The prokaryotes prokaryotic physiology and biochemistry

48 AbstractThis chapter circumscribes the acetogens, a physiologicallydefined group of the domain Bacteria that are anaerobes, usingthe acetyl-CoA pathway as a mechanism for the reductiv

The Prokaryotes

Eugene Rosenberg (Editor-in-Chief)

Edward F DeLong, Stephen Lory, Erko Stackebrandt and Fabiano Thompson (Eds.)

Eugene Rosenberg

Department of Molecular Microbiology and Biotechnology

Tel Aviv University

Tel Aviv, Israel

Editors

Edward F DeLong

Department of Biological Engineering

Massachusetts Institute of Technology

Cambridge, MA, USA

Stephen Lory

Department of Microbiology and Immunology

Harvard Medical School

Boston, MA, USA

Erko Stackebrandt

Leibniz Institute DSMZ-German Collection of Microorganisms

and Cell Cultures

Braunschweig, Germany

Fabiano ThompsonLaboratory of Microbiology, Institute of Biology, Center forHealth Sciences

Federal University of Rio de Janeiro (UFRJ)Ilha do Funda˜o, Rio de Janeiro, Brazil

ISBN 978-3-642-30142-1 (print and electronic bundle)

DOI 10.1007/978-3-642-30141-4

Springer Heidelberg New York Dordrecht London

Library of Congress Control Number: 2012955034

3rd edition: © Springer Science+Business Media, LLC 2006

4th edition: © Springer-Verlag Berlin Heidelberg 2013

This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifically for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer Permissions for use may be obtained through RightsLink at the Copyright Clearance Center Violations are liable to prosecution under the respective Copyright Law.

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Printed on acid-free paper

Springer is part of Springer Science þBusiness Media ( www.springer.com )

The purpose of this brief foreword is unchanged from the first edition; it is simply to make you, the reader, hungry for the scientificfeast that follows These 11 volumes (planned) on the prokaryotes offer an expanded scientific menu that displays the biochemicaldepth and remarkable physiological and morphological diversity of prokaryote life The size of the volumes might initially discouragethe unprepared mind from being attracted to the study of prokaryote life, for this landmark assemblage thoroughly documents thewealth of present knowledge But in confronting the reader with the state of the art, the Handbook also defines where more workneeds to be done on well-studied bacteria as well as on unusual or poorly studied organisms

This edition of The Prokaryotes recognizes the almost unbelievable impact that the work of Carl Woese has had in defining

a phylogenetic basis for the microbial world The concept that the ribosome is a highly conserved structure in all cells and that itsnucleic acid components may serve as a convenient reference point for relating all living things is now generally accepted At last, thephylogeny of prokaryotes has a scientific basis, and this is the first serious attempt to present a comprehensive treatise on prokaryotesalong recently defined phylogenetic lines Although evidence is incomplete for many microbial groups, these volumes make

a statement that clearly illuminates the path to follow

There are basically two ways of doing research with microbes A classical approach is first to define the phenomenon to be studiedand then to select the organism accordingly Another way is to choose a specific organism and go where it leads The pursuit of anunusual microbe brings out the latent hunter in all of us The intellectual challenges of the chase frequently test our ingenuity to thelimit Sometimes the quarry repeatedly escapes, but the final capture is indeed a wonderful experience For many of us, these simplerewards are sufficiently gratifying so that we have chosen to spend our scientific lives studying these unusual creatures In theseendeavors, many of the strategies and tools as well as much of the philosophy may be traced to the Delft School, passed on to us by ourteachers, Martinus Beijerinck, A J Kluyver, and C B van Niel, and in turn passed on by us to our students

In this school, the principles of the selective, enrichment culture technique have been developed and diversified; they have been

a major force in designing and applying new principles for the capture and isolation of microbes from nature For me, the ‘‘organismapproach’’ has provided rewarding adventures The organism continually challenges and literally drags the investigator into new areaswhere unfamiliar tools may be needed I believe that organism-oriented research is an important alternative to problem-orientedresearch, for new concepts of the future very likely lie in a study of the breadth of microbial life The physiology, biochemistry, andecology of the microbe remain the most powerful attractions Studies based on classical methods as well as modern genetictechniques will result in new insights and concepts

To some readers, this edition of The Prokaryotes may indicate that the field is now mature, that from here on it is a matter of filling

in details I suspect that this is not the case Perhaps we have assumed prematurely that we fully understand microbial life Van Nielpointed out to his students that—after a lifetime of study—it was a very humbling experience to view in the microscope a sample ofmicrobes from nature and recognize only a few Recent evidence suggests that microbes have been evolving for nearly 4 billion years.Most certainly, those microbes now domesticated and kept in captivity in culture collections represent only a minor portion of thespecies that have evolved in this time span Sometimes we must remind ourselves that evolution is actively taking place at the presentmoment That the eukaryote cell evolved as a chimera of certain prokaryote parts is a generally accepted concept today Higher as well

as lower eukaryotes evolved in contact with prokaryotes, and evidence surrounds us of the complex interactions between eukaryotesand prokaryotes as well as among prokaryotes We have so far only scratched the surface of these biochemical interrelationships.Perhaps the legume nodule is a pertinent example of nature caught in the act of evolving the ‘‘nitrosome,’’ a unique nitrogen-fixingorganelle The study of prokaryotes is proceeding at such a fast pace that major advances are occurring yearly The increase of thisedition to four volumes documents the exciting pace of discoveries

To prepare a treatise such as The Prokaryotes requires dedicated editors and authors; the task has been enormous I predict that thescientific community of microbiologists will again show its appreciation through use of these volumes—such that the pages willbecome ‘‘dog-eared’’ and worn as students seek basic information for the hunt These volumes belong in the laboratory, not in thelibrary I believe that a most effective way to introduce students to microbiology is for them to isolate microbes from nature, that is,from their habitats in soil, water, clinical specimens, or plants The Prokaryotes enormously simplifies this process and shouldencourage the construction of courses that contain a wide spectrum of diverse topics For the student as well as the advancedinvestigator, these volumes should generate excitement

Happy hunting!

