Chemical Biomarkers in Aquatic Ecosystems

Chemical Biomarkers

in Aquatic Ecosystems

Chemical Biomarkers

in Aquatic Ecosystems

     THOMAS S. BIANCHI AND ELIZABETH A. CANUEL

PRINCETON UNIVERSITY PRESS

PRINCETON AND OXFORD

Copyright © 2011 by Princeton University Press

Published by Princeton University Press, 41 William Street,

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In the United Kingdom: Princeton University Press, 6 Oxford Street,

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Library of Congress Cataloging-in-Publication Data

Bianchi, Thomas S.

Chemical biomarkers in aquatic ecosystems/Thomas S. Bianchi and Elizabeth A. Canuel.

p.   cm.

Includes bibliographical references and index.

ISBN 978-0-691-13414-7 (cloth : alk. paper)

1. Aquatic ecology.   2. Biochemical markers.   I. Canuel, Elizabeth A., 1959–   II. Title.

QH541.5.W3B53 2011

577.6—dc22           2010029921

British Library Cataloging-in-Publication Data is available

This book has been composed in Sabon and Din Pro

Printed on acid-free paper. ∞

Typeset by S R Nova Pvt Ltd, Bangalore, India

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1

We dedicate this book to our spouses, Jo Ann Bianchi and Emmett Duffy, and our children, Christopher Bianchi and Conor Duffy, for their unending support and patience, without which we could never have completed this book.

Several current characteristics of organic geochemical research serve to limit advances toward a deeper understanding of global biogeochemical cycles. For the most part, these problems are shared by all organic geochemists (author included), but are not unique to our guild. The first of these is that geochemical research is decidedly provincial. That is, functionally similar processes occurring in weathering rocks, soils, lakes, rivers and the ocean tend to be studied by different types of geochemists with dissimilar backgrounds, terminologies and methodologies. This geographic specialization has limited the scope of both our fieldwork and insight, and has resulted in lost research and interpretive opportunities. . . . At present, there is insufficient information to obtain unique solutions at the global scale, even for bulk carbon. The main constraint is that meaningful rate constants for in situ degradation within key environment are not known.

— John I. Hedges, 1992

Contents

Preface

Acknowledgments

1. Metabolic Synthesis

2. Chemical Biomarker Applications to Ecology and Paleoecology

3. Stable Isotopes and Radiocarbon

4. Analytical Chemical Methods and Instrumentation

5. Carbohydrates: Neutral and Minor Sugars

6. Proteins: Amino Acids and Amines

7. Nucleic Acids and Molecular Tools

8. Lipids: Fatty Acids

9. Isoprenoid Lipids: Steroids, Hopanoids, and Triterpenoids

10. Lipids: Hydrocarbons

11. Lipids: Alkenones, Polar Lipids, and Ether Lipids

12. Photosynthetic Pigments: Chlorophylls, Carotenoids, and Phycobilins

13. Lignins, Cutins, and Suberins

14. Anthropogenic Markers

Appendix I. Atomic Weights of Elements

Appendix II. Useful SI Units and Conversion Factors

Appendix III. Physical and Chemical Constants

Glossary

Bibliography

Index

Preface

Due to the complexity of organic matter sources in aquatic systems, the application of chemical biomarkers has become widespread in limnology and oceanography. We define biomarker molecules in this book as compounds that characterize certain biotic sources and selectively retain their source information, even after stages of decomposition and diagenesis (after Meyers, 2003). Hence, the term biomarker molecule should not be confused with the terms commonly used by ecotoxicologists or molecular biologists (see Timbrell, 1998; Wilson and Suk, 2002; Decaprio, 2006, and references therein). Early definitions of biological markers, or simply biomarkers or chemical biomarkers, described these compounds as molecular fossils derived from formerly living organisms (Eglinton et al., 1964; Eglinton and Calvin, 1967).

More recently, biomarkers have been defined as complex organic compounds composed of carbon, hydrogen, and other elements, which occur in sediments, rocks, and crude oils, and show little to no change in their chemical structure from their precursor molecules that once existed in living organisms (Hunt et al., 1996; Peters et al., 2005; Gaines et al., 2009). Biomarkers have also been defined recently as lipid components found within bitumen in coal as well as kerogens that provide an unambiguous link to biological precursor compounds, because of their stability through diagenesis and catagenesis (Killops and Killops, 2005; Gaines et al., 2009). Additionally, the term geolipid has commonly been used to describe decay-resistant biomarkers in sediments because lipids are typically recalcitrant compared with other biochemical components of organic matter, making them more long-lived in the sedimentary record (Meyers, 1997). The first convincing linkage made between geolipids and living organisms was made by Treibs (1934), when a correlation was made between chlorophyll a in photosynthetic organisms and porphyrins in petroleum. This important discovery essentially marked the beginning of organic geochemistry.

Chemical biomarkers have provided numerous insights about present and past aspects of Earth history, including (1) the food and energy sources available to microbes and higher organisms, (2) microbial chemotaxonomy, (3) sources of fossil fuels, and (4) the evolution of life on Earth. A greater knowledge of the biogeochemical cycling of organic matter, which involves the transformation, fate, and transport of chemical substances, is critical in understanding the effects of natural and human-induced environmental changes—from a regional and global context. Approaching aquatic sciences from a biogeochemical perspective requires a fundamental understanding in the biochemistry of organic matter. Our motivation for writing this book is that many of the books on chemical biomarkers are edited volumes, too diffuse and difficult to use as textbooks for advanced undergraduate and graduate courses. We also wanted to develop a book with examples of the applications of biomarkers to a range of aquatic environments, including riverine, estuarine, and oceanic ecosystems.

This book is designed for advanced undergraduate and graduate level classes in aquatic and marine biogeochemistry, organic geochemistry, and global ecosystem dynamics. Prerequisites for such a course may include introductory courses in inorganic and organic chemistry, oceanography and/or limnology, and environmental and/or ecosystem ecology. The book should also prove to be a valuable resource for researchers in limnological, estuarine, marine, and environmental sciences. The basic organization of the book is derived from classes we have taught in global biogeochemistry, organic geochemistry, and marine/estuarine biogeochemistry. It is structured around individual classes of biomarkers, with each chapter describing the structure and synthesis of each class of compounds, their reactivity, and examples of specific applications for each class of biomarkers.

