Geochimica et Cosmochimica Acta, Vol. 65, No. 19, pp. 3299 –3305, 2001
Copyright © 2001 Elsevier Science Ltd
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Preservation of proteinaceous material during the degradation of the green alga
Botryococcus braunii: A solid-state 2D 15N 13C NMR spectroscopy study
XU ZANG,1 RENO T. NGUYEN,1 H. RODGER HARVEY,2 HEIKE KNICKER,3 and PATRICK G. HATCHER1
1
Department of Chemistry, The Ohio State University, Columbus, Ohio 43210, USA
Chesapeake Biological Laboratory, University of Maryland, Center For Environmental Science, Solomons, Maryland 20688, USA
3
Lehrstuhl für Bodenkunde, Technische Universität München, 85350 Freising Weihenstephan, Germany
2
(Received November 10, 2000; accepted in revised form May 4, 2001)
Abstract—Using solid-state cross-polarization-magic-angle-spinning (CPMAS) 13C and 15N nuclear magnetic resonance (NMR) and 2-D double cross polarization (DCP) MAS 15N 13C NMR techniques, microbially
degraded Botryococcus braunii was analyzed to study the chemical nature of organic nitrogen in the algal
residue. The amide linkage, as found in protein, was observed as the major nitrogen component in 201-day-old
degraded algae. No significant amount of heterocyclic nitrogen, or evidence for melanoidin products, was
found. The results strongly suggest that proteinaceous material can survive early diagenesis and be preserved
via its encapsulation by refractory, macromolecular, organic matter. Copyright © 2001 Elsevier Science Ltd
to sorptive protection by minerals (Ensminger and Gieseking,
1942; Marshmann and Marshall, 1981; Mayer, 1994; Keil et
al., 1994; Hedges and Keil, 1995), evidence does exist that
proteinaceous material can survive in systems where minerals
are absent, or only present in low amounts (Knicker and
Hatcher, 1997; Nguyen and Harvey, 1997, 1998, 2001; Derenne et al. 1998). One hypothesis proposed for the protection
of “labile” protein is encapsulation (Knicker and Hatcher,
1997), which states that proteins incorporated within the macromolecular matrix forming sedimentary organic matter are
sterically protected from bacterial hydrolysis. A recent hypothesis suggests that proteins may also undergo hydrophobic and
other non-covalent aggregations that enhance preservation
(Nguyen and Harvey, 2001).
In this study, the green microalga Botryococcus braunii was
labeled with 13C and 15N isotopes during growth and subsequently degraded under oxic conditions in a flow-through system. B. braunii occurs in freshwater, brackish, and saline lakes
around the world, and is known to contain insoluble, nonhydrolyzable, and highly refractory cell wall material termed
algaenan (Berkaloff et al.1983; Largeau, 1995). Algaenan is
thought to originate from polymerization of high molecular
mass lipids, and appears as a trilaminar sheath surrounding the
classical, polysaccharidic cell wall (Gelin et al., 1999). The aim
of this research was to investigate the potential for preservation
of proteinaceous material during early diagenesis in an aquatic
system devoid of minerals, and thus to evaluate the role of
encapsulation by refractory, macromolecular, organic matter
(e.g., algaenan), or for abiotic reactions. The fate of organic
nitrogen was monitored by the solid-state 2-D DCP MAS 15N
13
C-NMR technique, as well as by traditional CPMAS 13C and
15
N NMR techniques.
