ofdefined shape, usually spherical (Kirk, 1998). Theinoculants of Eudorina unicocca and Volvox aureuswere supplied by the Culture Collection of Algaeand Protozoa (CCAP) in Cumbria, United King-dom (now located in Dunstaffnage Marine Labora-tory, Oban, Scotland). The two cultured strainswere Eudorina unicocca G.M. Smith 1930 (CCAP24/1C, originated from freshwater near Blooming-ton, Indiana, USA) and Volvox aureus Ehrenberg1838 (CCAP 88/6, originated from freshwater inMalham Tarn, Yorkshire, England).
We conducted the culture experiments in the labora-tory of Dr. Daniel Repeta at the Woods Hole Ocean-ographic Institution from July to November, 2003.
T207208209210211212
213214215216217218219220221222223224225226227228229230231232
233234235
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2.1.2. Preparation of deuterium-enriched water and
the determination of water dD value
The reference standard for D/H ratios in thiswork is Vienna Standard Mean Ocean Water(VSMOW) with D/H = 155.76 ± 0.05 · 10�6. dD isdefined as
dD ¼ ½ðD=HÞsample � ðD=HÞVSMOW�=ðD=HÞVSMOW � 1000‰
In order to investigate the relationship between lipiddD and water dD over a wide range of values, we setup five cultures for each species in waters with differ-ent deuterium concentrations. We mixed varyingamounts of Aldrich deuterium oxide (99.9% D) withdistilled water (dD = �65&) to produce mediumwith five different dD values, ranging from �65&
to +450&.Water dD values were measured on an H-
device-Thermo Finnigan Deltaplus XL mass spec-trometer at Dartmouth College. The H-Deviceconsists of a quartz reactor filled with chromiumpowder and a series of valves. The quartz reactorwas sealed at one end by a septum and at the otherend by a pneumatic valve. The reactor was held at850 �C and the air evacuated by the vacuum sys-tem in the mass spectrometer. 1 ll of water wasinjected through the septum and flash evaporated.The water was reacted for one minute and theresulting gases admitted to the inlet system of themass spectrometer. Precision of the water dD anal-yses was 0.5&.
236237238239240
2.1.3. Culture media
The medium for the culture experiments wasautoclaved before algal inoculants were introduced.
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Glassware was acid-leached overnight and rinsedwith deionized water, then autoclaved. Algal trans-fers were conducted in a Laminar flow bench withsterilized labware.
B. braunii was grown on a modified CHU 13medium (composition in mg/l): KNO3, 200; K2HPO4Æ3 H2O, 54; MgSO4 Æ 7H2O, 100; CaCl2 Æ 2H2O,52; FeNaEDTA, 10; plus 5 ml of a solution ofmicronutrients (mg/l): H3BO3, 286; MnSO4 Æ H2O,154; ZnSO4 Æ 7H2O, 22; CuSO4 Æ 5H2O, 8; Na2
MoO4 Æ 2H2O, 6; CoSO4 Æ 7H2O, 9. The pH wasapproximately 7.4.
Cultures of E. unicocca and V. aureus were grownin Jaworski’s Medium (JM). The stock compositionin g/200 ml was: (1) Ca(NO3)2 Æ 4H2O, 4.0; (2) K2-HPO4, 2.48; (3) MgSO4 Æ 7H2O, 10.0; (4) NaHCO3,3.18; (5) FeNaEDTA, 0.45; Na2EDTA, 0.45; (6)H3BO3, 0.496; MnCl2 Æ 4H2O, 0.278 g; (NH4)4Mo7-O24 Æ 4H2O, 0.20 g; (7) Cyanocobalamin, 0.008 g;Thiamine HCl, 0.008 g; Biotin, 0.008 g; (8) NaNO3,16.0 g; (9) Na2HPO4 Æ 12H2O, 7.2 g. Stock solutions1–9 were diluted to 1 l with deionized water.
2.1.4. Light intensity and temperature control
Continuous light was provided by two banks oftwo 25 W cool-white fluorescent tubes. Light inten-sity was measured with a Biospherical InstrumentsQSL-100 probe equipped with a QSP-170 sensor.The center of the chamber was �20 · 1015 quanta/scm2, or 320 lmol/m2 s. Illumination was on a12 h light:12 h dark photo cycle.
The 15 cultures of B. braunii (A1W1-5, A2W1-5and BBW1-5; A1: A race, Morocco strain; A2, Arace, Titicaca strain; BB: B race, Martinique strain)were grown at summer room temperature (July–September, 2003), which averaged 28 �C but variedfrom 26.5 �C to 30 �C.
E. unicocca and V. aureus were grown at 15 �C in aclosed chamber built from foam insulation andcooled with an air-conditioner (Haier, 5000 BTU).The temperature inside the chamber varied between14.5–16 �C during the course of the experiment(EUW1-5, VVW1-5; EU: E. unicocca; VV: V. aureus).
2.1.5. Aeration
To expedite algal growth the culture medium wasbubbled with 1% CO2 in purified air that had beenpassed through two 0.2 lm membrane filters. Metz-ger et al. (1985a) reported that hydrocarbon levelsof Botryococcus brauni were greatly affected by cul-ture conditions; air augmented with 1% CO2
increased the hydrocarbon contents from 5% in
rogen isotope fractionation in freshwater algae: ..., Org.
T
241242243244245246247248249250251252253254255256257258259260
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unaerated cultures to 20–61% in aerated cultures,depending on the strain.
All plastic tubing and filters were autoclavedbefore use. Air was introduced into the mediumnear the bottom of the flask so that bubbling keptthe algae in suspension. The flow rate was�250 ml/min. 1% CO2 air was made by mixing N2
gas and gas from tanks of 95% O2 and 5% CO2 ina cell filled with glass beads.
Evaporation during the course of the experi-ments caused deuterium enrichment in the mediumthat we evaluated with a control experiment. A cul-ture flask with 1.5 l water (dD = �67.7%) was aer-ated with the algal cultures and sampled every fivedays. After 45 days the water was enriched in deute-rium by 45&. Aliquots of medium were thereforecollected from every culture flask throughout thecourse of the experiments for D/H analysis. dDwater
values at the start and end of each culture experi-ment are shown in Tables 1–3.
2.1.6. Cell density monitoring and harvest
Cell density was monitored daily by measuring asmall aliquot of each culture on a HP8452A DiodeArray Spectrophotometer. Because the aliquotswere not returned to the culture flasks (in order toprevent contamination) they typically constitutedthe largest source of water loss during the experi-ments. Growth curves for each strain of B. braunii,
and for E. unicocca and V. aureus were obtained bymeasuring the absorption of light at 400, 500, 600,and 700 nm. Absorption curves at different wave-lengths are nearly identical. Representative absorp-tion curves for 600 nm light are shown in SFig. 1(supplementary material). The continuous increasein light absorption (and by extension, cell density)with time was partly caused by evaporative loss ofwater resulting from aeration. B. braunii, and E.
unicocca/V. aureus cultures were harvested after 45days and 31 days, respectively, during the exponen-tial-phase on the growth curves.
Cultures were harvested by helium-pressurized fil-tration through 293 mm diameter Whatman GF/Ffilters (pore size 0.7 lm), and subsequently kept at�20 �C until analysis. A small aliquot of filtratewas filtered through a 0.2 lm membrane filter toobtain water for dD analysis.
2.2. Lipid extraction and isolation
Harvested algae on GF/F filters were freeze-dried, cut into 0.5 · 0.5 cm pieces, and extracted
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on a Dionex ASE-200 pressurized fluid extractorwith dichloromethane (DCM) and methanol(MeOH) (9:1) at 1500 psi and 150 �C. The total lipidextract was fractionated on an aminopropyl car-tridge-style SPE column (Burdick & Jackson, size500 mg/4 ml) with DCM/isopropyl alcohol (IPA)(3:1). Retained fatty acids were recovered with 4%acetic acid in diethyl ether, methylated with 10%BF3 in MeOH and purified by urea adduction.The DCM/IPA fraction was fractionated by columnchromatography using 5% water-deactivated silicagel in a 29 cm · 1.2 cm glass column. Hydrocarbonswere eluted with hexane, FAMEs and phytadieneswith 10% ethyl acetate (EtOAc) in hexane, and alco-hols (including phytol and sterols) with MeOH.FAMES and phytadienes were purified further byurea adduction.
Each fraction was analyzed by gas chromatogra-phy–mass spectrometry (GC–MS) to positivelyidentify each lipid and determine its purity, thenby gas chromatography with flame-ionization detec-tion (GC-FID) to determine individual lipid concen-trations. Agilent 6890 gas chromatographs wereoperated with programmable temperature vaporiza-tion (PTV) inlets, 60 m Varian Chrompac CP-Sil 5capillary columns with 0.32 mm i.d. and 0.25 lmfilm thickness, and helium carrier gas. Oven temper-ature programs differed for each lipid class.
Alkadienes in the Titicaca strain and botryococc-enes in the Martinique strain in B. braunii cultureswere hydrogeated with Raney Nickel in order toelucidate their exact structures.
2.3. Determination of molecular D/H ratios
The hydrogen isotopic composition of each lipidfraction was measured by gas chromatography–isotope ratio-mass spectrometry (GC–IRMS) with aFinnigan Deltaplus XP mass spectrometer, equippedwith a Trace GC and a Combustion III interface.
The Trace GC was equipped with a PTV inletoperated in splitless mode, a 30 m DB-5 capillarycolumn (J&W Scientific) with 0.25 mm i.d. and0.25 lm film, and was operated at a constant heliumflow rate of 1 ml/min. The oven temperature pro-gram differed for each lipid class. For hydrocar-bons, the starting temperature was 90 �C, rising to230 �C at 13 �C/min, then to 325 �C at 5.5 �C/min,followed by 13 min at 325 �C. Effluent from theGC entered the GC-C III interface, a graphite-linedceramic tube at 1400 �C, where quantitative pyroly-sis to graphite, hydrogen gas and carbon monoxide
rogen isotope fractionation in freshwater algae: ..., Org.
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Tab
le1
Hyd
roge
nis
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and
D/H
frac
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inh
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carb
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7
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nd
ard
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iati
on
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010
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005
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98.
90.
006
5.8
0.00
55.
10.
003
3.0
Eac
hst
rain
was
gro
wn
infi
vem
edia
con
tain
ing
diff
eren
tco
nce
ntr
atio
ns
of
deu
teri
um
.T
he
dDva
lue
of
each
lip
idw
asm
easu
red
intr
ipli
cate
and
the
aver
age
and
stan
dar
dd
evia
tio
nar
ere
po
rted
.
Th
edD
valu
eo
fth
ecu
ltu
rem
ediu
mat
the
star
tan
dh
arve
star
eal
sosh
ow
n.
iso
,is
om
er;r
,st
and
ard
dev
iati
on
.
Fra
tio
nat
ion
fact
or,
a,is
defi
ned
as(D
/H) l
ipid
/(D
/H) w
ate
r=
(dD
lip
id+
1000
)/(d
Dw
ate
r+
1000
).
Fra
ctio
nat
ion
,e,
isd
efin
edas
(a�
1)*
1000
=[(dD
lip
id+
1000
)/(d
Dw
ate
r+
1000
)�
1]*1
000.
aT
he
pea
ks
of
C34
bo
tryo
cocc
ene
(co
mp
ou
nd
5),
C32
bo
tryo
cocc
ene
(co
mp
ou
nd
6)
and
C33
bo
tryo
cocc
ene
(co
mp
ou
nd
7)
coel
ute
into
two
pea
ks
and
itis
har
dto
dis
tin
guis
ham
on
gth
em.
Th
ep
eak
of
C34
bo
tryo
cocc
ene
iso
mer
(5)
+?
isli
kel
yth
eco
elu
tio
no
fC
34
iso
mer
(co
mp
ou
nd
5)
and
the
un
iden
tifi
edp
eak
bef
ore
it.
Th
eas
sign
edC
32
+C
33
bo
tryo
cocc
ene
cou
ldev
enin
clu
de
the
smal
lp
eak
of
C34
bo
tryo
cocc
ene
iso
mer
wh
ich
elu
tes
afte
rth
em.
bT
his
pea
kco
uld
be
ano
ther
C34
bo
tryo
cocc
ene
or
ano
ther
ho
mo
logu
ew
ith
mo
reca
rbo
nat
om
s.