In chapter 1, we provide a general background on the synthesis of chemical biomarkers and their association with key metabolic pathways in organisms, as related to differences in cellular structure and function across the three domains of life. We discuss photosynthesis, the dominant pathway by which biomass is synthesized. Additionally, we present information about chemoautotrophic and microbial heterotrophic processes. The holistic view of biosynthetic pathways of chemical biomarkers here also provides a roadmap for other chapters in the book, where more specific details on chemical pathways are presented for each of the respective biomarker groups. While other organic geochemistry books have generally introduced the concepts of chemical biomarkers in the context of physical and chemical gradients found in natural ecosystems (e.g., anaerobic, aerobic), we begin by examining biosynthetic pathways at the cellular level of differentiation.

Chapter 2 provides a brief historical account of the success and limitations of using chemical biomarkers in aquatic ecosystems. This chapter also introduces the general concepts of chemical biomarkers as they relate to global biogeochemical cycling. The application of chemical biomarkers in modern and/or ancient ecosystems is largely a function of the inherent structure and stability of the molecule, as well as the physicochemical environment of the system wherein it exists. In some cases, redox changes in sediments have allowed for greater preservation of biomarker compounds; in well-defined laminated sediments, for example, a strong case can be made for paleo-reconstruction of past organic matter composition sources. However, many of the labile chemical biomarkers may be lost or transformed within minutes to hours of being released from the cell from processes such as bacterial and/or metazoan grazing, cell lysis, and photochemical breakdown, just to name a few. The role of trophic effects versus large-scale physiochemical gradients in preserving or destroying the integrity of chemical biomarkers varies greatly across different ecosystems. These effects will be discussed as they relate to aquatic systems such as lakes, estuaries, and oceans.

In chapter 3, we discuss the basic principles surrounding the application of stable isotopes in natural ecosystems, which are based on variations in the relative abundance of lighter isotopes from chemical rather than nuclear processes. Due to faster reaction kinetics of the lighter isotope of an element, reaction products in nature can be enriched in the lighter isotope. These fractionation processes can be complex, as discussed below, but have proven to be useful in determining geothermometry and paleoclimatology, as well as sources of organic matter in ecological studies. The most common stable isotopes used in oceanic and estuarine studies are ¹⁸O, ²H, ¹³C, ¹⁵N, and ³⁴S. The preference for using such isotopes is related to their low atomic mass, significant mass differences in isotopes, covalent character in bonding, multiple oxidations states, and sufficient abundance of the rare isotope.

Living plants and animals in the biosphere contain a constant level of ¹⁴C, but when they die there is no further exchange with the atmosphere and the activity of ¹⁴C decreases with a half-life of 5730 ± 40 yr; this provides the basis for establishing the age of archeological objects and fossil remains. The assumptions associated with dating materials are (1) that the initial activity of ¹⁴C in plants and animals is a known constant and is independent of geographic location, and (2) that the sample has not been contaminated with modern ¹⁴C. The application of ¹⁴C measurements in organic carbon cycling studies has been extensive in oceanic environments. While only a few studies using such techniques in organic carbon cycling were applied early on in coastal and estuarine regions, there has been a considerable increase in recent years, with a general paradigm emerging in river/estuarine systems whereby dissolved organic carbon (DOC) is typically more ¹⁴C-enriched (or younger) than particulate organic carbon (POC).

Recently developed methods, such as automated preparative capillary gas chromatography (PCGC), now allow for separation of target compounds for ¹⁴C analysis based on acceleration mass spectrometry (AMS). In very general terms, compound-specific isotope analysis (CSIA) allows for accurate determination of ages of compounds that are specific to a particular source (e.g., phytoplankton) within the heterogeneous matrix of other carbon compounds commonly found in sediments (e.g., terrigenous sources). Similarly, the application of CSIA to stable isotopic work has also proven to be useful in distinguishing between different types of organic carbon sources in estuarine systems. Future work in this area along with the application of multiple isotopic mixing models, which in some cases include δD, in aquatic systems is discussed in this chapter. In recent years, application of CSIA has contributed new insights to the field of chemical biomarkers. Measurements of δ¹³C, δ¹⁵N, and δD stable isotope composition of lipid and amino acid biomarkers are covered within individual chapters about specific compound classes.

In chapter 4, we provide a background on the important role technology has played in the study of chemical biomarkers and the many advances in the field that have resulted from the development of new analytical tools. The reader is introduced to some of the classic analytical tools used in organic geochemistry, including gas chromatography–mass spectrometry (GC-MS), pyrolysis GC-MS, direct temperature-resolved MS (DT-MS), CSIA, high-performance liquid chromatography (HPLC), and nuclear magnetic resonance (NMR) spectroscopy. Additionally, characterization of dissolved organic matter (DOM) and chromophoric DOM (CDOM) by fluorescence, use of pulsed amperometric detector (PAD) detectors in the analysis of sugars, and capillary electrophoresis are introduced. Recent advances in the following areas are covered: (1) analysis of polar organic compounds utilizing liquid chromatography mass spectrometry (LC-MS), (2) multidimensional NMR, and (3) Fourier transform ion cyclotron resonance MS (FT-ICR-MS).

Future work will include discussion of the limitations of scaling-up from compound-level measurements to bulk carbon pools. Additionally, the challenges of moving from the laboratory benchtop to larger spatial and temporal scales, particularly as advances are made in observing/remote sensing platforms, are discussed.

Chapter 5 covers carbohydrates, the most abundant class of biopolymers on Earth and significant components of water column particulate organic matter (POM) and DOM in aquatic environments. Carbohydrates are important structural and storage molecules and are critical in the metabolism of terrestrial and aquatic organisms. Carbohydrates can be further divided into monosaccharides (simple sugars), disaccharides (two covalently linked monosaccharides), oligosaccharides (a few covalently linked monosaccharides), and polysaccharides (polymers made up of several mono- and disaccharide units). In phytoplankton, carbohydrates serve as important reservoirs of energy, structural support, and cellular signaling components. Carbohydrates make up approximately 20 to 40% of the cellular biomass in phytoplankton and 75% of the weight of vascular plants. Minor sugars, such as acidic sugars, amino sugars, and O-methyl sugars, tend to be more source-specific than major sugars and can potentially provide further information on the biogeochemical cycling of carbohydrates. As discussed in chapter 3, the application of CSIA on monosaccharides may prove useful in distinguishing between different types of organic carbon sources in aquatic systems.