1. INTRODUCTION
Proteins, the most abundant nitrogen-containing substances
in many organisms, traditionally have been considered relatively labile in the environment. The recent observation of
proteinaceous material preserved in terrestrial and aquatic systems, therefore, has drawn considerable attention (Yamamoto
and Ishiwatari, 1992; Mitterer, 1993; Keil and Kirchman, 1994;
Knicker et al., 1996; Nguyen and Harvey, 1997, 1998, 2001;
Schulten and Schnitzer, 1997; Bada, 1998; Stankiewicz et al.,
1998; Walton, 1998; Pantoja and Lee, 1999; Poinar and Stankiewicz, 1999; Zang et al., 2000). Several solid-state 15N crosspolarization-magic-angle-spinning (CPMAS) nuclear magnetic
resonance (NMR) spectroscopic studies have suggested that a
significant part of refractory organic nitrogen (RON) in peat
(Zang et al., 2000), algal-derived sapropel (Knicker and
Hatcher, 1997), humin (Knicker and Hatcher, 1997), and algaenan (Derenne et al., 1993), is bound in amide and amino
forms. Such findings challenge the traditional depolymerization-recondensation hypothesis, which argues that heterocyclic
nitrogen comprises a significant amount of the RON (Schulten
and Schnitzer, 1997). However, it has been argued that the
CPMAS 15N NMR spectroscopy technique underestimates the
amount of certain types of heterocyclic-nitrogen that have no
directly bound 1H (e.g., pyridine-type-nitrogen) (Knicker,
2000). The significant overlap of the amide-nitrogen signal
with some types of heterocyclic-nitrogen (e.g., pyrrole-nitrogen) signals also makes it difficult to distinguish different
nitrogen functionalities (Knicker, 2000). In a recent study
(Knicker, 2000), a solid-state double cross polarization (DCP)
MAS NMR technique was applied to a degraded algal sample,
which only exhibited signals of 13C or 15N that interact intra
and intermolecularly. DCP MAS NMR has demonstrated great
promise as a technique with which we can better understand the
chemical nature of RON in the environment.
While the preservation of organic matter could be attributed
2. MATERIAL AND METHODS
2.1. Algal Cultures and Degradation Experiment
The experimental design and sample-processing scheme is illustrated
in Figure 1. Two cultures of B. braunii race A were grown in modified
CHU medium (Largeau et al., 1980) in 20-L carboys. The culture
medium was supplemented with NaHCO3 at a final concentration of 1
*Author to whom correspondence should be addressed (hatcher@
chemistry.ohio-state.edu).
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X. Zang et al.
Fig. 1. Experimental design and sample processing scheme.
mM, representing a typical concentration of bicarbonate in river water.
One culture was grown in medium containing NaH13CO3 (98 atom%;
from Isotec, Inc., Miamisburg, OH, USA) which was added to obtain
a 13C isotopic enrichment of 20 atom%; the other was grown in
medium containing K15NO3 for a 15N enrichment of 99 atom% with no
carbon isotopic enrichment. To the former culture, NaH13CO3 was
added aseptically (by syringe through a septum vial inserted into the
carboy stopper) every 3 days during growth, with the total addition
estimated to yield a 10 to 20% 13C label. Total bicarbonate addition
was based on the following: (1) the culture would reach a maximum
dry biomass of 0.8 g L⫺1 and would contain 60% organic carbon by
weight, based on pilot growth experiments; (2) one mole of reduced
algal carbon (CH2O) is produced per mole of CO2 utilized; and (3) one
mole of HCO⫺
3 is formed per mole of CO2 dissolved. The cultures were
grown (110 –170 ␮E m⫺2 s⫺1; 12:12 L:D cycle; 25°C; ⬎ 6 L min⫺1
aeration with 0.2 ␮m filtered air) to late-stationary phase of growth,
with dry weights used to assess growth (Casadevall et al., 1985). The
pH of both cultures was initially 7.8 and increased to 9.5 ⫾ 0.2 during
13
growth; therefore, loss of H13CO⫺
CO2 was not expected to be
3 as
significant. The two cultures, one containing 13C- and the other 15Nlabeled algae, were subsequently mixed together in a 40-L carboy and
allowed to degrade as previously described using a flow-through system (Harvey et al., 1995; Nguyen and Harvey, 1997). This mixing
resulted in ca. 50% and 5 to 10% of the algae being 15N- and 13Clabeled, respectively.
To provide the microbial consortia needed to mediate decay of the
algae, input waters were supplemented with nutrients. Water from a
natural, freshwater lake (Lake Lariat, Lusby, MD, USA) was amended
with trypticase peptone and yeast extract at 0.1% w/v to encourage
bacterial growth overnight. The bacterial inoculum was prefiltered to 3
␮m, and ⬃3.5 L was introduced into the header vessel containing 35 L
of low dissolved organic carbon ground water for a 10% v/v inoculum.
The header vessel water was subsequently pumped at a flow rate of 2
L d⫺1 into the 40-L vessel. B. braunii was degraded for 201 d, with
sampling at predetermined time points. Additional lake and ground
water was added to the header vessel when necessary during the
degradation period. Late-stationary phase cells (day 0 of the degradation experiment) and detritus (day 201) were harvested by centrifuga-
tion (20000 ⫻ g, 30 min), rinsed in distilled water, frozen in liquid
nitrogen, and then lyophilized.