Z. Zhang, J.P. Sachs / Organic Geochemistry xxx (2007) xxx–xxx 5
OG 1956 No. of Pages 27, Model 3+
31 January 2007 Disk UsedARTICLE IN PRESS
Please cite this article in press as: Zhang, Z., Sachs, J.P., Hydrogen isotope fractionation in freshwater algae: ..., Org.Geochem. (2007), doi:10.1016/j.orggeochem.2006.12.004
UN
CO
RR
EC
TED
PR
OO
F
Tab
le2
Hyd
roge
nis
oto
pe
rati
os
and
D/H
frac
tio
nat
ion
infr
eefa
tty
acid
s,n
atu
ral
FA
ME
san
dp
hyt
adie
nes
inth
ree
stra
ins
of
B.b
raunii
Cu
ltu
res
H2O
dD
atst
art
H2O
dD
ath
arve
st
C16
fatt
yac
idC
18:1
fatt
yac
idC
20:1
fatt
yac
idC
26:1
fatt
yac
idC
28:1
fatt
yac
idC
30:1
fatt
yac
id
dDS
tdev
ae
dDS
tdev
ae
dDS
tdev
ae
dDS
tdev
ae
dDS
tdev
ae
dDS
tdev
ae
Botr
yoco
ccus
bra
unii
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race
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art
iniq
ue
stra
in
BB
W1
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2.1
3.4
0.82
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176.
6�
191.
63.
10.
824
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6.0
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7.9
0.3
0.83
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162.
1�
187.
91.
70.
828
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9.4
3.5
0.82
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173.
8�
191.
00.
90.
825
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5.4
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W2
102.
216
1.1
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50.
829
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852
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833
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40.
838
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2.0
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184.
123
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15.9
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174.
219
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50.
829
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1.3
33.2
5.8
0.84
0�
160.
212
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30.
823
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6.9
20.4
2.2
0.82
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170.
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DN
DN
D
BB
W4
273.
732
7.6
95.4
3.6
0.82
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174.
986
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90.
819
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1.3
115.
31.
30.
840
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9.9
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ND
ND
ND
96.8
1.2
0.82
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173.
8N
DN
DN
DN
D
BB
W5
455.
151
3.6
228.
80.
70.
812
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8.1
233.
73.
50.
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4.9
256.
21.
10.
830
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0.0
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ND
ND
ND
242.
73.
00.
821
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ND
ND
ND
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rag
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823
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175.
80.
840
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2.5
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1�
168.
7
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ard
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on
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76.
50.
008
7.8
0.00
87.
80.
005
4.9
0.00
55.
00.
009
9.5
Botr
yoco
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unii
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race
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itic
aca
stra
in
AZ
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185.
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205.
61.
90.
816
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5.9
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153.
9�
167.
25.
80.
855
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162.
4�
175.
60.
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846
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128.
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798
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ND
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810
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21.
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817
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176.
3
AZ
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280.
732
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85.4
4.7
0.82
2�
178.
472
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10.
812
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9739
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1�
168.
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10.
89�
170.
610
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5
AZ
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485.
049
9.7
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45.
50.
793
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74.
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229.
02.
80.
820
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187.
20.
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825
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0.83
3�
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4
Sta
nd
ard
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on
0.01
211
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005
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111
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022
22.3
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010
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011
11.1
Botr
yoco
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bra
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race
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oro
cco
stra
in
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1�
64.5
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219.
93.
10.
804
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0.0
3.4
0.81
4�
185.
9�
185.
53.
40.
839
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0.7
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7.5
0.5
0.82
7�
173.
0�
199.
81.
40.
825
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5.4
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6.4
4.3
0.81
8�
182.
0
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288
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7.1
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3.0
1.6
0.80
3�
197.
1�
91.0
2.8
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4�
186.
3�
67.2
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0.83
5�
165.
0�
77.5
3.6
0.82
6�
195.
95.
41.
10.
824
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6.5
0.2
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9�
180.
7
A1W
318
7.7
220.
8�
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1.8
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198.
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2�
187.
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829
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176.
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40.
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0.7
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428
5.6
316.
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812
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4.4
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179.
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DN
DN
DN
DN
DN
DN
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20.
821
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ND
ND
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547
2.2
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8.0
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5.7
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194.
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178.
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Ave
rag
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823
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9
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nd
ard
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iati
on
0.00
54.
60.
006
5.6
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55.
20.
011
10.6
0.00
54.
90.
004
4.3
Cu
ltu
res
H2O
dD
atst
art
H2O
dD
ath
arve
st
Ph
ytad
len
eN
atu
rall
yo
ccu
rin
gF
AM
EdD
(%)
dDS
tdev
ae
C16
Std
eva
eC
18:1
Std
eva
eC
20:1
Std
eva
eC
28:1
Std
eva
eC
30:1
Std
eva
e
Botr
yoco
ccus
bra
unii
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race
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arti
niq
ue
stra
in
BB
W1
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18.9
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1.2
7.8
0.72
2�
277.
6�
175.
51.
60.
840
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9.6
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2.3
0.1
0.83
3�
166.
6�
162.
45.
70.
854
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69.3
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9.7
3.5
0.83
6�
163.
9�
182.
83.
20.
833
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7.0
BB
W2
102.
216
1.1
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7.5
1.6
0.69
1�
308.
8�
49.1
1.0
0.81
9�
181.
0�
46.1
3.3
0.82
2�
178.
4�
22.5
3.9
0.84
2�
158.
1�
45.2
6.3
0.82
2�
177.
7�
49.4
2.4
0.81
9�
181.
2
BB
W3
184.
123
0.2
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0.1
1.4
0.69
1�
309.
27.
02.
70.
819
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1.5
1.3
2.7
0.81
4�
186.
025
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40.
833
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6.8
4.7
0.5
0.81
7�
183.
3N
DN
DN
DN
D
BB
W4
273.
732
7.6
ND
ND
ND
ND
69.3
6.9
0.80
5�
194.
579
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10.
813
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7.1
106.
24.
60.
833
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78.8
2.6
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187.
4N
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DN
DN
D
BB
W5
455.
151
3.6
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ND
ND
ND
205.
88.
50.
797
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3.3
208.
66.
50.
799
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1.5
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ND
ND
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217.
70.
70.
804
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5.5
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ND
ND
ND
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rag
e0.
702
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184.
00.
816
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0.84
1�
159.
50.
818
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1.6
0.82
6�
174.
1
Sta
nd
ard
dev
iati
on
0.01
818
.10.
017
16.6
0.01
312
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007
9.7
0.01
211
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010
10.1
Botr
yoco
ccus
bra
unii
,A
race
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itic
aca
stra
in
AZ
W1
�64
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26.0
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ND
ND
ND
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4.2
0.81
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180.
8�
214.
31.
20.
807
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3.3
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2.7
1.9
0.82
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171.
1�
208.
03.
30.
813
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6.8
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4.7
5.5
0.81
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183.
4
AZ
W2
87.6
128.
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DN
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78.9
0.9
0.81
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183.
8�
83.8
2.3
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2�
188.
2�
69.1
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0.82
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175.
1�
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3.4
0.82
2�
178.
3N
DN
DN
DN
D
AZ
W3
182.
122
1.3
ND
ND
ND
ND
8.5
4.6
0.82
6�
174.
3�
0.5
0.9
0.81
8�
181.
627
.84.
70.
842
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8.5
1.8
2.8
0.82
0�
179.
7N
DN
DN
DN
D
AZ
W4
280.
732
1.1
ND
ND
ND
ND
69.9
1.5
0.81
0�
190.
172
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60.
812
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8.0
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N.D
ND
ND
82.3
7.2
0.81
9�
180.
8N
DN
DN
DN
D
AZ
W5
485.
049
9.7
ND
ND
ND
ND
208.
02.
10.
805
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4.5
206.
21.
00.
804
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5.7
245.
20.
10.
830
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9.7
ND
ND
ND
ND
ND
ND
ND
ND
Ave
rag
e0.
815
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4.7
0.81
1�
189.
40.
831
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8.6
0.81
9�
181.
40.
817
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3.4
Sta
nd
ard
dev
iati
on
0.00
87.
90.
005
5.5
0.00
77.
20.
004
3.7
ND
ND
Eac
hst
rain
was
gro
wn
infi
vem
edia
con
tain
ing
diff
eren
tco
nce
ntr
atio
ns
of
deu
teri
um
.T
he
dDva
lue
of
each
lip
idw
asm
easu
red
intr
ipli
cate
and
the
aver
age
and
stan
dar
dd
evia
tio
nar
ere
po
rted
.dD
valu
eso
ffr
eefa
tty
acid
sw
ere
mea
sure
do
nm
eth
yles
ter
der
ivat
ives
and
corr
ecte
dfo
ris
oto
pic
com
po
siti
on
of
the
add
edm
eth
ylgr
ou
p.
Th
edD
valu
eo
fth
ecu
ltu
rem
ediu
mat
the
star
tan
dh
arve
star
eal
sosh
ow
n.
std
ev,
stan
dar
dd
evia
tio
n;
ND
,n
ot
det
erm
ined
du
eto
insu
ffici
ent
qu
anti
tyo
fm
ater
ial.
ais
defi
ned
as(D
/H) l
ipid
/(D
/H) w
ate
r=
(dD
lip
id+
1000
)/(d
Dw
ate
r+
1000
);e
isd
efin
edas
(a�
1)*1
000
=[(dD
lip
id+
1000
)/(d
Dw
ate
r+
1000
)�
1]*1
000.
6 Z. Zhang, J.P. Sachs / Organic Geochemistry xxx (2007) xxx–xxx
OG 1956 No. of Pages 27, Model 3+
31 January 2007 Disk UsedARTICLE IN PRESS
Please cite this article in press as: Zhang, Z., Sachs, J.P., Hydrogen isotope fractionation in freshwater algae: ..., Org.Geochem. (2007), doi:10.1016/j.orggeochem.2006.12.004
UN
CO
RR
EC
TED
PR
OO
F
Tab
le3
Hyd
roge
nis
oto
pe
rati
os
and
D/H
frac
tio
nat
ion
infr
eefa
tty
acid
s,F
AM
Es
and
ph
ytad
ien
ein
E.
unic
occ
aan
dV
.aure
us
Cu
ltu
res
H2O
dDat
star
tH
2O
dDat
har
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Eac
hst
rain
was
gro
wn
infi
vem
edia
con
tain
ing
diff
eren
tco
nce
ntr
atio
ns
of
deu
teri
um
.T
he
dDva
lue
of
each
lip
idw
asm
easu
red
intr
ipli
cate
and
the
aver
age
and
stan
dar
dd
evia
tio
nar
ere
po
rted
.dD
valu
eso
ffr
eefa
tty
acid
sw
ere
mea
sure
do
nm
eth
yles
ter
der
ivat
ives
and
corr
ecte
dfo
ris
oto
pic
com
po
siti
on
of
the
add
edm
eth
ylgr
ou
p.
Th
edD
valu
eo
fth
ecu
ltu
rem
ediu
mat
the
star
tan
dh
arve
stis
also
sho
wn
.
ND
,n
ot
det
erm
ined
du
eto
insu
ffici
ent
amo
un
tS
tdev
,st
and
ard
dev
iati
on
,b
ased
on
trip
lem
easu
rem
ents
un
less
no
ted
oth
erw
ise.
a,is
defi
ned
as(D
/H) l
ipid
/(D
/H) w
ate
r=
(dD
lip
id+
1000
)/(d
Dw
ate
r+
1000
);e,
isd
efin
edas
(a�
1)*1
000
=[(dD
lip
id+
1000
)/(d
Dw
ate
r+
1000
)�
1]*
1000
.
aA
llfa
tty
acid
sw
ith
C18
skel
eto
nar
em
erge
din
totw
op
eak
so
nly
,u
nsa
tura
ted
and
satu
rate
d.
Un
satu
rate
dar
em
ain
lyC
18:1
and
C18:3
,b
ut
po
ssib
lyin
clu
des
smal
lp
eak
of
C18:2
.
bA
llfa
tty
acid
sw
ith
C18
skel
eto
nar
em
erge
din
totw
op
eak
so
nly
,u
nsa
tura
ted
and
satu
rate
d.
Un
satu
rate
dis
pre
do
min
ated
by
C18:3
,fo
llo
wed
by
C18:1
and
,to
am
uch
less
exte
nt,
C18:2
.In
VV
W5,
un
satu
rate
dis
on
eh
um
p.
cF
or
EU
W1,
2an
d3,
all
fatt
yac
ids
wit
hC
18
skel
eto
nar
em
erge
din
totw
op
eak
so
nly
,u
nsa
tura
ted
and
satu
rate
d.