In chapter 6, we discuss proteins, which make up approximately 50% of organic matter and contain about 85% of the organic N in marine organisms. Peptides and proteins comprise an important fraction of the POC (13–37%) and particulate organic nitrogen (PON) (30–81%), as well as dissolved organic nitrogen (DON) (5–20%) and DOC (3–4%) in oceanic and coastal waters. In sediments, proteins account for approximately 7 to 25% of organic carbon and an estimated 30 to 90% of total N.

Amino acids are the basic building blocks of proteins. This class of compounds is essential to all organisms and represents one of the most important components in the organic nitrogen cycle. Amino acids represent one of the most labile pools of organic carbon and nitrogen. The typical mole percentage of protein amino acids in different organisms shows considerable uniformity in compositional abundance. In aquatic systems, amino acids have typically been analyzed in characteristic dissolved and particulate fractions. For example, the typical pools of amino acids commonly measured in coastal systems are divided into the total hydrolyzable amino acids (THAA) in water column POM and sedimentary organic matter (SOM), and dissolved free and combined amino acids (DFAA and DCAA) in DOM.

Another process that can account for the occurrence of D-amino acids in organisms is racemization, which involves the conversion of L-amino acids to their mirror-image D form. Estuarine invertebrates have been found to have D-amino acids in their tissues as a result of this process. Rates of racemization of amino acids in shell materials, calibrated against radiocarbon measurements, have also been used as an index for historical reconstruction of coastal erosion. Future work on amino acids and proteins as biomarkers is discussed in the context of C and N isotopic analyses of D- and L-amino acids, as well as advances in the area of proteomics.

Chapter 7 examines nucleic acids, polymers of the nucleotides ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), which act as the templates for protein synthesis. High levels of nucleic acids in microbes contribute to their elevated nitrogen and phosphorus contents. In recent years, isotopic signatures of nucleic acids have provided new insights about the sources of carbon-supporting bacterial activity.

This chapter discusses recent efforts to bridge the fields of organic geochemistry and molecular ecology. The coupling of biomarker information to molecular (genetic) data has the potential to provide new insights about specific sediment microbial communities and their effects on sediment organic matter. Recent studies have provided information about the evolutionary basis of biosynthetic pathways, which influence the capabilities of microorganisms to utilize specific substrates and the synthesis of unique biomarkers. Such efforts demonstrate the impact that microorganisms have on organic geochemistry.

Future directions will discuss attempts to characterize isotopic signatures of DNA. Progress is also being made in the application of proteomics to more fully characterize organic matter. While many of the major advances in biochemistry have been in protein chemistry, limitations remain in applying these methods to aquatic systems.

Chapter 8 covers fatty acids, the building blocks of lipids, which represent a significant fraction of the total lipid pool in aquatic organisms. We explore how chain length and levels of unsaturation (number of double bonds) have been shown to be correlated to decomposition, indicating a pre- and postdepositional selective loss of short-chain and polyunsaturated fatty acids. In contrast, saturated fatty acids are more stable and typically increase in relative proportion to total fatty acids with increasing sediment depth. Polyunsaturated fatty acids (PUFAs) are predominantly used as proxies for the presence of fresh algal sources, although some PUFAs also occur in vascular plants and deep-sea bacteria. Thus, these biomarkers represent a very diverse group of compounds present in aquatic systems.

This chapter discusses the numerous applications of fatty acid biomarkers to identifying the sources of organic matter in lakes, rivers, estuaries, and marine ecosystems. Fatty acid biomarkers have been applied to ultrafiltered dissolved organic matter (UDOM), POM, and sediments. Additionally, fatty acids have been used in ecological studies examining the dietary requirements of aquatic organisms and trophic relationships (e.g., work by Tenore and Marsh on effects of fatty acid composition on growth/reproduction of Capitella sp.) (Tenore and Chesney, 1985; Marsh and Tenore, 1990). Fatty acids have also been widely used in chemotaxonomic studies aimed at understanding microbial community structure. Recent studies have documented the influence of essential fatty acids in the transfer of algal organic matter to higher trophic levels (e.g., Müller-Navarra and colleagues) and trophic upgrading of food quality by protozoans and heterotrophic protists (e.g., work by Klein Breteler et al. and Veloza et al.).

In chapter 9 we discuss several classes of cyclic isoprenoids and their respective derivatives, which have proven to be important biomarkers that can be used to estimate algal and vascular plant contributions as well as diagenetic proxies. Sterols are a group of cyclic alcohols (typically between C26 and C30) that vary structurally in the number, stereochemistry, and position of double bonds as well as methyl- and ethyl-group substitutions on the side chain. Sterols are biosynthesized from isoprene units using the mevalonate pathway and are classified as triterpenes (i.e., consisting of six isoprene units). Marine organisms such as phytoplankton and zooplankton tend to be dominated by C27 and C28 sterols. Vascular plants have been shown to have a relatively high abundance of C29 sterols, such as 24-ethylcholest-5-en-3β-ol (sitosterol) (C29Δ⁵) and 24-ethylcholest-5,22E-dien-3β-ol (stigmasterol, C29 A⁵,²²), as well as the C28 sterol 24-methylcholest-5-en-3β-ol (campesterol) (C28Δ⁵). Finally, we show that although C29 sterols are considered to be the dominant sterols found in vascular plants, these compounds may be present in certain epibenthic cyanobacteria and phytoplanktonic species, indicating that these compounds represent a very diverse and powerful suite of chemical biomarkers in aquatic ecosystems.

Di- and triterpenoids are useful biomarkers for determining the sources and transformations of natural organic matter in aquatic ecosystems. Like sterols, these terpenoids are formed from isoprene units using the mevalonate pathway. The sources and diagenetic formation of molecules like retene, chrysene, picene, and perylene are discussed. Many of these compounds are natural products derived from plants, while others are formed from diagenesis. Tetraterpenoids (e.g., carotenoids) are covered in chapter 13.