2.2. Extraction of Organic Components from Algal Cells and
Detritus
Stationary-phase B. braunii and 201-d old detritus were treated with
multiple solvents to remove lipids, pigments, and extractable proteins.
Lyophilized material (500 mg) was weighed into 50-mL Teflon centrifuge tubes. All extractions were performed with 35-mL solvent
volumes, unless noted otherwise, using gas chromatography grade
reagents, and with the assistance of ultra-sonication (30 W). Centrifugation following each extraction was at 20000 ⫻ g for 30 min. Initial
extraction was with ice-cold 10% (w/v) trichloroacetic acid in acetone
(containing 0.1% ␤-mercaptoethanol) as described by Nguyen and
Harvey (1998). Extraction was continued with acetone (containing
0.1% ␤-mercaptoethanol), and this was performed until the supernatant
was clear (three times). Since pigments could not be removed completely with the previous treatments, extraction with 1:1 CH2Cl2:
methanol was performed (six times). The organic solvent treated residue was then dried under nitrogen. Removal of “easily-extractable”
protein was achieved with extraction (1 h; 25°C) in 15 mL of 0.1 N
NaOH, with tubes placed on a rotary shaker. A short, room temperature
extraction was performed to avoid protein hydrolysis as well as artifactual melanoidin formation, which likely would have occurred at
higher temperatures and with longer extraction times (Hedges, 1978).
The insoluble residues remaining after NaOH extraction were washed
thoroughly with deionized water until the pH of the supernatant was
neutral. A final extraction with 1:1 CH2Cl2: methanol was performed
(two times) on the residues. The postsolvent residues were finally dried
at 50°C under a stream of nitrogen gas before analysis by NMR
spectroscopy.
2.3. Solid-State CPMAS
13
13
C NMR
Solid-state C NMR spectroscopy utilizing cross polarization magic
angle spinning (CP-MAS), a ramp-CP procedure, and TPPM (two
pulse phase modulation) was performed using a Bruker DMX-300
2D NMR study of protein during algal decay
MHz NMR spectrometer with a 1H frequency of 300 MHz or a 13C
resonance frequency of 75.48 MHz. Spectra were obtained with a pulse
delay of 1 s, a contact time of 2 ms, and a magic angle spinning speed
of 13 kHz. Chemical shifts were calibrated using the carboxyl signal of
glycine as an external standard (176.03 ppm).
2.4. Solid-State CPMAS
15
N NMR
15
Solid-state N NMR spectroscopy utilizing CP-MAS and TPPM
was performed using a Bruker DMX-400 MHz NMR spectrometer
with a 1H frequency of 400 MHz, or a 15N resonance frequency of 40.5
MHz. Spectra were obtained with a pulse delay of 250 ms, a contact
time of 2 ms, and a magic angle spinning speed of 5 kHz. Chemical
shifts were calibrated using glycine as a reference (⫺347.6 ppm).
2.5. Solid-State 2-D DCP MAS
15
N
13
C NMR
For the solid-state 2-D DCP MAS 15N 13C NMR spectra, a Bruker
DMX 400 instrument with a triple resonance 7 mm probe was used.
The 15N resonance frequency was 40.54 MHz; that of the 13C was
100.61 MHz. A consecutive matched spin-lock transfer was used, first
from 1H to 15N (contact time t1 ⫽ 0.7 ms) and then from 15N to 13C
(contact time t2 ⫽ 3 ms). During the latter, protons were decoupled
using a frequency-shifted Lee-Goldburg sequence. During t1 a ramped
1
H-pulse was used, and during t2 a ramped 15N-pulse was used. Both
pulses were shaped from 100% down to 50% (Peersen et al., 1993). For
postsolvent residues of stationary-phase B. braunii and 201-d detritus,
spectra were obtained with mixing times increasing at an increment of
0.062 ms and at a spinning speed of 5.5 kHz. For the stationary-phase
algal residue, 9216 scans with a pulse delay of 1.5 s were accumulated
for each spectrum. For the degraded algal residue, 61440 scans with a
pulse delay of 1.5 s were accumulated for each spectrum. The 15N
chemical shifts are referenced to the nitromethane scale (⫽ 0 ppm) and
were calibrated with glycine (⫽ ⫺347.6 ppm). The 13C chemical shifts
are referenced to the tetramethysilane (⫽ 0 ppm) and were also calibrated with glycine (carboxylic carbon ⫽ 176.04 ppm).