Un
satu
rate
dar
em
ain
lyC
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and
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,b
ut
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ud
essm
all
pea
ko
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;In
EU
W4
and
5,u
nst
aru
ated
iso
ne
hu
mp
.
dF
or
EU
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4an
d5,
satu
rate
dF
AM
Ed
oes
no
th
ave
suffi
cien
tam
ou
nt
for
dDm
easu
rem
ent.
eA
llF
AM
Es
wit
hC
18
skel
eto
nar
em
erge
din
totw
op
eak
so
nly
,u
nsa
tura
ted
and
satu
rate
d.
Un
satu
rate
dis
pre
do
min
ated
by
C18:3
,fo
llo
wed
by
C18:1
and
,to
am
uch
less
exte
nt,
C18:2
.In
VV
W4
and
5,u
nst
aru
ated
iso
ne
hu
mp
.
fV
VW
3n
atu
ral
FA
ME
and
hep
tad
ecen
efr
acti
on
has
on
lyo
ne
mea
sure
men
td
ue
toli
mit
edam
ou
nt
afte
rp
roce
ssin
gan
dn
ot
incl
ud
edin
aver
age
calc
ula
tio
n.
Z. Zhang, J.P. Sachs / Organic Geochemistry xxx (2007) xxx–xxx 7
OG 1956 No. of Pages 27, Model 3+
31 January 2007 Disk UsedARTICLE IN PRESS
Please cite this article in press as: Zhang, Z., Sachs, J.P., Hydrogen isotope fractionation in freshwater algae: ..., Org.Geochem. (2007), doi:10.1016/j.orggeochem.2006.12.004
T
340341342343344345346347348349350351352353354355356357358359360361362363364365366367368369370371372373374375376377378379380
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8 Z. Zhang, J.P. Sachs / Organic Geochemistry xxx (2007) xxx–xxx
OG 1956 No. of Pages 27, Model 3+
31 January 2007 Disk UsedARTICLE IN PRESS
UN
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RR
EC
occurred (Burgoyne and Hayes, 1998). The hydro-gen gas stream was introduced to the mass spec-trometer via an open split, where a flow of heliumcarried the H2 gas into the mass spectrometer.
Sensitivity of the instrument was monitored withsix pulses of commercial H2 gas (ultra high puritygrade) via a second open split, four at the beginningand two at the end of each run (SFig. 2). Interfer-ence from Hþ3 , which is formed in the ion sourcevia ion-molecule reactions between Hþ2 and neutralH2, impedes the accurate determination of HD+
(Sessions et al., 1999). A calibration curve, or an‘‘Hþ3 factor’’, was determined daily to correct forthe Hþ3 interference. Hþ3 was determined by measur-ing the (m/z 3)/(m/z 2) response of 10 injections ofH2 reference gas. A low and stable value of less thanseven was typically achieved.
To every sample was added a set of co-injectionstandards with known dD values that were chosento bracket the peaks of interest in the chromato-grams. All isotopic standards were obtained fromDr. Arndt Schimmelmann at the BiogeochemicalLaboratory, Indiana University. For hydrocarbons,a mixture of C14 n-alkane, C16 n-alkane, 5a-andro-stane, and C40 n-alkane was used. C14 and C16 n-alk-anes were used as ‘‘throw-away’’ peaks to avoid anypossible hydrogen isotope memory effect, while 5a-androstane and C40 n-alkane were used as isotopicstandards for the computation of lipid dD valueswith IsoDat 2.0 software (Thermo Finnigan)(SFig. 2). A different mixture of standards was pre-pared for each lipid fraction.
Each sample was run in triplicate and the stan-dard deviation was usually less than 5& (Tables1–3), similar to values reported by Sessions et al.(1999) and Englebrecht and Sachs (2005). A set of15 n-alkanes with known dD values (Mixture A orB) acquired from Indiana University were injectedevery 6–9 runs to ensure the accuracy of our data.dD values are reported with reference to theVSMOW standard.
2.4. Correction of dD contribution from –CH3 added
during methylation
Fatty acid dD values were measured on methylester derivatives. The dD value of the three H atomsadded during the methylation reaction with BF3 inMeOH was determined by experimentation withphthalic acid (Dr. Arndt Schimmelmann, IndianaUniversity) having a known dD value. Three exper-iments performed over the course of this study, each
Please cite this article in press as: Zhang, Z., Sachs, J.P., HydGeochem. (2007), doi:10.1016/j.orggeochem.2006.12.004
ED
PR
OO
F
measured in triplicate, indicated a dD value for thethree derivative H atoms of �115.7 ± 0.5&. Thisvalue was used to correct the measured dD valuesof fatty acid methyl esters for the added hydrogenatoms by mass balance.
3. Results
3.1. Algal growth
Growth curves for all cultures indicated exponen-tial growth and, by extension, healthy cultures(SFig. 1). With a relatively small (but uncertain)contribution to light absorption caused by evapora-tion, most of the progressive increase in lightabsorption can be attributed to an increasing bio-mass. Growth rates were determined using the end-points of the exponential portion of the growthcurves (by curve fitting, SFig. 1) as k (divisions/d) =log2(N1/N0)/(T1 � T0), where N1 and N0 are theabsorption values at the end and beginning, respec-tively, and T1 and T0 are the time (days) at the endand beginning, respectively, of the exponentialgrowth period (Adolf et al., 2003). Calculatedgrowth rates for the Morocco, Titicaca and Marti-nique strains of B. braunii were 0.128–0.138,0.120–0.133 and 0.124–0.135 divisions/day, respec-tively. E. unicocca and V. aureus had growth ratesof 0.156–0.166 and 0.159–0.170 divisions/day,respectively.
3.2. Hydrocarbon distributions within B. braunii, E.
unicocca and V. aureus
3.2.1. B. braunii, A race (Titicaca and Morocco)
A race cultures contained substantial quantitiesof odd carbon-numbered hydrocarbons in the rangeof C23–C33, each with a terminal double bond(Metzger et al., 1985a, 1986; Metzger and Largeau,1999). Hydrocarbons in the Titicaca strain consistedalmost exclusively of C25–C31 odd n-alkadienes:C27H52(1, 18; E/Z), C29H56 (1, 20; E/Z), C31H60
(1, 22; E/Z) (SFig. 3a) (structure 1 in Appendix A),in accord with previous reports (Metzger et al.,1986). Minor alkenes included C23H44 (1, 14; E/Z), and C25H48 (1, 16; E/Z), and C29 alkatrienes(SFig. 3a). On GC-IRMS, the Z/E isomers co-eluted, resulting in a single isotopic value.
Hydrocarbons in the Morocco strain differedfrom those in the Titicaca strain in that only oneisomer of each odd C25–C31 n-alkadiene wasobserved and an appreciable concentration of the
rogen isotope fractionation in freshwater algae: ..., Org.
T
437438439
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515516517518519520521522523524525526527528529530531532533534535536537
Z. Zhang, J.P. Sachs / Organic Geochemistry xxx (2007) xxx–xxx 9
OG 1956 No. of Pages 27, Model 3+
31 January 2007 Disk UsedARTICLE IN PRESS
UN
CO
RR
EC
C29H54 alkatriene eluted between the C29 and C31
alkadienes (SFig. 4a), as previously reported byMetzger et al. (1986).
3.2.2. B. braunii, B race (Martinique)
3.2.2.1. Botryococcenes. Polymethylated triterpenesof the generalized formula CnH2n�10, where30 6 n 6 37, termed botryococcenes, are producedby the B race (Metzger et al., 1985a,b, 1988; Metz-ger and Largeau, 1999). Botryococcene mixturesexhibit a large range of molecular mass and isomer-ism related to genetic and physicochemical factors(Metzger et al., 1985b). Seven botryococcenes wereidentified by mass spectrometry and coinjectionwith authentic standards provided by Dr. PierreMetzger (SFig. 5a and Appendix A), the most abun-dant of which were two isomers of the C34 botryo-coccene (structures 4 and 5 in Appendix A), withlesser quantities of C30, C31, C32 and C33 botryo-coccenes (structures 2, 3, 6 and 7 in Appendix A).
Positive identification of all botryococcenes dur-ing GC–IRMS was usually not possible due to inad-equate peak separation caused by the largeinjections required for dD determinations. Forexample, in GC–IRMS chromatograms, com-pounds #5–7, which were baseline-separated onthe GC-FID (SFig. 5a), were not well-resolved,co-eluting in as few as two peaks. The last peak inGC–IRMS chromatograms may be another isomerof the C34 botryococcene or it may be a differenthomologue altogether. In either case, pending posi-tive structural identification by GC–MS, we refer tothat compound as C34 iso in Table 1 and SFig. 5a.
The C30 botryococcene is the precursor for theC31–C34 botryococcenes which are synthesized bymethylation on positions 3, 7, 16 and/or 20 of theC30 backbone (Metzger et al., 1987; Okada et al.,2004). The relative abundance of C30 botryococcenein a population results from a balance between itsproduction and its loss via methylation to formC31–C34 botryococcenes (Okada et al., 2004). As aculture ages and botryococcenes accumulate, thesynthesis of botryococcenes is shifted toward thelonger homologues, resulting in minor amounts ofC30 botryococcene late in a culture cycle (Okadaet al., 2004). Growing for 45 days, C34 botryococc-enes were most abundant in our cultures and C30
abundances were low (SFig. 5a).
3.2.2.2. Phytadiene. Quantities of phytadiene suffi-cient for D/H analysis existed in only three culturesowing to their loss during urea adduction of
Please cite this article in press as: Zhang, Z., Sachs, J.P., HydGeochem. (2007), doi:10.1016/j.orggeochem.2006.12.004
ED
PR
OO
F
FAMEs (the two co-eluted during column chroma-tography). Four isomers were observed, each with aparent ion at m/z 278. Structure determination wasbased on the comparison of our electron impactmass spectra and gas chromatographic retentiontimes with published data (Fukushima et al., 1992).
Phytadienes are degradation products of phytol,the ester-linked side-chain of chlorophyll-a. Theycan be produced (i) at elevated temperatures fromchlorins, (ii) during GC analysis of underivatizedphytol, and (iii) by acidic dehydration of phytol(Grossi et al., 1996). However, a controlled experi-ment in which we injected underivatized phytol(Sigma-Aldrich) onto the GC demonstrated thatless than 1% was converted to phyadienes, and thoseproduced had a different distribution of isomers.Furthermore, chlorophyll (Pfaltz & Bauer) sub-jected to identical ASE extraction conditionsresulted in no production of phytadienes. We there-fore conclude that phytadienes occurred naturally inthe B Race of B. braunii, probably by dehydrationof phytol (Volkman and Maxwell, 1986; Fukushimaet al., 1992; Grossi et al., 1996). Here, we reportonly the dD values of the most abundant isomer,neophytadiene (structure 8 in the Appendix A)(Table 2). Phytols were likely still present, alongwith sterols, in the alcohol fractions which werenot analyzed.
3.2.3. Hydrocarbon distributions in Eudorina
unicocca and Volvox aureus
Both E. unicocca and V. aureus had similarhydrocarbon distributions characterized almostexclusively by 8-heptadecene (C17:1) (structure 9 inthe Appendix A) with minor amounts of heptade-cane (C17) and 10-nonadecene (C19:1) (SFig. 6a).This distribution of hydrocarbons differs from theshort-to-mid chain length alkane distributionsfound in many algae (Gelpi et al., 1970). Becausethe hydrocarbon fraction contained a single abun-dant compound it was combined with the FAMEfraction for dD analysis (SFigs. 6c and 7b). Thoughno published reports of lipid distributions in E. unic-
occa or V. aureus exist, and few reports of 8-hepta-decene are in the literature, it is likely derived fromglycerol monoolein in the cell membrane (Peterson,1980).
Phytadienes occurred in E. unicocca and V. aur-
eus (SFigs. 6c and 7b), but loss during sampleworkup prevented D/H analysis of all but the mostabundant isomer (compound 8 in the Appendix A)in two cultures of V. aureus (Table 3).
rogen isotope fractionation in freshwater algae: ..., Org.