Recent applications of δ¹³C and δD to steroid and triterpenoid biomarker compounds will continue to help resolve complex mixtures of organic matter in natural systems. Compound-specific isotope analysis provides an opportunity to better understand the sources of these compounds in the environment and extend their application to ecological and paleoecological studies investigating changes in environmental and climatic conditions.

In chapter 10 we examine hydrocarbons present in the environment and derived from natural and anthropogenic sources. The abundance of hydrocarbons derived from anthropogenic sources (petroleum hydrocarbons) has increased significantly in aquatic systems since the industrial revolution (this addressed in hapter 14). Natural oil seeps and erosion of bitumen deposits can also contribute to hydrocarbon abundance and composition in systems. These petroleum hydrocarbons can be distinguished from biological hydrocarbons by their absence of odd-carbon chain lengths commonly found in biological hydrocarbons and the greater structural diversity found in petroleum hydrocarbons.

This chapter focuses on naturally produced hydrocarbons. We provide examples of how aliphatic and isoprenoid hydrocarbons have been successfully used to distinguish between algal, bacterial, and terrigenous vascular plant sources of carbon in aquatic systems. We discuss how pristine and phytane are formed from phytol under oxic vs. anoxic conditions, respectively. We also introduce the reader to highly branched isoprenoids and their use as algal biomarkers.

Chapter 11 focuses on several classes of polar lipids, including alkenones, which are di-, tri-, and tetra-unsaturated long-chain ketones. These compounds are produced by a restricted number of species of prymnesiophyte algae (coccolithophorid alga Emiliania huxleyi), living over a wide temperature range (2–29 °C). Prymnesiophytes are able to live under different temperature regimes because they are able to regulate the degree of unsaturation of these compounds; as ambient water temperature decreases, unsaturation increases. Long-chain ketones are more stable than most unsaturated lipids and can survive diagenesis. Because of these properties, alkenones have been used widely as paleothermometers.

Paleoclimate studies of continental environments have been hampered by the lack of a useful temperature proxy. Glycerol dialkyl glycerol tetraethers (GDGTs) occur ubiquitously, including sites where alkenones are not produced due to the absence/low abundance of alkenone-producing algae. The TEX86 index, based on the number of cyclopentane rings in the GDGTs, provides a useful paleotemperature index for lakes and other sites where alkenones are not produced.

The analysis of intact polar molecules is becoming more widespread with the advent of LC-MS techniques. In some cases, these compounds are proving to be more useful for identifying microbial communities associated with soils and sediments (e.g., intact polar lipids), than the more traditional approaches that involve hydrolysis of polar compounds and analysis of their subcomponents (e.g., phospholipid linked fatty acids [PLFA]). This compound class shows great promise for identifying microbes associated with soils and marsh sediments and the delivery of soil-derived organic matter to coastal regions (e.g., BIT index).

In chapter 12 we examine the primary photosynthetic pigments used in absorbing photosynthetically active radiation (PAR), which incluide chlorophylls, carotenoids, and phycobilins—with chlorophyll representing the dominant photosynthetic pigment. Although a greater amount of chlorophyll is found on land, 75% of the annual global turnover (ca. 10⁹ Mg) occurs in oceans, lakes, and rivers/estuaries. All of the light-harvesting pigments are bound to proteins making up distinct carotenoid and chlorophyll–protein complexes. In this chapter, we examine the chemistry and application of these very important chemical biomarkers and discuss their limitations in aquatic systems. The matrix factorization program CHEMical TAXonomy (CHEMTAX) was introduced to calculate the relative abundance of major algal groups based on concentrations of diagnostic pigments and is also discussed.

The combined method of selective photopigment HPLC separation coupled with in-line flow scintillation counting using the chlorophyll a radiolabeling (¹⁴C) technique can also provide information on phytoplankton growth rates under different environmental conditions. Fossil pigments have also been shown to be useful paleotracers of algal and bacterial communities.

As discussed in chapter 3, the application of CSIA to plant pigments may prove useful in distinguishing between different types of organic carbon sources in aquatic systems. We also address the application of satellite imagery in determining chlorophyll concentrations in natural waters, as well as new ideas for developing in situ HPLC systems for real-time data gathering in coastal observatories.

In chapter 13 we examine lignin, which has proven to be a useful chemical biomarker for tracing vascular-plant inputs to aquatic systems. Cellulose, hemicellulose, and lignin generally make up >75% of the biomass of woody plant materials. Lignins are a group of macromolecular heteropolymers (600–1000 kDa) found in the cell wall of vascular plants that are made up of phenylpropanoid units. The shikimic acid pathway, which is common in plants, bacteria, and fungi, is the pathway for synthesis of aromatic amino acids (e.g., tryptophan, phenylalanine, and tyrosine), thereby providing the parent compounds for the synthesis of the phenylpropanoid units in lignins. Specifically, the primary building blocks for lignins are the following monolignols: p-coumaryl alcohol; coniferyl alcohol; and sinapyl alcohol. Oxidation of lignin using the CuO oxidation method yields eleven dominant phenolic monomers, which can be separated into four families: p-hydroxyl, vanillyl (V), syringyl (S), and cinnamyl (C) phenols.

Cutins and suberins, which are lipid polymers in vascular plant tissues and serve as a protective layer (cuticle) and as cell wall components of cork cells, respectively, are also examined in this chapter. This chapter describes how cutins have been shown to be an effective biomarker for vascular plants in aquatic systems. When cutin is oxidized, using the CuO method commonly used for lignin analyses, a series of fatty acids are produced that can be divided into three groups: C16 hydroxy acids, C18 hydroxy acids, and Cn hydroxyl acids. Since cutins are not found in wood, they are similar to the p-hydroxyl lignin-derived cinnamyl phenols (e.g., trans- p-coumaric and ferulic acids) and serve as biomarkers of nonwoody vascular plant tissues.