3. RESULTS AND DISCUSSION
The 2-D DCP MAS NMR technique used here has previously been applied to a mixed algal culture that has been
degraded under aerobic conditions for two months (Knicker,
2000). The B. braunii samples are different from the previous
mixed algal culture in several ways. Firstly, the mixed algal
culture was comprised of strains of 13C and 15N dually labeled
Chlamydomonas, Chlorella, Closterium, and Scenedesmus. B.
braunii for our degradation experiment was generated by mixing two cultures that were labeled separately with 13C and 15N.
Such an approach allows testing of the depolymerization-recondensation hypothesis (Tissot and Welte, 1984; Bada, 1998).
The identification of enriched levels of 13C’s bonded to enriched 15N’s would confirm condensation reactions, whereas
lack of such bonding would indicate that the biopolymers in
each algal pool have maintained their original structural integrity. The power of the isotopic label approach for NMR spectroscopy is that the sensitivity of detection of any potential
melanoidin reaction can be improved by one to greater than two
orders of magnitude over natural abundance studies. A second
difference between our experimental design and that of Knicker
(2000) is that our algae were degraded for 201 d in a flowthrough system, with active microbial consortia present to
mediate the decay. The decay time used is three times longer
than that of the previous study of Knicker (2000). Importantly,
the B. braunii degradation was carried out in a mineral-free
medium; consequently, we can rule out any observed protein
preservation as due to sorptive protection by minerals. Finally,
we performed multiple solvent extractions on the fresh algae
3301
and the detritus to remove soluble lipids and unshielded proteins to evaluate the potential role of encapsulation by the
refractory cell wall.
Solid-state 13C NMR spectra of the stationary-phase- and
201-d-degraded B. braunii residues are shown in Figure 2. The
dominant signals in the 13C NMR spectrum of the stationaryphase algal residue (Fig. 2 a) are those for carbohydrates (74
and 105 ppm), paraffinic carbons (0 – 45 ppm), olefinic carbons
(129 ppm), and carboxyl and amide carbons (173 ppm), which
corresponds to the primary composition of algae, including
proteins, carbohydrates, and lipids. Our spectrum for the stationary-phase alga is not directly comparable to other spectra
obtained for various green algal species (e.g., Zelibor et al.,
1988), since we performed solvent extractions. Varied growth
conditions, species composition, and NMR acquisition parameters would also affect comparisons.
Degradation of B. braunii under oxic conditions in a flowthrough system significantly alters the carbon component (Fig.
2 b). The signals from aliphatic carbon components (30 ppm
and 129 ppm) that are inherently chemically resistant to biodegradation become more intense relative to other components
at 74, 105, and 173 ppm. These changes suggest that biologically “labile” substances, such as proteins and carbohydrates,
are preferentially lost during oxic degradation. The peak at 129
ppm can mainly be assigned to an isolated unsaturated carbon
in a long paraffinic chain, e.g., those in algaenan, as this signal
has been observed for chemically isolated algaenan (Derenne et
al., 1991; Gelin et al., 1996; Blokker et al., 1998) and a 4
million year old kerogen derived primarily from the diagenesis
of Botryococcus (Derenne et al., 1997). The ultimate product of
the biodegradation of B. braunii can be speculated to be the
algaenan residue that is selectively enriched, as other more
labile carbon components are lost.
15
N CPMAS NMR spectra of the postsolvent residues of
stationary-phase algae and algal detritus are illustrated in Figure 3. Although the algae have been degraded under oxic
conditions for 201 d, both of the spectra show almost identical
signals, namely an intense peak at ⫺257 ppm and minor peaks
at ⫺294, ⫺304, and ⫺345 ppm. The predominant amide signal
at ⫺257 ppm corresponds to peptide-nitrogen, with proteinaceous materials constituting the bulk of algal biomass. The
signals denoted by asterisks are spinning side bands of the
amide signal, arising due to incomplete removal of chemical
anisotropy. The resonance signals at ⫺294 ppm and ⫺304 ppm
are mostly assigned to the amino groups of basic amino acids
or nucleosides (Witanowski et al., 1993). The resonance signal
corresponding to the free amino group at ⫺345 ppm is also
resolved. The presence of the amide signal in the spectra
indicates that some proteins are not released upon NaOH treatment. A significant amount of proteins in the solvent treated,
stationary-phase B. braunii residue was unexpected. More surprisingly, occurrence of proteins in the detritus residue indicates that some proteins are intimately associated with the
refractory, aliphatic cell wall material.