T
538
539540541542543544545546547548549550551552553554555556557558559560561562563564565566567568569570571
572573
574575576577578579580581582583584585586
587588589590591592593594595596597598599600601602603604605606607608609610611612613614615616617618619620621622623624625626627628629630631
632
633634635636
10 Z. Zhang, J.P. Sachs / Organic Geochemistry xxx (2007) xxx–xxx
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3.3. Fatty acid distributions
The B race (Martinique) contained even num-bered monocarboxylic acids ranging from n-C14 ton-C30, with C16, C18:1 and C28:1 predominating(SFig. 5b). Except for the C16 fatty acid (palmitic),all other fatty acids were unsaturated. Linoleic acid(C18:2x6) and linolenic acid (C18:3x3) eluted closelywith monosaturated C18:1. Of the longer homo-logues, C28:1 was most abundant, followed by sub-stantial quantities of C20:1, C26:1 and C30:1, andtrace amounts of C22:1 and C24:1. Such distributionsare in agreement with previous report (Douglaset al., 1969).
Unlike the hydrocarbons, fatty acid distribu-tions were similar between A and B races. A racefatty acids were primarily even numbered, varyingin length from C16 to C32, with C16, C18:1 and C28:1
predominating (SFig. 3b). The only major differ-ences between the A and B race fatty acids werea higher abundance of C16 relative to C18:1and amore pronounced even-over-odd predominance inthe A race.
Compared to fatty acids in B. braunii, E. unicocca
and V. aureus produced fatty acids with substan-tially shorter chain lengths (SFig. 6b). The mostabundant fatty acid in both species was palmiticacid, C16:0. Other saturated fatty acids includedC18, C14, and trace amounts of C17 (SFig. 6b). Theprimary unsaturated fatty acids in the two specieswere C18:2, C18:3 and C18:1, with lesser amounts ofC20:1 (SFig. 7a). Differences between the fatty aciddistributions in the two species included relativelylarger amounts of C18 in V. aureus, and relativelylarger quantities of C20:1in E. unicocca.
3.4. Naturally occurring fatty acid methyl esters
(FAMES)
Though not previously reported, our cultured B.
braunii contained large quantities of naturally occur-ring FAMEs. Both A (Titicaca) and B (Martinique)races had FAME distributions that closely followedtheir fatty acid (FA) distributions, with C16, C18:1
and C28:1 predominating and a pronounced even-over-odd preference (SFigs. 3c and 5c).
A control experiment was conducted to ensurethat our lipid extraction procedure (DCM/MeOH(9:1 v/v), 150 �C, 1,500 psi) did not cause methyla-tion of fatty acids. C16 and C18 fatty acids (Sigma-Aldrich) were subjected to ASE extraction usingthree solvent systems: 100% DCM, 10% MeOH in
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DCM (1:9 v/v) and 50% MeOH in DCM (1:1 v/v).Extractions with DCM and 10% MeOH in DCMproduced no measurable fatty acid methyl esters.Extraction with 50% MeOH in DCM convertedless than 0.1% of both C16 and C18 FAs to FAMEs,substantially less than observed in our cultures.
While we recognize that the control experimentswith pure fatty acids lacked the potential catalyticand matrix effects other compounds and compo-nents of cultured algae (on a filter) may providewe have no evidence to indicate that the FAMEsin the cultures were produced during sampleworkup. Furthermore, when the Martinique andTiticaca cultures were extracted at room tempera-ture by ultrasonication in 100% DCM—a gentleextraction procedure–significant quantities ofFAMEs were observed.
Fatty acid methyl/ethyl esters have been reportedin the extracts of algae (Weete, 1976), fungus (Laseteret al., 1968; Laseter and Weete, 1971), mammaliantissues (Saladin and Napier, 1967), pollen (Fathipouret al., 1967), and protozoans (Chu et al., 1972).Hydroxy fatty acid methyl esters were found in mar-ine algae (Sinninghe Damste et al., 2003).
Though, we cannot exclude the possibility that asmall fraction of the FAMEs in our B. braunii wereproduced during sample preparation, the bulk ofthem are likely natural, begging the question whythey have not previously been reported in algal cul-tures. We hypothesize that the common procedureof methylation of fatty acids or hydrolysis of totallipid extracts before column chromatography andGC–MS analysis destroyed or masked any naturalFAMEs. The biosynthesis of FAMEs was investi-gated in the bacterium Mycobacterium phlei: S-adenosylmethionine as the most effective methyldonor and fatty acids as acyl acceptors (Akamatsuand Law, 1970).
E. unicocca and V. aureus also produced appre-ciable amounts of FAMEs. As with B. braunii,FAMEs in both species had very similar distribu-tions to the free fatty acids, with C16 FAME mostabundant, followed C18 and C18:1/3/2 FAMEs, anda pronounced even-over-odd preference (SFigs. 6cand 7b).
3.5. Hydrogen isotope fractionation in hydrocarbons
In quantifying D/H fractionation during lipidsynthesis we adopt the traditional definition of ‘‘frac-tionation factor’’ for reactions under thermodynamicequilibrium, i.e., alipid–water = (D/H)lipid/(D/H)water =
rogen isotope fractionation in freshwater algae: ..., Org.
T
637638639640641642643644645646647648649650651652653654655656657658659660661662663664665666667668669670671672673674675676677678679680681682683684685686687688
689690691692
693
694695696697698699700701702703704705706707708709710711712
713714715716717718719720721722723724725726727728729730
731732
733734735
Z. Zhang, J.P. Sachs / Organic Geochemistry xxx (2007) xxx–xxx 11
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(dDlipid + 1000)/(dDwater + 1000). the natural vari-ability of a is small, an approximation of ‘‘fraction-ation,’’ termed an ‘‘enrichment factor’’, is oftenreported, with geochemists tending to use eA–B �103 · lnaA–B (Hoefs, 2004), and ecologists, e =(aA–B � 1) · 1000 (Lajtha and Marshall, 1994). Bothquantities were calculated for each lipid in eachculture and in the ensuing discussion we use the‘‘ecologist’s’’ definition, elipid–water = (alipid–water� 1) ·1000 = [(dD
lipid+ 1000)/(dDwater + 1000) � 1] · 1000
(primarily because the e values in five culturescalculated this way are more consistent). Becauselipids are depleted in deuterium relative to waterthe value of fractionation is always negative. A ‘‘lar-ger’’ isotope fractionation means the absolute valueof e is larger.
For continuity with recent literature we reportthe linear regression equations for five cultures ofeach species, y = slope · dDwater + intercept. Theobservation that the intercept 5(slope � 1) · 1000,a point discussed by Sessions and Hayes (2005), willbe discussed in detail in a subsequent paper. Fornow we simply note that the y-intercept of the linearregression of dDlipid versus dDwater is the D/H frac-tionation when dDwater = 0.
dD values of C27, C29 and C31 n-alkadienes in B.
braunii, A Race (Titicaca and Morocco) closelytracked water dD values, with R2 > 0.99 (Table 1;Fig. 1a and d). In spite of the large water dD range,a for C29 n-alkadiene in the Titicaca strain wasnearly constant in five cultures, varying from 0.754to 0.779, and averaging 0.769 with a standard devi-ation (r) of 0.01. Similarly, a for the C29 n-alkadienein the Morocco strain averaged of 0.779 ± 0.007.Therefore, a simple approximation of a = (D/H)lipid
/(D/H)water = (dDlipid + 1000)/(dDwater + 1000) issuitable for the experiments reported here.
The e values for the C29 n-alkadienes varied from�220.6& to �246.0& in the Titicaca strain, averag-ing �231.5 ± 10.3&, and varied between �212.4&
to �228.5&, averaging �221.0 ± 7.3& in the Mor-occo strain (Table 1). Average a values were 0.769and 0.779, respectively. The dD values of C27 alkadi-enes in two of the Morocco strain cultures (A1W1and A1W2) differed substantially from the C29 alk-adiene dD values (Table 1), an observation forwhich we have no explanation.
dD values of C30–C34 botryococcenes in the Brace, Martinique strain, of B. braunii closely trackedwater dD values, with R2 > 0.99 (Fig. 2a). The e val-ues of C30, C31 and C34 averaged �273&, �293&,and �310& to �359&, respectively (Table 1).
Please cite this article in press as: Zhang, Z., Sachs, J.P., HydGeochem. (2007), doi:10.1016/j.orggeochem.2006.12.004
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The dD values of 8-heptadecene in E. unicocca
and V. aureus closely tracked water dD values, withR2 > 0.99 (Fig. 3b and d). The e values averaged�111.6& and �106.4&, respectively (Table 3).
3.6. Hydrogen isotope fractionation in free fatty acids
3.6.1. Fatty acid dD values in B. braunii
Fatty acid dD values in all three A and B race B.
braunii strains closely tracked water dD values, withR2 > 0.99 (Figs. 1b, e and 2b). In the A race Titicacastrain, e values of C16, C18:1 and C28:1 fatty acidsaveraged �192.4&, �187.2& and �174.6&,respectively, while e values of C20:1 fatty acids aver-aged �168.1& (Table 2). In the A race Moroccostrain, e values of C16 and C18:1 fatty acids averaged�195.9& and �186.8&, respectively (Table 2),while e values of C20:1 and C28:1 fatty acids averaged�167.0& and �177.5&, respectively. In the B race(Martinique), e values of C16, C18:1 and C28:1 fattyacids averaged �177.0&, �175.8& and �172.5&,respectively, compared to �160.1& for the C20:1
fatty acid. Taken together, the C16, C18:1 and C28:1
fatty acids of A and B race B. braunii had e valuesof �174& to �196&, while the C20:1 fatty acidwas consistently enriched in deuterium by �15&.
3.6.2. Fatty acid dD values in E. unicocca and V.aureus
Fatty acid dD values in both E. unicocca and V.
aureus closely tracked water dD values, withR2 > 0.99 (Fig. 3a and c). The e values of the C16
fatty acid were �49.8& in E. unicocca and�58.1& in V. aureus, significantly less than in B.
braunii and, for E. unicocca, about 50& enrichedin deuterium relative to the C14 fatty acid (Table 3).
Some uncertainty in the absolute dD values ofC18 fatty acids exists as a result of partial co-elutionof C18:2, C18:3, C18:1, etc. (see notes in Table 3), attimes requiring the integration of all unsaturatedC18 acids as a single peak. Nevertheless, excellentlinear relationships between C18 fatty acid (satu-rated and unsaturated) and water dD values, andsimilar e values for the C18 and C16 fatty acids sug-gests that the uncertainty is relatively small.
3.7. Hydrogen isotope fractionation in FAMEs and
phytadienes
3.7.1. FAME and phytadiene dD values in B. braunii
dD values of naturally occurring C16, C18:1 andC28:1 FAMEs in B. braunii A (Titicaca) and B (Mar-
rogen isotope fractionation in freshwater algae: ..., Org.
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-300
-200
-100
0
100
200δD
of a
lkan
dien
es (
‰)
0 100 200 300 400 500
δD of waters at harvest (‰)
y = 0.708x - 219.016
y = 0.691x - 207.429
(A race, Titicaca strain)
C31- diene
C29-diene
C27- diene
(C27:2)
(C29:2 )
-300
-200
-100
0
100
200
δD o
f alk
adie
nes
and
alka
trie
nes
(‰)
0 100 200 300 400 500
δD of waters at harvest (‰)
y = 0.750x - 215.365
y = 0.661x - 169.218
(A race, Morocco strain)
C31-diene
C29-triene
C29-diene
C27- diene (C27:2)
(C29:2)
-200
-100
0
100
200
300
δD o
f fre
e fa
tty a
cids
(co
rrec
ted,
‰)
0 100 200 300 400 500
δD of waters at harvest (‰)
y = 0.776x - 157.036
y = 0.773x - 163.884
y = 0.782x - 180.955
y = 0.768x - 184.316
(A race, Titicaca strain)
C20:1
C28:1
C18:1
C16
(C20:1 )
(C16 )
(C18:1 )
(C28:1 )
-300
-200
-100
0
100
200
300
δD o
f fre
e fa
tty a
cids
(‰
)
0 100 200 300 400 500
D of waters at harvest (‰ )
y = 0.808x - 162.687
y = 0.795x - 171.904
y = 0.801x - 195.320
(A race, Morocco strain)
C20:1
C28:1
C18:1
C16
(C20:1)
(C28:1)
(C16)
-300
-200
-100
0
100
200
300
D o
f nat
ural
occ
urin
g F
AM
E (
‰)
0 100 200 300 400 500D of waters at harvest (‰)
y = 0.837x - 169.571
y = 0.836x - 183.940
y = 0.800x - 187.147
y = 0.778x - 177.098
(A race, Titicaca strain)
C20:1
C28:1
C18:1
C16(C20:1 )
(C16 )
(C18:1 )
(C28:1 )
a
d
e
b
c
Fig. 1. Relationships between lipid and water dD values in cultures ofB. braunii, A race, Titicaca (A–C) and Morocco (D–E) strains. Allalkadiene, alkatriene, fatty acid and FAME dD values were highly correlated with water dD values (R2 > 0.99). (a) C27, C29 and C31
alkadienes in the Titicaca strain. C27 alkadienes were deuterium-enriched compared to other homologues. (b) C16, C18:1, C20:1 and C28:1
fatty acids in the Titicaca strain. C20:1 was most deuterium enriched, followed by C28:1 and C16. (c) C16, C18:1, C20:1 and C28:1 FAMES inthe Titicaca strain. Chain-length-dependant dD variations were identical to the fatty acids. (d) C27, C29 and C31 alkadienes and C29
alkatriene in the Morocco strain. C27 was most deuterium-enriched. (e) C16, C18:1, C20:1 and C28:1 fatty acids in the Morocco strain. C20:1
was most deuterium-enriched, with C28:1 slightly enriched compared to C16.