We further explain how different analytical methods for lignin detection have been developed for use in aquatic systems. There are new microwave extraction techniques as well as applications of CSIA of lignin monomers using stable isotopes and radiocarbon. Finally, new techniques in multidimensional NMR have allowed for better elucidation of aromatic structures potentially derived from lignins in natural waters.

Chapter 14 examines the application of anthropogenic compounds as biomarkers. Since World War II, human activities have introduced a wide array of compounds into the environment, including insecticides such as dichloro-diphenyl-trichloroethane (DDT) and pesticides, halocarbons (chlorofluorocarbons), sewage products (coprostanol), and polycyclic aromatic hydrocarbons (PAHs). This chapter introduces structural features of these compounds, their distribution and transformation in the environment, and their potential use(s) as tracers. A focus of this chapter is to present examples of how relationships between anthropogenic markers and biomarkers can be used to provide information about the sources, delivery, and fate of natural organic matter in aquatic ecosystems.

This chapter introduces various emerging contaminants (personal care pharmaceutical products (PCPPs), caffeine, and flame retardants) and their potential use as tracers for anthropogenic organic matter in aquatic ecosystems. We describe how δ¹³C, stable isotopes of Cl and Br, and radiocarbon can be used to apportion sources of organic contaminants (e.g., PAHs and PCBs).

Acknowledgments

Over the past two years it has taken to write this book, many people have helped along the way, and we are eternally grateful for their input. We thank our friends and colleagues for sharing new and unpublished work with us as well as their support during the writing and editing phases. Their positive comments and encouragement were a great help. In particular, E. Canuel would like to acknowledge the importance of the Gordon Research Conference on Organic Geochemistry for providing a stimulating, intellectual climate in which new ideas can be exchanged freely and warm friendships developed and nurtured. We would also like to thank our colleagues, postdocs, students, and family members who reviewed individual chapters of this book, including Jo Ann Bianchi, Emmett Duffy, Amber Hardison, Yuehan Lu, Christie Pondell, and Stephanie Salisbury. We also appreciate the dedicated and heroic efforts of Erin Ferer, Emily Jayne, and Stephanie Salisbury for their contributions to the glossary.

Throughout our careers, our curiosity and research interests in organic geochemistry have evolved through interactions with our students and colleagues. E. Canuel would like to acknowledge collaborations with past and present students, including Krisa Arzayus, Amber Hardison, Emily Jayne, Elizabeth Lerberg, Leigh Mccallister, John Pohlman, Christie Pondell, Stephanie Salisbury, Sarah Schillawski, Amanda Spivak, Craig Tobias, and Andy Zimmerman. T. Bianchi would like to thank the following past and present students who contributed to development of this book: Nianhong Chen, Shuiwang Duan, Richard Smith, and Kathyrn Schreiner. These interactions have encouraged us to venture into new areas of research and provided a constant stream of ideas and intellectual stimulation. We are indebted to our parents and mentors, who showed us the way and kept us on the path to becoming successful scientists. Sadly, T. Bianchi’s father, Thomas Bianchi, passed away in 2008; while he never understood the life of his son as an academic, his blue-collar work ethic and kindness to others are traits that will be remembered for many years to come. E. Canuel thanks Stuart Wakeham for his friendship and intellectual input, and the many opportunities he has provided to her over 20+ years. T. Bianchi thanks many of his close friends for their intellectual discussions over the years, such as Mead Allison, Mark Baskaran, James Bauer, Robert Cook, Michael Dagg, Rodger Dawson, Ragnar Elmgren, Tim Filley, Patrick Hatcher, Franco Marcantonio, Mark Marvin-DiPasquale, Sid Mitra, Brent McKee, Hans Pearl, Rodney Powell, Eric Roden, Peter Santschi, Pichan Sawangwong, and the late Robert Wetzel. T. Bianchi acknowledges his friendship and collaborations with John Morse who passed away in 2009 after a long and distinguished career—he will be missed by his family and his many geochemical colleagues around the world. Finally, we also thank our muse, John Hedges. John’s insights, friendship, and generosity continue to inspire us and serve as a role model for our personal and professional interactions with students and colleagues.

We thank Ingrid Gnerlich and the team at Princeton University Press for their patience and dedication to this project. We are also grateful to the two anonymous reviewers who provided many useful comments that contributed to the improvement of the book. We also are grateful for continued support from the National Science Foundation.

1. Metabolic Synthesis

1.1 Background

In this chapter, we begin by providing a brief background on the classification of organisms. We then provide a general background on the synthesis of chemical biomarkers and their association with key metabolic pathways in organisms, as they relate to differences in cellular structure and function across the three systematic domains of life. We also discuss photosynthesis, the dominant pathway by which biomass is synthesized, and provide information about chemoautotrophic and microbial heterotrophic processes. This holistic view of biosynthetic pathways of chemical biomarkers provides a roadmap for other chapters in this book, where more specific details on chemical pathways are presented for each of the respective classes of biomarkers. While other excellent books in the area of organic geochemistry have effectively introduced the concepts of chemical biomarkers in the context of physical and chemical gradients found in natural ecosystems (e.g., anaerobic, aerobic) (Killops and Killops, 2005; Peters et al., 2005), we begin by first examining biosynthetic pathways at the cellular level of differentiation. We believe that an understanding of the general biosynthetic pathways of these chemical biomarkers is critical when examining the complexity of rate-controlling processes that determine their production and fate in aquatic systems. We also provide the major features of different cell membrane structures, since these membranes play an important role in the transport of simple molecules in and out of the cell and influence the preservation of chemical biomarkers in sediments.

1.2 Classification of Organisms

The taxonomic classification of all living organisms is shown in fig. 1.1. The former five-Kingdom classification system consisted of the following phyla: Animalia, Plantae, Fungi, Protista, and Bacteria. It was replaced by a three-domain system—primarily derived from the phylogenetic analysis of base sequences of nucleic acids from rRNA (Woese et al., 1990) (fig. 1.1). These domains can be further divided into heterotrophs (e.g., animals and fungi) and autotrophs (e.g., vascular plants and algae), which are either prokaryotes (unicellular organisms that do not possess a nuclear membranes [e.g., just a nucleoid, or DNA in the form of chromosomes] or eukaryotes (unicellular and multicellular organisms with nuclear membranes and DNA in the form of chromosomes) (fig. 1.2). The bacteria (eubacteria) and archaea (archaebacteria), both prokaryotes, represent important microbial groups and are involved in many of the biogeochemical cycling processes of aquatic ecosystems.