Although the 15N NMR spectrum of the biodegraded algae
indicates a predominant proteinaceous nature (Fig. 3 b), conclusive exclusion of heterocyclic nitrogen, such as nitrogen in
purines, indoles, imidazoles, pyrrole-like compounds, and carbazoles from the nitrogenous components in the degraded algae
is arguable. A previous study pointed out that the chemical shift
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X. Zang et al.
Fig. 2. Solid-state 13C CPMAS NMR spectra of 15N- (⬃ 50%) and 13C- (⬃ 5–10%) enriched, (A) stationary-phase- and
(B) 201-d-degraded Botryococcus braunii residues remaining after sequential solvent extractions.
region of some of these heterocyclic compounds can expand to
⫺270 ppm (Knicker, 2000). Consequently, the ability to determine their contribution may be diminished by the broad amide
signal. To distinguish the contribution of amide-nitrogen and
heterocyclic-nitrogen to the total signal intensity of the peak at
⫺257 ppm, we have applied a solid-state two-dimensional
double-cross-polarization magic angle spinning (2-D DCP
MAS) NMR technique to the solvent extracted, stationaryphase- and 201-d-degraded algal residues. During the 2-D DCP
MAS NMR experiment, magnetization is transferred first from
1
H to 15N and then to 13C (Knicker, 2000). The resulting
spectrum shows cross peaks correlating 15N signal intensity to
13
C chemical shifts of carbons that have strong dipolar interactions with neighboring 15N. Solid-state 2-D DCP MAS 15N
13
C NMR spectra of the stationary-phase algae and the degraded algae are shown in Figures 4 and 5. In both cases, cross
peaks are observed only for carboxyl/amide-C (185 – 160 ppm)
and N-substituted alkyl-C (60 – 45 ppm). The cross peaks at
⬃230 ppm and 120 ppm of the 13C dimension are spinning side
bands of the signal at 173 ppm. No cross peaks are detected for
C in the sp2 13C chemical shift region that could give direct
evidence for pyrrole-type nitrogen (⫺145 ppm). Thus, the peak
at ⫺257 ppm in the CPMAS 15N NMR spectrum of the
degraded algal residue (Fig. 3 b) can be assigned solely to
amide-nitrogen. A 1-D spectrum version of the 2-D DCP MAS
15
N 13C NMR experiment is also shown in Figures 4 and 5.
Two major signals at 185 – 160 ppm and 60 – 45 ppm with
comparable intensities are observed, indicating almost all of the
nitrogen in amide bonds is additionally bound to alkyl carbon,
as would be expected for peptides.
A further examination of the 1-D spectrum of the degraded
algal residue revealed a poor signal to noise ratio, demonstrating a lack of extensive resonance signal from direct bonding
between 13C–15N (Fig. 5). In the case of the stationary-phase
sample (Fig. 4), the 13C NMR spectrum shows poor signal-tonoise because the 13C enriched structure are bound to 15N
present only at natural abundance levels (0.36 atom%); this is
due to the fact that the isotopic labels were applied to separate
B. braunii cultures which were then mixed before initiating the
oxic decomposition. The enriched 15N-containing stationaryphase algae are not 13C enriched, and the enriched 13C-containing stationary-phase algae are not 15N enriched. If crosscoupling reactions were occurring during diagenesis, we would
expect the 15N-enriched proteins in one subculture to react with
the 13C-enriched components of the other subculture, thereby
showing a significant gain in signal-to-noise ratio. The fact that
a higher signal-to-noise ratio was not observed in the case of
the degraded sample indicates that 13C-enriched components
do not cross couple to 15N-enriched components; thus, the
signals observed are from residual proteinaceous materials
rather than newly synthesized material produced during the
decomposition process. Since the number of scans accumulated
for the spectrum of the degraded algal residue is 6.7 times more
than that accumulated for the spectrum of the stationary-phase
algal residue, we should observe a decrease in the signal to
noise ratio if the number of scans for both samples were
2D NMR study of protein during algal decay
Fig. 3. Solid-state 15N CPMAS NMR spectra of 15N- and 13C- enriched, (A) stationary-phase- and (B) 201-d-degraded
B. braunii residues. Asterisks indicate spinning side bands of the signal at ⫺257 ppm.
Fig. 4. Solid-state 2-D DCP MAS
residues.