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736737738739740741742743744745746747748749
750751752753754755756757758
759760761762
-300
-200
-100
0
100
δD o
f ph
ytad
iene
s (
‰)
0 100 200 300 400 500
δD of waters at harvest (‰)
y = 0.557x - 282.096
-200
-100
0
100
200
300
δD o
f nat
ural
ly o
ccur
ing
FA
ME
(‰
)
0 100 200 300 400 500
δD of waters at harvest (‰)
y = 0.770x - 148.222
y = 0.746x - 165.893
y = 0.715x - 162.069
C20:1
C28:1
C18:1
C16
(C20:1 )
(C28:1 )
(C16 )
-200
-100
0
100
200
300
δD o
f fre
e fa
tty a
cids
(co
rrec
ted,
‰)
0 100 200 300 400 500
δD of waters at harvest (‰)
y = 0.810x - 153.424
y = 0.807x - 167.963
y = 0.791x - 169.872
C20:1
C28:1
C18:1
C16
(C20:1 )
(C28:1 )
(C16 )
-400
-300
-200
-100
0
100
200δD
of b
otry
ococ
cene
s (
‰)
0 100 200 300 400 500
δD of waters at harvest (‰)
y = 0.647x - 359.821
y = 0.726x - 329.067
y = 0.730x - 297.445
y = 0.751x - 277.948(Martinque)
C34bot iso
C34bot(5)+?
C34bot(4)
C32+C33 bot
C31bot
C30bot(C30 bot)
(C34 bot [4])
(C31 bot)
(C34 bot iso ?)
a
c d
b
Fig. 2. Relationships between lipid and water dD values in cultures of B. braunii, B race, Martinique strain. All botryococcene, fatty acid,FAME and phytadiene dD values were highly correlated with water dD values (R2 > 0.99). (a) C30–C34 botryococcenes (see structures inAppendix A). Deuterium-depletion increased with increasing carbon number. The compound labeled ‘‘C34 bot iso?’’ (i.e., the last peak inSFig. 5a) may be an isomer of the C34 botryococcene, or possibly a longer homologue. (b) C16, C18:1, C20:1 and C28:1 fatty acids. C20:1 wasconsistently the most deuterium enriched homologue. (c) FAMEs. (d) Phytadiene.
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COtinique) races closely tracked water dD values, with
R2 > 0.99 (Figs. 1c and 2c). The e values of the Arace (Titicaca) FAMEs were �181.4& to�189.4& for C16, C18:1 and C28:1FAMEs (Table 2).The C20:1 FAME was �20& enriched in deuteriumrelative to other homologues, as observed for thefree fatty acids. The e values of B race (Martinique)FAMEs were from �181.6& to �184.0& for C16,C18:1 and C28:1 (Table 2). And, similar to the A race,the C20:1 FAME was enriched in deuterium relativeto other FAMEs by �20&, with an e value of�159.5&.
Phytadiene dD values were measured in three Brace (Martinique) cultures that had sufficient mate-
Please cite this article in press as: Zhang, Z., Sachs, J.P., HydGeochem. (2007), doi:10.1016/j.orggeochem.2006.12.004
rial. dD values of phytadiene were linearly correlatedwith water dD values, with R2 > 0.99 (Fig. 2d). The evalues averaged �298.5 ± 18.1& (Table 2), substan-tially greater than for fatty acids and FAMEs. Wealso note that the slope of the regression was low,0.557, compared to 0.73–0.83 for fatty acids(Fig. 2d; Table 4). With only three cultures to definethe slope, however, we are unable to evaluate itssignificance.
3.7.2. FAME and phytadiene dD values in E.
unicocca and V. aureusdD values of C16 and C18:1 FAMEs from E. unic-
occa closely tracked water dD values, with R2 > 0.99
rogen isotope fractionation in freshwater algae: ..., Org.
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PR
OO
F
763764765766767768769770771772773774775
776777778
779
780
781782783784785
-100
0
100
200
300
400
-50 50 150 250 350 450
δD of waters at harvest (‰ )
y = 0.895x - 63.069
y = 0.849x - 104.543
(Eudorina unicocca)
C18 FAME-unsat
C16 FAME
C17:1-ene
( C17:1-ene )
(C18-unsat )
-100
0
100
200
300
400
δD o
f fre
e fa
tty a
cids
(co
rrec
ted,
‰)
-50 50 150 250 350 450
δD of waters at harvest (‰ )
y = 0.970x - 67.527
y = 0.858x - 93.322
y = 0.949x - 49.435
(Eudorina unicocca)
C18 FA
C14 FA
C16 FA
(C18 )
(C16 )
(C14 )
-150
-50
50
150
250
350
450
δD o
f fre
e fa
tty a
cids
(co
rrec
ted,
‰)
-50 50 150 250 350 450
δD of waters at harvest (‰)
y = 0.948x - 45.885
y = 0.931x - 56.259
(Volvox aureus)
C18FAunsat
C18 FA
C16 FA
(C16 )
(C18 unsat)
-300
-200
-100
0
100
200
300
400
δD o
f FA
ME
s, C
17:1
-ene
and
phy
tadi
ene
(‰)
δD o
f FA
ME
s, C
17:1
-ene
and
phy
tadi
ene
(‰)
-50 50 150 250 350 450
δD of waters at harvest (‰ )
y = 0.925x - 59.797
y = 0.740x - 93.548
y = 0.840x - 71.505
(Volvox aureus)
phytadiene
C18 FAME
C17:1-ene
C16 FAME(C18 FAME )
(C16 FAME )
(C17:1-ene )
a c
dc
Fig. 3. Relationships between lipid and water dD values in cultures ofEudorina unicocca and Volovx aureus. All fatty acid, FAME and n-alkene dD values were highly correlated with water dD values (R2 > 0.99). (a) C14, C16 and C18 fatty E. unicoccacultures. C14 wasdeuterium-depleted compared to both C16 and C18. (b) Heptadecenes and C16 and C18 FAMEs in E. unicocca. Heptadecene is substantiallydepleted in deuterium relative to fatty acids and FAMEs. (c) C16 and C18 fatty acids in V. aureus. (d) FAMEs, heptadecene andphytadienes in V. aureus. dD values of heptadecene (in three cultures for which we have data) were depleted in deuterium relative to fattyacids and FAMEs. Although only two analyses of phytadienes were performed both were significantly depleted in deuterium relative to allother lipids.
14 Z. Zhang, J.P. Sachs / Organic Geochemistry xxx (2007) xxx–xxx
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CO(Fig. 3b). The e values for two FAMEs in E. unicocca
were�69.5& and�78.3&, somewhat more negativethan the corresponding FAs (Table 3). dD values ofthe C16 FAME in V. aureus also closely tracked waterdD values, with R2 = 0.99 (Fig. 3d). The e value forthe C16 FAME in V. aureus, �78&, was more nega-tive than in E. unicocca. The slope of the water–lipiddD regression for the C18:1 FAME was larger than forthe C16 FAME (Fig. 3d; Table 5).
Two V. aureus cultures had sufficient phytadienefor dD determination. The e values of �246.6& and�257.2& indicate substantial deuterium depletionin phytadiene relative to FAs and FAMEs. A simi-
Please cite this article in press as: Zhang, Z., Sachs, J.P., HydGeochem. (2007), doi:10.1016/j.orggeochem.2006.12.004
lar isotopic depletion in phytadiene relative to FAsand FAMEs was observed in B. braunii (Marti-nique) (Fig. 2d; Table 2).
4. Discussion
4.1. Hydrogen isotopes in lipids from B. braunii
Lipids within a single class generally fell within anarrow dD range of <50&. Between lipid classeshydrogen isotopic differences of 50–200& werecommon (Tables 1–3). In B. braunii A (Titicaca)and B (Martinique) races fatty acids and FAMEs
rogen isotope fractionation in freshwater algae: ..., Org.
NC
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786787788789790791
792793794795796797
Table 4Summary of D/H fractionation parameters in lipids from B. braunii
Strain Analyte R2 Slopea Intercepta ab Stdevc ed
eHydrocarbon
Titicaca Strain C27-alkadiene 0.999 0.691 �207.4 0.7755 0.0141 �224.5
C29-alkadiene 0.999 0.708 �219.0 0.7685 0.0103 �231.5
C31-alkadiene 0.999 0.709 �219.0 0.7688 0.0098 �231.2
Morocco strain C27-alkadiene 0.995 0.661 �169.2 0.8028 0.0259 �197.2
C29-alkadiene 0.998 0.750 �215.4 0.7790 0.0073 �221.0
C29-alkatriene 0.998 0.723 �213.9 0.7757 0.0112 �224.3
C31-alkadiene 0.998 0.744 �207.1 0.7849 0.0094 �215.1
Martinique strain C30 botryococcene 0.994 0.751 �277.9 0.7272 0.0103 �272.8
C31 botryococcene 1.000 0.730 �297.4 0.7075 0.0046 �292.5
C34 botryococcene 0.997 0.726 �329.1 0.6808 0.0089 �319.2
C34 bot isomer + ?e 0.999 0.724 �316.3 0.6909 0.0058 �309.1
C32 + C33 bot (?)e 0.999 0.699 �318.3 0.6847 0.0051 �315.3
C34 bot (?)e 0.999 0.647 �359.8 0.6413 0.0030 �358.7
Fatty acids
Titicaca strain C16 fatty acid 0.992 0.768 �184.3 0.8076 0.0118 �192.44
C18:1 fatty acid 0.999 0.782 �181.0 0.8128 0.0052 �187.2
C20:1 fatty acidf 0.999 0.776 �157.0 0.8319 0.0111 �168.1
C28:1 fatty acid 0.997 0.773 �163.9 0.8254 0.0103 �174.6
Morocco strain C16 fatty acid 0.999 0.801 �195.3 0.8041 0.0046 �195.9
C18:1 fatty acid 0.998 0.797 �183.5 0.8132 0.0056 �186.8
C20:1 fatty acidf 1.000 0.808 �162.7 0.8330 0.0052 �167.0
C28:1 fatty acid 0.999 0.795 �171.9 0.8225 0.0049 �177.5
Martinique strain C16 fatty acid 0.998 0.791 �169.9 0.8230 0.0065 �177.0
C18:1 fatty acid 0.998 0.790 �168.2 0.8242 0.0078 �175.8
C20:1 fatty acid 0.997 0.810 �153.4 0.8399 0.0078 �160.1
C28:1 fatty acid 0.999 0.807 �168.0 0.8275 0.0050 �172.5
Titicaca strain C16 FAME 0.998 0.778 �177.1 0.8153 0.0079 �184.7
C18:1 FAME 0.998 0.800 �187.1 0.8106 0.0055 �189.4
C20:1 FAMEf 0.998 0.837 �169.6 0.8314 0.0072 �168.6
C28:1 FAMEf 0.999 0.836 �183.9 0.8186 0.0037 �181.4
C16 FAME 1.000 0.715 �162.1 0.8160 0.0166 �184.0
C18:1 FAME 1.000 0.736 �166.4 0.8161 0.0128 �183.9
C20:1 FAMEf 0.999 0.770 �148.2 0.8405 0.0097 �159.5
C28:1 FAME 1.000 0.746 �165.9 0.8184 0.0118 �181.6
Phytadiene
Martinique strain Phytadieneg 0.996 0.557 �282.1 0.7015 0.0181 �298.5
a The values of slope and intercept are from the linear regression equations are derived from the measurements of dD values of abiomraker in five cultures and dD values of waters at harvest; n is typically 5, unless noted otherwise. The slope and intercept are ratherempirical values, and can be applied for reconstruction of paleoenvironmental water, given all correlation coefficient R2 > 0.99. However,such values should not be deemed as fractionation factor, a, and fractionation, e, i.e. 1000 · (a � 1).
b Fractionation factor, a, is determined for each individual culture based on the D/H ratios measured of biomarkers and waters atharvest, i.e. alipid–water = (D/H)lipid/(D/H)water = (dDlipid + 1000)/(dDwater + 1000). Reported is average value of five cultures with stan-dard deviation given.
c Stdev, standard deviation for measured fractionation factors in cultures for a specific bomarker.d Fractionation, e, is defined as 1000 · (a � 1) = [(dDlipid + 1000)/(dDwater + 1000) � 1] · 1000, where a is fractionation factor.e Peak identification with certain uncertainty. See text for details.f Where n = 4.g Where n = 3.