Figure 1.1. The three-domain system derived from the phylogenetic analysis of base sequences of nucleic acids from rRNA. Phylogenetically the Archaea fall into two distinct groups as shown by the gray line: the methanogens (and their relatives) and the thermophiles. (Adapted from Woese et al.,1990.)

Photosynthesis is the dominant process by which organic matter is synthesized. During photosynthesis, organisms synthesize simple carbohydrates (carbon fixation) using light energy, water and an electron donor. The earliest photosynthetic organisms are thought to have been anoxygenic, likely using hydrogen, sulfur, or organic compounds as electron donors. Fossils of these organisms date to 3.4 billion years (3.4 × 10⁹ years or 3.4 Gyr) before present (BP). Oxygenic photosynthesis evolved later; the first oxygenic photosynthetic organisms were likely the cyanobacteria, which became important around 2.1 Gyr BP (Brocks et al., 1999). As oxygen accumulated in the atmosphere through the photosynthetic activity of cyanobacteria (Schopf and Packer, 1987), life on Earth needed to quickly adapt. In fact, it is believed that a transfer of bacterial genes was responsible for the development of the first eukaryotic cell (Gypania spiralis), estimated, based on the fossil record, to be 2.1 Gyr old (Han and Runnegar, 1992). When a cell consumed aerobic (oxygen-using) bacteria, it was able to survive in the newly oxygenated world. Today, the aerobic bacteria have evolved to include mitochondria, which help the cell convert food into energy. Hence, the appearance of mitochondria and chloroplasts are believed to have evolved in eukaryotes as endosymbionts (Thorington and Margulis, 1981). The current theory of endosymbionts is based on the concept that an earlier combination of prokaryotes, once thought to be living together as symbionts, resulted in the origin of eukaryotes (Schenk et al., 1997; Peters et al., 2005). This theory suggests that some of the organelles in eukaryotes (e.g., mitochondria, kinetosomes, hydrogenosomes, and plastids) may have begun as symbionts. For example, it is thought that mitochondria and chloroplasts may have evolved from aerobic nonphotosynthetic bacteria and photosynthetic cyanobacteria, respectively. Endosymbiotic theory also explains the presence of eubacterial genes within eukaryotic organisms (Palenik, 2002). The important energy-transforming processes that directly or indirectly occur in association with these organelles, such as photosynthesis, glycolysis, the Calvin cycle, and the citric acid cycle, are discussed in more detail later in this chapter.

Figure 1.2. The basic cellular design of Eukaryotes, unicellular and multicellular organisms with nuclear membranes and DNA in the form of chromosomes, and Prokaryotes, unicellular organisms that do not possess a nuclear membrane (e.g., just a nucleoid, or DNA in the form of chromosomes).

While the origins of life on early Earth remain controversial, experimental evidence for the possible evolution of early life began back with the work of Miller and Urey (Miller, 1953; Miller and Urey, 1959). The basic premise of this work, which has remained central in many current studies, is that simple organic compounds, including amino acids, formed after a spark was applied to a flask containing constituents thought to be present in the Earth’s early atmosphere (e.g., methane, ammonia, carbon dioxide, and water). It is thought that these simple organic compounds provided the seeds for the synthesis of more complex prebiotic organic compounds (fig. 1.3). In addition to the in situ formation of these simple molecules on Earth (based on the aforementioned lab experiments in the 1950s), extraterrestrial sources, such as comets, meteorites, and interstellar particles, have also been posited as possible sources for seeding early Earth with these simple molecules. There is now strong evidence for the presence of these prebiotic compounds in these extraterrestrial sources (Engel and Macko, 1986; Galimov, 2006; and references therein). One particular theory focuses on the notion that the synthesis of adenosine triphosphate (ATP) was most critical in the early stages of prebiotic evolution (Galimov, 2001, 2004). The hydrolysis of ATP to adenosine diphosphate (ADP) is critical in the ordering and assembly of more complex molecules. For example, the formation of peptides from amino acids, and nucleic acids from nucleotides, is inherently linked to the ATP molecule (fig. 1.3). Another important step in prebiotic chemical evolution of life was the development of a molecule that allowed for genetic coding in primitive organisms, and transfer ribonucleic acid (tRNA). Many scientists now suspect that all life diverged from a common ancestor relatively soon after life began (figs. 1.1 and 1.3). Based on DNA sequencing, it is believed that millions of years after the evolution of archaebacteria and eubacteria, the ancestors of eukaryotes split off from the archaebacteria. The Earth is at least 4.6 Gyr old (Sogin, 2000), but it was the microbial organisms that dominated for the first 70 to 90 percent of Earth’s history (Woese, 1981; Woese et al., 1990). While there has been considerable debate about the composition of the atmosphere of early Earth, it is now generally accepted that it was a reducing environment (Sagan and Chyba, 1997; Galimov, 2005). Kump et al. (2010) state the atmosphere is now thought to have been composed primarily of N2 and CO2. Banded iron formations (BIF) first appear in sediments deposited 3 Gyr BP, during the early history of the Earth. These formations include layers of iron oxides, either magnetite or hematite, alternating with iron-poor layers of shale and chert. It is thought that these iron oxide formations were formed in seawater from the reaction between oxygen produced during photosynthesis by cyanobacteria with dissolved reduced iron. The subsequent disappearance of banded iron formations in the geologic record approximately 1.8 Gyr BP is believed to have resulted following a phase of rising oxygen levels in the atmosphere that began about 2.4 to 2.3 Gyr BP (Farquhar et al., 2000; Bekker et al., 2004). Other recent work has suggested that even during the mid-Proterozoic (ca. 1.8 to 0.8 Gyr ago) the oceans were either anaerobic (no oxygen) or dysaerobic ) into biomass using photosynthesis; many of these early photoautotrophs were likely cyanobacteria (Brocks et al., 1999).