15
N
13
C-NMR spectrum of
15
N- and
13
C- enriched, stationary-phase B. braunii
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X. Zang et al.
Fig. 5. Solid-state 2-D DCP MAS
residues.
15
N
13
C-NMR spectrum of
equivalent. A decrease in the signal to noise ratio would be mainly
due to the loss of proteinaceous material during diagenesis.
Since bacteria are reliant on the algal derived C and N to
synthesize compounds necessary for their survival, we would
expect the 15N- and 13C-labeled organic matter to be hydrolyzed and recombined. Thus, in the 2-D experiment we might
expect to observe an increased resonance signal from direct
bonding between 13C–15N. Several reasons could explain why
recombination (due to bacterial metabolism) was not observed.
Firstly, past studies of oxic algal decay have indicated that
bacteria generally contribute less than 10% of total particulate
organic carbon by the end of the incubations (Harvey and
Macko, 1997). With the current B. braunii degradation experiment, bacteria appear to contribute only 1 to 2% of the total
particulate organic carbon content during the entire degradation
experiment (Nguyen et al., manuscript in prep.). We hypothesize that some 15N- and 13C-labeled amino acids from algae
were incorporated by bacteria (for de novo synthesis of proteins
containing both 15N and 13C labels), but the bacterial contribution was not significant enough to observe an increased
resonance signal from direct bonding between 13C–15N. Secondly, although the decomposer organisms may have used
much of the biologically available (“un-encapsulated”) algal
protein, most of the newly synthesized protein was likely
degraded near the end of the degradation experiment. Finally,
another explanation for the observed lack of increased bonding
between 13C–15N lies in the fact that we are looking at the
residue remaining after organic solvent and NaOH extraction. It
15
N- and
13
C- enriched, 201-d-degraded B. braunii
is possible that only minor amounts of bacterial proteins are
associated with the solvent-treated residue, due to the bacteria’s
lack of refractory, algaenan-like material. We hypothesize that
most of the solvent-treated residues (stationary phase algae and
detritus) are comprised of algal proteins intimately associated
with algaenan.
The results strongly suggests that biopolymers in the 15N and
13
C labeled algal pools have maintained their original structural
integrity and that depolymerization-recondensation reactions
among biopolymers were very unlikely during the microbiallymediated degradation of B. braunii. The NMR data support
other evidence indicating the minor importance of the melanoidin reaction during early diagenesis, for example, in recent
work applying a cleaving agent (N-phenylacylthiazolium bromide) for glucose-derived protein-protein cross-links (Nguyen
and Harvey, 2001).
4. CONCLUSIONS
(1) Proteinaceous material remains the major organic nitrogen component in B. braunii residual organic matter after over
200 d of oxic degradation by natural microbial assemblages.
(2) Results of 2-D DCP MAS 15N 13C NMR do not support
the formation of heterocyclic-nitrogen compounds resulting
from depolymerization-recondensation reactions during the microbial degradation of B. braunii. The finding of both proteinaceous and highly aliphatic materials in the degraded algae
supports the “encapsulation” hypothesis of Knicker and
2D NMR study of protein during algal decay
Hatcher (1997), whereby the highly aliphatic macromolecular
network (e.g., algaenan) physically protects intrinsically labile
proteins from bacterial hydrolysis.
Acknowledgments—We thank Claude Largeau for the Botryococcus
stock culture, and three anonymous reviewers for comments which
improved the final manuscript. Financial support for this work was
provided by the National Science Foundation (OCE-9711596 to PGH
and OCE-9907069 to HRH), the donors of the Petroleum Research
Fund of the American Chemical Society (HRH), and the Ohio State
University through a Graduate Student Award to X. Zang.
Associate editor: J. I. Hedges
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