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with the same carbon skeleton had similar dD values(Fig. 4a and c), suggesting that the hydrogen atomson the FAME were derived from the same pool ofhydrogen from which fatty acids were producedand that the esterification reaction imparted littleor no hydrogen isotope fractionation.
Please cite this article in press as: Zhang, Z., Sachs, J.P., HydGeochem. (2007), doi:10.1016/j.orggeochem.2006.12.004
Hydrocarbons were depleted in deuterium rela-tive to fatty acids in all cultures: Alkadienes in theA race (Titicaca and Morocco) by about 40&,and botryococcenes in the B race (Martinique) by100–180& (Fig. 4a–c; Tables 1 and 2). While inagreement with Estep and Hoering (1980), who
rogen isotope fractionation in freshwater algae: ..., Org.
TED
PR
OO
F798799800801802803804805806807808809810811812813814815816817818819820821
822823824825826827828829830831832833834835836837838839840841842843844845
Table 5Summary of D/H fractionation parameters in lipids from E. unicocca and V. aureus
Strain Analyte na R2 Slopeb Interceptb ac Stdevd ee
Hydrocarbon
Eudorina unicocca 8-Heptadecene 5 0.993 0.849 �104.5 0.8884 0.0146 �111.6
Fatty acids
Eudorina unicocca C14 fatty acid 4 0.997 0.858 �93.3 0.8991 0.0103 �100.9
C16 fatty acid 5 0.999 0.949 �49.4 0.9502 0.0053 �49.8
C18 FA-unsaturated 4 0.999 0.984 �47.0 0.9571 0.0067 �42.9
C18 fatty acid 4 0.999 0.970 �67.5 0.9373 0.0076 �62.7
Volvox aureus C16 fatty acid 5 0.994 0.931 �56.3 0.9419 0.0141 �58.1
C18 FA-unsat 4 0.992 0.948 �45.9 0.9536 0.0164 �46.4
C18 fatty acid 3 0.996 0.978 �47.9 0.9562 0.0128 �43.8
Natural occurring fatty acid methyl esters (FAME)
Eudorina unicocca C16 FAME 5 0.998 0.895 �63.1 0.9305 0.0097 �69.5
C18 FAME-unsat 5 0.999 0.898 �57.6 0.9355 0.0075 �64.5
Volvox aureus C16 FAME 5 0.988 0.840 �71.5 0.9146 0.0212 �78.1
C18 FAME-unsat 5 0.982 0.838 �55.4 0.9280 0.0262 �63.9
C18 FAME 4 0.998 0.925 �59.8 0.9380 0.0102 �62.0
Notes:a n indicates the numbers of cultures in which dD values of a biomarker were measured.b The values of slope and intercept are from the linear regression equations are derived from the measurements of dD values of a
biomarker in five cultures and dD values of waters at harvest; The slope and intercept are rather empirical values, and can be applied forreconstruction of paleoenvironmental water, given all correlation coefficient R2 > 0.98. However, such values should not be deemed asfractionation factor, a, and fractionation, e, i.e. 1000 · (a � 1).
c Fractionation factor, a, is determined for each individual culture based on the D/H ratios measured of biomarkers and waters atharvest, i.e. alipid–water = (D/H)lipid/(D/H)water = (dDlipid + 1000)/(dDwater + 1000). Reported is average value of five cultures with stan-dard deviation given.
d Stdev, standard deviation for measured fractionation factors in cultures for a specific bomarker.e Fractionation, e, is defined as 1000 · (a � 1) = [(dDlipid + 1000)/(dDwater + 1000) � 1] · 1000, where a is fractionation factor.
16 Z. Zhang, J.P. Sachs / Organic Geochemistry xxx (2007) xxx–xxx
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ECreported deuterium enrichments in fatty acids rela-
tive to hydrocarbons in higher plants, these findingsare at odds with Sessions et al. (1999) who reporteddeuterium depletions of �50–100& in fatty acidsrelative to hydrocarbons in both a higher plant Dau-
cus carota (carrot) and a microalga Isochrysis gall-
ana (haptophyte).Carbon chain length had a minor and variable
influence on dD values of fatty acids, FAMEs and alk-adienes in B. braunii, but a significant influence on dD
values of botrycoccenes. In the Martinique strain, dD
values of C16 and C18:1 fatty acids were very similarwhile in both the Titicaca and Morocco strains dD
values of C18:1 fatty acids were slightly elevated com-pared to those of C16 fatty acids. However, in all threestrains, dD values of C28:1 were about 10& more posi-tive than those of C16 and C18:1. The C20:1 fatty acid, aminor component, was always the most deuterium-enriched fatty acid, typically by �20& relative toC16, C18:1 and C28:1 FAs. Similar isotopic relation-ships were observed for FAMEs (Table 2).
The trend toward deuterium enrichment withincreasing fatty acid chain length reported by Ses-sions et al. (1999) is not borne out by our data.
Please cite this article in press as: Zhang, Z., Sachs, J.P., HydGeochem. (2007), doi:10.1016/j.orggeochem.2006.12.004
Nor are the large deuterium enrichments of 112–163& in C18 relative to C16 FA in marine red andbrown macroalgae reported by Chikaraishi et al.(2004c). Chikaraishi et al. (2004c) also reported aprogressive deuterium depletion of �117& to�181& with increasing degree of unsaturation inC18:0 to C18:4 FAs. One possible explanation forthe discordant findings might be isotopic fraction-ation during lipid purification when argentationchromatography was used. Chikaraishi et al.(2004c) converted FAs into FAMEs and then usedAgNO3-impregnated silica gel to separate unsatu-rated from saturated FAs, a procedure likely tocause D/H fractionation in carbon double bonds,particularly when multiple unsaturations exist (deLigny, 1976). Alternatively, there might be real dif-ferences in isotopic fractionations during fatty acidsynthesis between the green algae we cultured andthe red/brown algae Chikaraishi et al. (2004c) inves-tigated. Additional culture experiments and stan-dardized lipid purification procedures are requiredto address this discrepancy.
Hydrogen isotope fractionation in alkadienesfrom the A race may have a slight carbon-chain-
rogen isotope fractionation in freshwater algae: ..., Org.
RR
EC
TED
PR
OO
F
846847848849850851852853854855856857858859860
861862863864865866867868869870871872873874875
-300
-200
-100
0
100
200
300δD
of a
lgal
lipi
d (‰
)
0 100 200 300 400 500
δD of waters at harvest (‰)
y = 0.782x - 180.955
y = 0.715x - 220.492
(Titicaca strain)
C18:1 FAME
C18:1 FA
C29-diene
(C18:1 FA )
(C29:2 diene)
-300
-200
-100
0
100
200
300
δD o
f alg
al li
pids
(‰
)
0 100 200 300 400 500
δD of waters at harvest (‰)
y = 0.797x - 183.499
y = 0.752x - 215.536
(Morocco strain)
C29:1 triene
C18:1 FA
C29-diene
(C18:1 FA )
(C29:2 diene)
-400
-300
-200
-100
0
100
200
300
δD o
f alg
al li
pids
(‰
)
0 100 200 300 400 500
δD of watesr at harvest (‰ )
y = 0.730x - 297.445
y = 0.726x - 329.067
y = 0.791x - 169.872
(Martinique strain)
phytadiene
C30bot
C34 bot
C16 FAME
C16 FA
(C30 bot)
(C34 bot)
(C16 FA)
a b
c
Fig. 4. D/H fractionation in different lipid classes within three strains of B. braunii. (a) A race, Titicaca strain. Alkadienes were �40&
depleted in deuterium relative to fatty acids. (b) A race, Morocco strain. Alkadienes and alkatrienes were �40& depleted in deuteriumrelative to fatty acids. (c) B Race, Martinique strain. Botryococcenes and phytadienes were >110& depleted in deuterium relative to fattyacids and FAMEs.
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COlength dependence. In the Titicaca strain, dD values
of C29 and C31 alkadienes were almost identical,while the C27 alkadiene was enriched in deuteriumby �7& relative to C29and C31 (Fig. 1a; Table 1).In the Morocco strain dD values of C27 alkadieneswere enriched by �24& relative to C29 (Table 1).The one alkatriene analyzed, C29:3, had a dD valuevery close to the C29:2-diene (Table 1), suggestingfurther desaturation did not cause D/Hfractionation.
Botryococcene dD values had a discernible car-bon-chain-length dependence, with longer chain-lengths generally associated with more negative dD
values (Fig. 2a; Table 1). As such, the C30 botryo-coccene—the precursor to all other botryococc-
Please cite this article in press as: Zhang, Z., Sachs, J.P., HydGeochem. (2007), doi:10.1016/j.orggeochem.2006.12.004
enes—had the least negative dD value (�272.8&),followed by the C31(�292.5&), and C34 botryococc-enes (�319.2&) (Table 1). We hypothesize that themethyl donor in botryococcene synthesis is depletedin deuterium relative to the C30 botryococcene,resulting in increasingly negative dD values withincreasing carbon number. Nevertheless, our con-clusion must be considered tentative due to co-elu-tion of the C32 and C33 botryococcenes on theGC–IRMS (see Section 3.2.2.1).
Phytadienes in the B race had e averaging�298.5&, significantly deuterium-depleted relativeto fatty acids (Table 2), consistent with prior studiesof D/H fractionation in phytol from marine algae(Estep and Hoering, 1980; Sessions et al., 1999)
rogen isotope fractionation in freshwater algae: ..., Org.
T
876877878879
880881
882883884885886887888889890891892893894895
896897
898899900901902903904905906907908909910911912913914915916917918919920921922923
924925926927928929
930931932933934935936937938939940941942943944945946947948949950951952953954955956957958959960961962963964965966967968969970971972973974
18 Z. Zhang, J.P. Sachs / Organic Geochemistry xxx (2007) xxx–xxx
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and vascular plants (Chikaraishi et al., 2004a).Hydrogen isotope fractionation between phytadieneand water was similar to that between C34 botryo-coccenes and water (Fig. 4c; Tables 1 and 2).
4.2. Hydrogen isotopes in lipids from E. unicocca and
V. aureus
FAMEs in both E. unicocca and V. aureus cul-tures were 20–27& depleted in deuterium relativeto fatty acids (Table 3). The only dominant hydro-carbon in both species, 8-heptadecene, was 48–62& more negative than the C16 FAs.
A chain length effect may exist for fatty acids inE. unicocca cultures in which the C14 FA was 50&
more negative compared to the C16 and C18 fattyacids (Table 3).
Two analyses of phytadiene dD values were madein V. aureus, yielding deuterium depletions of�246& and �257& relative to water (Table 3), sim-ilar to the deuterium depletions in B. braunii
(Martinique).
4.3. Hydrogen isotope fractionation during lipid
synthesis
During photosynthesis water is oxidized to O2
and NADP+ is reduced to NADPH (Raven et al.,1999), with the latter associated with a large D/Hfractionation (Estep and Hoering, 1980; Luo et al.,1991). 3-Phosphoglyceric acid (PGA) (also calledglycerate 3-phosphate) is produced when ribulose-1,5-bisphosphate and CO2 are catalyzed by theRubisco enzyme. The PGA is then converted to gly-ceraldehydes-3-phosphate (G3P) using ATP asenergy and NADPH as reductant. During this con-version, NADPH yields hydrogen that is incorpo-rated into organic matter.