Figure 1.3. A scenario for the evolution of life focused on the notion that the synthesis of adenosine triphosphate (ATP) was most critical in the early stages of prebiotic evolution. (Adapted from Galimov, 2001, 2004.)

Figure 1.4. Schematic depicting the redox conditions during the mid-Proterozoic (ca. 1.8 to 0.8 Gyr ago) in the oceans, which are believed to have been considerably more anaerobic (no oxygen) and dysaerobic (low oxygen) than the oceans of today. (Adapted from Arnold et al., 2004.)

Archaebacteria were originally thought to live only in extreme environments (e.g., high temperatures, pH extremes, and high radiation levels), but have recently been found in a variety of habitats (Delong and Pace, 2001; Giovannoni and Stingl, 2005; Delong, 2006; Ingalls et al., 2006). Examples of archaebacteria include anaerobic methanogens and halophilic bacteria, and consist of organisms living both in cold (e.g., Antarctica) and hot (e.g., springs of Yellowstone Park [USA]) environments. The fact that these organisms can be found in extreme environments is consistent with the ancient heritage of this domain. Early Earth was likely a very hot environment, with many active volcanoes and an atmosphere composed mostly of nitrogen, methane, ammonia, carbon dioxide, and water—with little to no oxygen present. It is believed that the archaebacteria, and in some cases bacteria, evolved under these conditions, allowing them to live in harsh conditions today. For example, thermophiles live at high temperatures, of which the present record is 121°C (Kashefi and Lovley, 2003). In contrast, no known eukaryote can survive over 60°C. Another example is the psychrophiles, which live in extremely cold temperatures—there is one species in the Antarctic that grows best at 4°C. As a group, these hard-living archaebacteria are called extremophiles. The archaebacteria include other kinds of extremophiles, such as acidophiles, which live at pH levels as low as 1. Alkaliphiles thrive at high pH levels, while halophiles live in very salty environments. It should be noted that there are also alkaliphilic, acidophilic, and halophilic eukaryotes, and that not all archaebacteria are extremophiles. It has also been suggested that the thermophilic archaebacteria that live around deep-sea volcanic vents may represent the earliest life on Earth (Reysenbach et al., 2000). These thermophilic archaea harvest their energy very efficiently from chemicals (e.g., H2,CO2, and O2) found at the vents using a process called chemosynthesis. These organisms are not greatly impacted by surface environmental changes. As such, thermophilic organisms living around deep-sea volcanic vents may have been the only organisms able to survive the large, frequent meteor impacts of Earth’s early years.

Figure 1.5. Differences in the composition of the membrane lipids in bacteria and eukarya. Membrane lipids in bacteria are composed of ester linkages, while membrane lipids in Archaea, have ether linkages. Glycerol (H2OC–CHO–CH2O) is identified in the gray boxes.

Archaea are believed to be the most primitive forms of life on Earth, as reflected by their name, which is derived from archae meaning ancient (Woese, 1981; Kates et al., 1993; Brock et al., 1994; Sogin, 2000). Archaea are divided into two main phyla: the Euryarchaeota and Crenarchaeota. Like bacteria, archaebacteria have rod, spiral, and marble-like shapes. Phylogenetic trees also show relationships between archaea and eukarya and some have argued that the archaebacteria and eukaryotes arose from specialized eubacteria. One of the distinguishing features of the archaea is the composition of the lipids comprising their cell membranes; membrane lipids are associated with an ester linkage in bacteria, while those in archaebacteria are associated with an ether linkage (fig. 1.5). This is described further below and in subsequent chapters (e.g., chapter 8). The most striking chemical difference between archaea and other living cells is their cell membrane. There are four fundamental differences between the archaeal membrane and those of other cells: (1) chirality of glycerol, (2) ether linkage, (3) isoprenoid chains, and (4) branching of side chains. These are discussed in more detail below. In other words, archaebacteria build the same structures as other organisms, but they build them from different chemical components. For example, the cell walls of all bacteria contain peptidoglycan, while the cell walls of archaea are composed of surface-layer-proteins. Also, archaebacteria do not produce cell walls of cellulose (as do plants) or chitin (as do fungi); thus, the cell wall of archaebacteria is chemically distinct.

Glycerol is an important building block of lipids. This compound has three carbon atoms, each with a hydroxyl (–OH) group attached (fig. 1.5). When considering the chirality of glycerol, we need to begin with the basic unit from which cell membranes are built in bacteria and eukaryotes—phospholipids. Phospholipids are composed of a molecule of glycerol with fatty acids esterified to the C-1 and C-2 positions of the glycerol and a phosphate group attached to C-3 position by an ester bond (fig. 1.5). In the cell membrane, the glycerol and phosphate end of the molecules are at the surface of the membrane, with the long chains in the middle (fig. 1.6). This layering provides an effective chemical barrier around the cell, which helps to maintain chemical equilibrium. In archaea, the stereochemistry of the glycerol is the reverse of that found in bacteria and eukaryotes (i.e., the glycerol is a stereoisomer of the glycerol present in phospholipids), suggesting that archaea synthesize glycerol by a different biosynthetic pathway than bacteria and eukaryotes. Stereoisomers are optical isomers of one other, meaning there are two possible forms of the molecule and they are mirror images of each other. While eubacteria and eukaryotes have dextrorotary (D) D-glycerol in their membranes, archaeans have levorotary (L) L-glycerol. Chemical components of the cell have to be built by enzymes, and the handedness (chirality) of the molecule is determined by the shape of the enzymes.

Figure 1.6. The structure of the eubacteria and eukaryotic cell membrane, showing the glycerol and phosphate end of the molecules at the surface of the membrane, with the long chains in the middle.

In most organisms, side chains added to the glycerol are bonded using an ester linkage. Esters are acids where one of the hydroxyl (–OH) groups has been replaced by an O-alkyl group (fig. 1.5). The general formula for esters is: R¹–C(=O)–O–R². In the case of phospholipids, R¹ is the carboxyl carbon (C-1) on the fatty acid side chain and R² is the C-1 or C-2 position in glycerol. By contrast, membrane lipids in archaea are bound using an ether linkage, in which an oxygen atom is connected to two alkyl groups (general formula is R–O–R′). This causes the chemical properties of membrane lipids in archaea to differ from the membrane lipids of other organisms.