Hydrogen isotopic variations within and betweenlipids in a single cell, and between lipids in differentspecies, come from three sources: (1) the isotopiccomposition of biosynthetic precursors, (2) isotopeeffects during biosynthetic reactions, and (3) the iso-topic composition of added hydrogen, primarilyfrom NADPH and NADH (Smith and Epstein,1970). The third source of isotopic variation is notrelated to the flow of substrates used in the assemblyof carbon skeletons, providing a distinct differencefrom carbon isotope studies (Sessions et al., 1999).
Lipids in plants derive from three primary biosyn-thetic pathways (Chikaraishi et al., 2004a). Straight-chain (n-alkyl) lipids are produced via the acetogenic
Please cite this article in press as: Zhang, Z., Sachs, J.P., HydGeochem. (2007), doi:10.1016/j.orggeochem.2006.12.004
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pathway using acetyl coenzyme-A (acetyl-CoA). Iso-prenoid (i.e., branched) lipids are synthesized via themevalonic acid (MVA) or non-mevalonic-acid path-way (DOXP/MEP, or 1-deoxy-D-xylulose 5-phos-phate/2-C-methylerythritol 4-phosphate) usingisopentenyl pyrophosphate (IPP).
4.3.1. D/H fractionation in acetogenic lipids: fatty
acids
Fatty acids are the precursor to all other aceto-genic lipids and acetyl-CoA is the direct precursorto fatty acids (Harwood, 1988). The principle pho-tosynthate in the plant cell, sucrose, is convertedto glucose 1-phosphate and further degraded inthe cytoplasm to either malic or pyruvic acid(Stumpf, 1980). These two respiratory substratesenter the mitochondria where pyruvate is oxida-tively decarboxylated to form acetyl-CoA and CO2
by the pyruvate dehydrogenase complex (Fig. 5a).Palmitic acid (C16) is synthesized from acetyl-
CoA and malonyl-CoA via several enzymatic reac-tions involving acyl carrier protein (ACP), NADPHand NADH (Fig. 5a) (Stumpf, 1980; Ohlrogee,1987). Its immediate precursor is palmitoyl-ACP,which can have three fates: elongation to stearoyl-ACP, use in glycerolipid synthesis, or hydrolysis topalmitic acid. Hydrogen isotope fractionation inC16 fatty acid in algae is the sum of these processes.
Elongation of palmitoyl-ACP to stearoyl-ACP,and the subsequent desaturation of stearoyl-ACPto form oleoyl-ACP do not, in all likelihood, causemuch hydrogen isotope fractionation, as evidencedby similar dD values in C16 and C18:1 fatty acids inthree strains of B. braunii (Table 2). This is consis-tent with the findings of Behrouzian et al. (2001)who reported that desaturation by stearoyl-ACPD9 desaturase proceeded without a measurablekinetic isotope effect. Stearoyl-ACP desaturase hassuch a high activity that stearoyl-ACP rarely accu-mulates, with near complete conversion to oleate(Harwood, 1997). Supporting that notion is ourobservation of much higher concentrations ofC18:1than C18:0 fatty acids in B. braunii (SFigs. 3b,4b, and 5b).
Longer FA homologues are synthesized fromoleic acid by two different elongases, one in theendoplasmic reticulum (C18-CoA elongase, catalyz-ing C18:1! C20:1), the other in the Golgi apparatus(C20-CoA elongase, catalyzing C20:1! C22:1! C30:1)(Agrawal and Stumpf, 1985a,b; Lessire et al., 1985).Agrawal and Stumpf (1985b) further demonstratedthat both the C18:1! C20:1 and C20:1! C22:1 elon-
rogen isotope fractionation in freshwater algae: ..., Org.
OR
REC
TED
PR
OO
F
975976977978979980981982983984985986
987988
989990991992993994995996997
Fig. 5. Lipid biosynthetic pathways in B. braunii with estimated D/H fractionation. (a) Fatty acids and alkadienes in the A race (Titicaca)are produced via the acetogenic pathway using acetyl coenzyme-A as a precursor. (b) Botryococcenes and phytadiene in the B race(Martinique) are isoprenoid (i.e., branched) lipids synthesized via the DOXP/MEP pathway using isopentenyl pyrophosphate as aprecursor (IPP). D values shown represent D/H fractionation when dDwater = 0. Acronyms are as follows, Co-A: coenzyme A; ACP: acylcarrier protein; DMAPP: dimethylallyl diphosphate; DOXP: 1-deoxy-D-xylulose 5-phosphate; FPP: farnesyl diphosphate; GPP: geranyldiphosphate; GGPP: geranylgeranyl diphosphate; IPP: isopentenyl diphosphate; MEP: 2-C-methyl-D-erythritol 4-phosphate; PSPP:presqualene diphosphate. (Referred from Pollard et al., 1979; Stumpf, 1980, 1987; Templier et al., 1984, 1991; Agrawal and Stumpf, 1985;Harwood, 1988; Schwender et al., 1997, 2001; Lichtenthaler, 1999; Rohmer, 1999; Charon et al., 1999, 2000; Szkopinska, 2000; Wankeet al., 2001; Okada et al., 2004; Sato et al., 2004).
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Cgations can proceed with NADPH as a reductant,but that NADH can only be used for theC18:1! C20:1 elongation (Fig. 5a).
We hypothesize that consistently higher dD val-ues of C20:1 relative to all other fatty acids (Table2; Figs. 1b, e and 2b) resulted from either or bothits synthesis from C18:1 in the endoplasmic reticulumor NADH serving as the sole reductant. Likewise,similar dD values of both C28:1 and C30:1 fatty acids(Table 1) may result from their synthesis in theGolgi apparatus. The hydrogen isotope data thussupport the notion that the C20:1 fatty acid is synthe-
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sized from C18:1 in a different location than that inwhich subsequent elongations occur.
4.3.2. D/H fractionation in acetogenic lipids:
alkadienes
The observed �40& deuterium depletion of alk-adienes relative to fatty acids (Fig. 4a, b) can beattributed to D/H fractionation during elongationand decarboxylation of C18:1 fatty acids during alk-adiene (and alkatriene) biosynthesis (Templier et al.,1984, 1991). Considering that D/H fractionationduring elongation of fatty acids is significantly less
rogen isotope fractionation in freshwater algae: ..., Org.
CO
RR
EC
TED
PR
OO
F
998999
100010011002100310041005
1006100710081009
101010111012
Fig. 5 (continued)
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than 40& it is possible that the decarboxylationprocess is characterized by a large kinetic isotopeeffect (Fig. 5a).
Furthermore, we note that the C29 and C31 alk-adienes had similar dD values in both (Titicacaand Morocco) strains of A race, whereas the C27
alkadiene was enriched in deuterium (Fig. 1a andd; Table 1), suggesting a common biosynthetic route
Please cite this article in press as: Zhang, Z., Sachs, J.P., HydGeochem. (2007), doi:10.1016/j.orggeochem.2006.12.004
for C29 and C31 that differed from that for C27. TheC29 alkatriene dD values were similar to the C29 alk-adiene values suggesting that further desaturationresulted in little or no D/H fractionation.
4.3.3. D/H fractionation in isoprenoid lipids
dD values of isoprenoid (i.e., branched) lipids, suchas botryococcenes and phytadienes, were much lower
rogen isotope fractionation in freshwater algae: ..., Org.
T
1013101410151016101710181019102010211022102310241025102610271028102910301031103210331034103510361037103810391040104110421043104410451046104710481049105010511052105310541055105610571058105910601061106210631064
1065106610671068106910701071107210731074107510761077
1078107910801081108210831084108510861087108810891090
10911092
109310941095109610971098109911001101110211031104110511061107110811091110111111121113
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than dD values of acetogenic lipids (Tables 1–3).This pattern has been observed previously by sev-eral researchers and attributed to the different bio-synthetic pathways for these two classes of lipids(c.f., Sessions et al., 1999; Hayes, 2001).
Green algae use the DOXP/MEP pathway exclu-sively for isoprenoid synthesis (Lichtenthaler, 1999;Schwender et al., 2001; Sato et al., 2003). Hydrogenatoms in isoprenoids have two sources. The decar-boxylation of pyruvate and G3P yields DOXP(Schwender et al., 1997; Charon et al., 1999,2000). A rearrangement followed by a reductionleads to the formation of MEP, which possessesthe C5 isoprene backbone (Fig. 5b) (Charon et al.,1999, 2000). This step uses NADPH as a hydrogendonor, thus incorporating deuterium-depletedhydrogen to C1 of MEP (corresponding to C-4 ofIPP) (Fig. 5b). Conversion of MEP into IPP resultsin the elimination of three water molecules via tworeductions and one phosphorylation (Lichtenthaler,1999; Rohmer, 1999). Hydrogen on C2 and C4 ofIPP and/or DMAPP (dimethylallyl diphosphate)comes from NADH or NADPH (Charon et al.,1999), while the remaining hydrogen comes fromDOXP/MEP. It is likely that the IPP synthesis isresponsible for much of the deuterium-depletion inisoprenoids from green algae (Fig. 5b).
IPP and farnesyl diphosphate (FPP), the directprecursor of C30 botryococcene, are synthesizedvia the DOXP/MEP pathway in B. braunii (B Race)(Sato et al., 2003) via a 2-step reaction in the chlo-roplast (Okada et al., 2004) (Fig. 5b). In the firststep, two molecules of FPP are condensed to formpresqualene diphosphate (PSPP). In the secondstep, the cycloprapane ring in PSPP is cleaved, fol-lowed by reduction with NADPH (Fig. 5b) (Okadaet al., 2004).
C30 botryococcene is rapidly converted to highermolecular weight (C31–C34) botryococcenes by suc-cessive methylation reactions (Metzger et al.,1987). The source of the additional methyl groupsis presumably methionine, the origin of whichremains unknown (Metzger et al., 1987). The factthat we observed progressively more deuterium-depletion with increasing carbon number (i.e.,C30–C34) in botryococcenes suggests either that themethionine is depleted in deuterium relative to theC30 botryococcene and/or there is a kinetic isotopeeffect associated with the methylation reaction.The position of methylation on the C30 botryococ-cene may also influence the dD values of differentisomers.
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Phytadiene (specifically, neophytadiene (Com-pound 8 in the Appendix A), the most abundant iso-mer) is produced by dehydration of phytol(Volkman and Maxwell, 1986; Grossi et al., 1996),which in turn is synthesized from GGPP (geranyl-geranyl diphosphate) during three hydrogenationreactions using NADPH (Chikaraishi et al.,2004a). IPP and DMAPP serve as precursors toGGPP (Fig. 5b). Though we did not measure thedD value of phytol we did observe e values of�251& to �298& in phytadienes, relative to water,in three species (B. braunii (B race), E. unicocca andV. aureus), in accord with Chikaraishi et al. (2004a).
4.3.4. Summary of D/H fractionation during lipid
synthesis
Though fatty acids, botryococcenes and phytadi-enes in the Martinque strain of B. braunii, and fattyacids and phytadiene in V. aureus are all synthesizedin the chloroplast (cf. Sato et al., 2003), presumablywith the same pool of NADPH, significant hydro-gen isotopic differences exist between botryococc-enes/phytadienes and fatty acids in B. braunii, andphytadiene and fatty acids in V. aureus. Thesehydrogen isotopic differences between acetogenicand isoprenoid lipids result from different biosyn-thetic pathways for the two types of lipids.
4.4. Species-dependence of hydrogen isotope
fractionation in green algae
A small but significant difference of�10–15& in D/H fractionation during lipid synthesis was observed indifferent species, while a large difference of�90–100&was observed between families of green algae (B. brau-
nii, from the Trebouxiophyceae; E. unicocca and V.aureus from the Chlorophyceae) (Fig. 6a and b).
The water–lipid isotopic difference for two com-pounds (C16 FA and FAME) from four speciesand two families was compared (Fig. 6a and b).Notwithstanding a small influence of temperatureon D/H fractionation during C16 FA and FAMEsynthesis of �–3&/�C (Zhang and Sachs, unpub-lished results), deuterium depletion in C16 FAs(Fig. 6a) and FAMEs (Fig. 6b) was much greater(i.e., >110&) in B. braunii (Trebouxiophyceae) com-pared to either V. aureus or E. unicocca (Chlorophy-ceae), suggesting a large inter-Family difference inD/H fractionation.
Between species the water-C16 FA isotopic differ-ence was much smaller, amounting to 15& betweenthe B (Martinique) (�177&) and A (Titicaca)
rogen isotope fractionation in freshwater algae: ..., Org.
TD
PR
OO
F
11141115111611171118111911201121112211231124112511261127112811291130113111321133
11341135
1136113711381139
1140114111421143114411451146114711481149115011511152115311541155115611571158115911601161116211631164116511661167
-200
-100
0
100
200
300
400δD
of C
16
fa
tty a
cids
(co
rrec
ted,
‰)
0 100 200 300 400 500
δD of waters at harvest (‰)
y = 0.768x - 184.3 r2 = 0.992
y = 0.791x - 169.9 r2 = 0.998
y = 0.869x - 74.6 r2 = 1.000
y = 0.931x - 56.3 r2 = 0.994EU
A2
BB
VVHT
VV
(VV at 15˚C)
(VVHT at 25˚C)
( BB at 28˚C )
(A2 at 28˚C )-200
-100
0
100
200
300
400
δD o
f C16
fat
ty a
cid
met
hyl e
ster
(‰
)
0 100 200 300 400 500
δD of waters at harvest (‰ )
y = 0.778x - 177.1 r2 = 0.998
y = 0.772x - 70.6 r2 = 1.000
y = 0.715x - 162.1 r2 = 1.000
y = 0.840x - 71.5 r2 = 0.988
(VVHT at 25˚C)
(VV at 15˚C)
(BB at 28˚C)
(A2 at 28˚C)
a b
Fig. 6. Species and family dependence of D/H fractionation during synthesis of (a) palmitic acid (C16:0) and (b) C16 FAME. Large(�100&) differences in D/H fractionation in a single lipid are observed between families of green algae. The effect is much larger than fortemperature, which has a small influence (see, for example, the lower slope for the high-temperature (VVHT, 25 �C) experiment with V.
aureus compared to the 15 �C experiment (VV)). Acronyms are as follows: EU: E. unicocca grown at 15 �C. VV: V. aureus grown at 15 �C.VVHT1 and VVHT2: V. aureus grown at 25 �C. BB: B. braunii, B race, Martinique strain grown at 28 �C. A2: B. braunii, A race, Titicacastrain, grown at 28 �C.
22 Z. Zhang, J.P. Sachs / Organic Geochemistry xxx (2007) xxx–xxx
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(�192&) races of B. braunii (Table 2), and 8&
between E. unicocca (�50&) and V. aureus
(�58&) (Table 3). Similarly, the water-C16 FAMEisotopic difference was 1& between Martinique(�184&) and Titicaca (�185&) strains of B. brau-
nii, and 8& between E. unicocca (�70&) and V.
aureus (�78&) (Tables 2 and 3).Schouten et al. (2006) studied the effect of growth
rate on D/H fractionation in alkenones from marinecoccolithophorids. In a subsequent paper, we willexplore the role that growth rate may play in D/Hfractionation of lipids. Here, we note that a 5-folddifference in growth rate (at constant temperature)caused little change in e. So although the large dif-ference in D/H fractionation during C16 FA andFAME synthesis in B. braunii vs. E. unicocca andV. aureus may result in part from differing growthrates, there are likely to be large differences in thehydrogen isotopic fractionation during synthesis ofa single lipid by different families of algae.
4.5. Implications for paleohydrologic reconstructions
from lipid D/H
The near-perfect linear correlation (R2 > 0.99)between all lipids studied and water dD values infive species of freshwater microalgae, and in alke-nones from the marine phytoplankton Emiliania
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Ehuxleyi (Englebrecht and Sachs, 2005), provides asound basis for using sedimentary algal lipid dD val-ues to reconstruct water dD values through time. Animportant caveat to doing so, however, is that verylittle is known yet about the role environmentalparameters such as nutrients, light, temperatureand salinity may play in influencing D/H fraction-ation during lipid synthesis. Studies we and otherresearchers are conducting ought to shed light onthese potential influences in the near future.
Tables 4 and 5 list the linear regression equationsfor all lipids we have studied as well as a and e values.Though the empirical linear regression equations canusually be used to derive water dD values from lipiddD values, neither the slope nor the intercept areequivalent to D/H fractionation as expressed by aand e. Furthermore, simply adding e to lipid dD val-ues to reconstruct water dD values could be inappro-priate if water dD values span a large range. Wetherefore recommend reconstructing water dD valuesfrom lipid dD values using a, which accounts for theD/H discrimination between lipid and water, usingthe equation: dDwater = (dDlipid + 1000)/a � 1000.
To demonstrate the utility of this approach wecalculated water dD values from palmitic acid dD
values in five B. braunii (Martinique) cultures usingthe average a value (0.816) reported in Table 4, andcompared this value to the measured dDwater values
rogen isotope fractionation in freshwater algae: ..., Org.
T
11681169117011711172117311741175117611771178117911801181118211831184118511861187118811891190119111921193119411951196119711981199120012011202120312041205120612071208120912101211
1212
12131214121512161217
1218121912201221122212231224122512261227122812291230123112321233123412351236123712381239124012411242124312441245124612471248124912501251125212531254125512561257125812591260126112621263
1264
126512661267
Z. Zhang, J.P. Sachs / Organic Geochemistry xxx (2007) xxx–xxx 23
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in those cultures at the time of harvest. The differ-ences were 0.5&, 8.1&, 4.2&, 3.4&, and �20.4&
for BBW1 to BBW5, respectively. Except forBBW5, which had dDwater = 500&, the recon-structed dDwater values are satisfactory.
Furthermore, if non source-specific lipids, such asmost fatty acids and sterols, are used to reconstructwater dD values, the influence of changing organicmatter inputs to the particular lake or ocean sedimentmust be considered. Compounds such as palmiticacid (C16) and oleic acid (C18:1) are ubiquitous, beingfound in higher plants, algae and bacteria. Becauselarge (>90&) differences in D/H fractionation wereobserved in palmitic acid in different families of greenalgae (Fig. 6a) it is prudent to assume similarly largedifferences occur in other types of algae. Conse-quently, a change in the proportion of different algaein a lake would be expected to alter the dD value ofpalmitic acid deposited in sediment, absent anychange in the lake water D/H ratio.
Palmitic acid and other lipids that derive fromboth aquatic and terrestrial plants and algae fur-ther confound the interpretation of down-corechanges in dD measured on a non source-specific(i.e., ubiquitous) lipid because higher plant lipidsare enriched in deuterium by �30–60& relativeto aquatic lipids (Sachse et al., 2004). Thus anychange in the proportion of palmitic acid derivedfrom aquatic algae and higher plants would beexpected to alter the dD value of sedimentary pal-mitic acid in the absence of any change in lake ormeteoric water dD values.
Clearly the use of non-specific lipids in lake sed-iment, such as palmitic acid, for reconstructing lakewater dD values and paleohydrology is susceptibleto misinterpretation (cf Huang et al., 2002). A betterapproach for down-core lake water dD reconstruc-tions is the use of a lipid biomarker specific to afamily of plants or algae, if not a species (the inter-species differences being rather small at �5–15&),and for which the fractionation factor, a, or anempirical water–lipid dD calibration has been devel-oped. An example would be the C34 botryococcene,produced solely by the B race of B. braunii.
5. Conclusions
Here, we have shown that lipids from five species ofcultured green algae, including E. unicocca, V. aureus
and three strains of B. braunii, were near-perfect record-ers (R2 > 0.99) of water D/H ratios. Barring any as yetunknown environmental influences on D/H fraction-
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ation during lipid synthesis, algal lipid dD values cantherefore be used as surrogates for water dD valueswhen the lipids derive from a single species or family.
All algal lipids were highly depleted in deuteriumrelative to environmental water, the result of kineticisotope effects during enzymatic processes. In thegreen algae, we studied deuterium depletion in lipidsvaried with the biosynthetic pathways that pro-duced them. As observed previously in other plantsand algae, isoprenoid lipids (botryococcenes andphytadiene), which are synthesized via the DOXP/MEP pathway, were highly depleted in deuteriumrelative to acetogenic lipids (straight-chain fattyacids, alkadienes, etc.), which are synthesized fromvia the acetogenic pathway.
Systematic hydrogen isotopic differences wereassociated with carbon chain length. The C20:1 fattyacid was consistently deuterium-enriched relative tothe C28:1 and C30:1 fatty acids, both of which wereenriched in deuterium relative to the C16 andC18:1fatty acids. We attribute these differences tothe site of elongation, which differs for C18:1–C20:1,and for C20:1–C28:1/C30:1fatty acids. Similarly, theC30 botryococcene is enriched in deuterium relativeto longer (C31–C34) homologues, implicating themethylation reactions as an additional source ofhydrogen isotopic fractionation.
Alkadienes, the products of elongation anddecarboxylation of the C18:1 fatty acid, are depletedin deuterium by 40& relative to that precursor, pre-sumably the result of isotopic discrimination duringthe decarboxylation reaction.
D/H fractionation in lipids also varies betweenspecies of green algae. Deuterium depletion in a sin-gle compound, such as the C16 fatty acid (palmiticacid), was approximately 100& greater in B. brauniithan in both E. unicocca and V. aureus.
Attempts at reconstructing water dD valuesthrough time using non-source-specific lipids, suchas palmitic acid, therefore run the risk of misinter-preting changes in the source of sedimentary lipidsfor changes in water D/H ratios. Lipid biomarkersunique to a family (or genus) should be targetedfor down-core reconstructions of water D/H ratiosusing empirically derived water–lipid fractionationfactors (a) established from culture experiments.
Acknowledgements
We are indebted to Dr. Pierre Metzger in the Lab-oratoire de Chimie Bioorganique et Organique Phy-sique (France) for supplying Botryococcous braunii
rogen isotope fractionation in freshwater algae: ..., Org.
1268126912701271127212731274127512761277127812791280
1281128212831284128512861287
1288
128912901291
24 Z. Zhang, J.P. Sachs / Organic Geochemistry xxx (2007) xxx–xxx
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inocculants and for sharing his wisdom on culturingand understanding this alga, and to Dr. Daniel Repe-ta at the Woods Hole Oceanographic Institution forassisting with the culture experiments. We thank Drs.Stefan Schouten, Alex Sessions and Mark Pagani forconstructive reviews that greatly improved this man-uscript, and Amy Cash for useful editorial com-ments. We are grateful to Carolyn Colonero andRoger Summons for assistance with GC–IRMSinstrumentation at MIT, Anthony Faiia for waterdD analyses at Dartmouth College, and Yanek Heb-ting for assistance in hydrogenating botryococcenesand alkadienes. We thank the Culture Collection of
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T1
23
4
5
24
7
8
9
10
11
12
1314
15
16
23 6
25
2627
28 2
CH3(CH2)7CH CH(CH2)xCH CH21.
2.
3.
4.
5.
6.
7.
8.
CH3(CH2)6CH CH(CH2)7CH3
Wh
9.
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OF
Algae and Protozoa in Scotland, UK for providinginoculants of E. unicocca and V. aureus. Fundingfor this research was provided in part by the GaryComer Science and Education Foundation (J.S.),the Jeptha H. and Emily V. Wade Award for Re-search at MIT (J.S.), and a Henry L. and Grace Doh-erty Professorship at MIT (J.S.).
Appendix A
Molecular structures of hydrocarbons discussedin this paper. Compounds 2–7 are botryococcenescontaining 30–34 carbon atoms.
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1718
19
2021
229
30
C30 bot
C31 bot
C32 bot
C34 bot
C34 bot-iso
C33 bot
ere x=15, 17 or 19
Phytadiene
rogen isotope fractionation in freshwater algae: ..., Org.
T
1292
129312941295
12961297
1298
1299
1300
13011302130313041305130613071308130913101311131213131314131513161317131813191320132113221323132413251326132713281329133013311332133313341335133613371338133913401341134213431344134513461347134813491350
13511352135313541355135613571358135913601361136213631364136513661367136813691370137113721373137413751376137713781379138013811382138313841385138613871388138913901391139213931394139513961397139813991400140114021403140414051406140714081409141014111412
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Appendix B. Supplementary data
Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.orggeochem.2006.12.004.
Associate Editor—S. Schouten
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14751476147714781479148014811482148314841485148614871488148914901491149214931494149514961497149814991500150115021503150415051506150715081509151015111512151315141515151615171518151915201521152215231524152515261527152815291530153115321533153415351536
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