The side chains in the phospholipids of eubacteria and eukaryotes are fatty acids of usually 16 to 18 carbon atoms (fig. 1.5) (Killops and Killops, 2005; Peters et al., 2005) (see chapter 8 for more details about fatty acids). Archaebacteria do not use fatty acids to build their membrane phospholipids. Instead, they have side chains of 20 carbon atoms built from isoprene (2-methylbuta-1,3-diene or C5 H8)—a C5 compound, which forms the building block for a class of compounds called terpenes. By definition, terpenes are a class of compounds constructed by connecting isoprene molecules together (C5H8)n, where n is the number of linked isoprene units (see chapters 9 and 12 for more details).

The membrane lipids of archaea also differ from those of eubacteria or eukaryotes, because they have side chains off the main isoprene structure (fig. 1.5) (Kates et al., 1993). This results in some interesting properties in archaeal membranes. For example, isoprene side chains can be joined together, allowing side chains of the membrane lipids to join together, or become joined with side chains of other compounds on the other side of the membrane. No other group of organisms can form such transmembrane lipids. Another interesting property of the side chains is their ability to form carbon rings. This happens when one of the side branches curls around and bonds with another atom down the chain to make a five-carbon ring. These carbon rings are thought to provide structural stability to the membrane, which may allow archaebacteria to be more tolerant of high temperatures. They may work in the same way that cholesterol (another terpene) does in eukaryotic cells to stabilize membranes (fig. 1.6).

Cellular membranes of eukaryotes have diverse functions in the different regions and organelles of a cell (Brock et al., 1994). Membranes are vital because they separate the cell from the outside world. They also separate compartments inside the cell to protect important processes and events. In water, phospholipids are amphipathic: the polar head groups are attracted to water (hydrophilic) and the tails are hydrophobic or oriented away from water, creating the classic lipid bilayer (fig. 1.6). The tail groups orient against one another, forming a membrane with hydrophilic heads on both ends. This allows liposomes or small lipid vesicles to form, which can then transport materials across the cell membrane. Various micelles, such as spherical micelles, also allow for a stable configuration for amphipathic lipids that have a conical shape, such as fatty acids. As mentioned earlier, cholesterol is an important constituent of cell membranes. It has a rigid ring system, has a short, branched hydrocarbon tail, and is largely hydrophobic. However, it has one polar group, a hydroxyl group, making it amphipathic. Cholesterol inserts into lipid bilayer membranes with its hydroxyl group oriented toward the aqueous phase and its hydrophobic ring system adjacent to fatty acid tails of phospholipids. The hydroxyl group of cholesterol forms hydrogen bonds with polar phospholipid head groups. It is thought that cholesterol contributes to membrane fluidity by hindering the packing together of phospholipids.

1.3 Photosynthesis and Respiration

The sum of all biochemical processes in organisms is called metabolism, which can further be divided into catabolic and anabolic pathways. These processes are responsible for the formation of many of the chemical biomarker compounds discussed in this book, which occur through an intermediary metabolism via glycolysis and the citric acid cycle (Voet and Voet, 2004). Kossel (1891) first noted the distinction between primary and secondary metabolism. Primary metabolism, also called basic metabolism, includes all the pathways and products that are essential for the cell itself. Secondary metabolism produces molecules that are not necessarily important for the survival of the cell itself, but are important for the whole organism. To understand biochemical and molecular biological processes such as metabolism, differentiation, growth, and inheritance, knowledge of the molecules involved is imperative. Cell-specific molecules are generated via a number of intermediate products from simple precursors, while others are broken down or rearranged. During evolution, pathways have been developed and maintained that produce functional molecules needed by the cell or the organism.

The chemical reactions of oxygenic photosynthesis (primary production) and oxidation (respiration or decomposition) of organic matter are described in equation 1.1:

Figure 1.7. The pathways of photosynthesis and respiration, which, in part, produce oxygen and carbon dioxide, respectively, and occur in the chloroplasts and mitochondria.

The end products of electron transport during photosynthesis are NADH or NADPH and ATP, which can be used for carbon or nitrogen fixation and for intermediary metabolism. An important aspect of photosynthesis is the integration of carbon dioxide into organic compounds, called carbon fixation. (Ehleringer and Monson, 1993; Ehleringer et al., 2005). The pathways of photosynthesis and respiration, which, in part, produce oxygen and carbon dioxide, respectively, occur in the chloroplasts and mitochondria (fig. 1.7). If we look more closely at photosynthesis we find that photosystems I and II are located in the chloroplasts (figs. 1.7 and 1.8) (see chapter 12 for more details). The chlorophylls, also found in the chloroplast, serve as the principal antenna pigments for capturing sunlight in the photosystems (described later in this chapter). In higher plants, assimilation tissues are those tissues that are made from chloroplast-containing cells and are able to perform photosynthesis (fig. 1.9). The leaves of higher plants are by far the most important production centers—if you disregard unicellular aquatic algae. Leaves usually consist of the following three tissues: the mesophyll, epidermis, and vascular tissues. The mesophyll is a parenchyma tissue that is an important location for the reduction of carbon dioxide, which enters through the stomata in the epidermis in the carbon-fixation reactions of the Calvin cycle (Monson, 1989, and references therein). The Calvin cycle is the assimilatory path that is involved in all autotrophic carbon fixation, both photosynthetic and chemosynthetic.

Finally, it important to note that while the previous section was focused on oxygenic photosynthesis, bacteria are also capable of performing anoxygenic photosynthesis. In general, the four types of bacteria that perform this process are the purple, green sulfur, green-sliding, and gram-positive bacteria (Brocks et al., 1999). Most of the bacteria that perform anoxygenic photosynthesis live in environments where oxygen is in low supply as is discussed in later chapters. In anoxygenic photosynthesis, chemical species such as hydrogen sulfide and nitrite substitute for water as the electron donor.

Figure 1.8. A plant cell and the associated organelles illustrating the chlorophylls (found in the chloroplast), which serve as the principal antenna pigments for capturing sunlight in the photosystems.

1.3.1 C3, C4, and CAM Pathways

The