Ralph S WolfeDepartment of MicrobiologyUniversity of Illinois at Urbana-Champaign

During most of the twentieth century, microbiologists studied pure cultures under defined laboratory conditions in order to uncoverthe causative agents of disease and subsequently as ideal model systems to discover the fundamental principles of genetics andbiochemistry Microbiology as a discipline onto itself, e.g., microbial ecology, diversity, and evolution-based taxonomy, has onlyrecently been the subject of general interest, partly because of the realization that microorganisms play a key role in the environment.The development and application of powerful culture-independent molecular techniques and bioinformatics tools has made thisdevelopment possible The fourth edition of the Handbook of the Prokaryotes has been updated and expanded in order to reflect thisnew era of microbiology

The first five volumes of the fourth edition contain 34 updated and 43 entirely new chapters Most of the new chapters are in thetwo new sections: Prokaryotic Communities and Bacteria in Human Health and Disease A collection of microorganisms occupyingthe same physical habitat is called a ‘‘community,’’ and several examples of bacterial communities are presented in the ProkaryoticCommunities section, organized by Edward F DeLong Over the last decade, important advances in molecular biology andbioinformatics have led to the development of innovative culture-independent approaches for describing microbial communities.These new strategies, based on the analysis of DNA directly extracted from environmental samples, circumvent the steps of isolationand culturing of microorganisms, which are known for their selectivity leading to a nonrepresentative view of prokaryotic diversity.Describing bacterial communities is the first step in understanding the complex, interacting microbial systems in the natural world.The section on Bacteria in Human Health and Disease, organized by Stephen Lory, contains chapters on most of the importantbacterial diseases, each written by an expert in the field In addition, there are separate general chapters on identification of pathogens

by classical and non-culturing molecular techniques and virulence mechanisms, such as adhesion and bacterial toxins In recognition

of the recent important research on beneficial bacteria in human health, the section also includes chapters on gut microbiota,prebiotics, and probiotics Together with the updated and expanded chapter on Bacterial Pharmaceutical Products, this section is

a valuable resource to graduate students, teachers, and researchers interested in medical microbiology

Volumes 6–11, organized by Erko Stackebrandt and Fabiano Thompson, contain chapters on each of the ca 300 knownprokaryotic families Each chapter presents both the historical and current taxonomy of higher taxa, mostly above the genus level;molecular analyses (e.g., DDH, MLSA, riboprinting, and MALDI-TOF); genomic and phenetic properties of the taxa covered;genome analyses including nonchromosomal genetic elements; phenotypic analyses; methods for the enrichment, isolation, andmaintenance of members of the family; ecological studies; clinical relevance; and applications

As in the third edition, the volumes in the fourth edition are available both as hard copies and e-books, and as eReferences Theadvantages of the online version include no restriction of color illustrations, the possibility of updating chapters continuously and,most importantly, libraries can place their subscribed copies on their servers, making it available to their community in offices andlaboratories The editors thank all the chapter authors and the editorial staff of Springer, especially Hanna Hensler-Fritton, IsabelUllmann, Daniel Quin˜ones, Alejandra Kudo, and Audrey Wong, for making this contribution possible

Eugene RosenbergEditor-in-Chief

About the Editors

Eugene Rosenberg (Editor-in-Chief)Department of Molecular Microbiology and BiotechnologyTel Aviv University

Tel AvivIsrael

Eugene Rosenberg holds a Ph.D in biochemistry from Columbia University (1961) where he described the chemical structures of thecapsules of Hemophilus influenzae, types B, E, and F His postdoctoral research was performed in organic chemistry under theguidance of Lord Todd in Cambridge University He was an assistant and associate professor of microbiology at the University ofCalifornia at Los Angeles from 1962 to 1970, where he worked on the biochemistry of Myxococcus xanthus Since 1970, he has been inthe Department of Molecular Microbiology and Biotechnology, Tel Aviv University, as an associate professor (1970–1974), fullprofessor (1975–2005), and professor emeritus (2006–present) He has held the Gol Chair in Applied and Environmental Micro-biology since 1989 He is a member of the American Academy of Microbiology and European Academy of Microbiology He has beenawarded a Guggenheim Fellowship, a Fogarty International Scholar of the NIH, the Pan Lab Prize of the Society of IndustrialMicrobiology, the Proctor & Gamble Prize of the ASM, the Sakov Prize, the Landau Prize, and the Israel Prize for a ‘‘Beautiful Israel.’’His research has focused on myxobacteriology; hydrocarbon microbiology; surface-active polymers from Acinetobacter; biore-mediation; coral microbiology; and the role of symbiotic microorganisms in the adaptation, development, behavior, and evolution ofanimals and plants He is the author of about 250 research papers and reviews, 9 books, and 16 patents

Edward F DeLongDepartment of Biological EngineeringMassachusetts Institute of TechnologyCambridge, MA

USA

Edward DeLong received his bachelor of science in bacteriology at the University of California, Davis, and his Ph.D in marinebiology at Scripps Institute of Oceanography at the University of California, San Diego He was a professor at the University ofCalifornia, Santa Barbara, in the Department of Ecology for 7 years, before moving to the Monterey Bay Aquarium Research Institutewhere he was a senior scientist and chair of the science department, also for 7 years He now serves as a professor at the MassachusettsInstitute of Technology in the Department of Biological Engineering, where he holds the Morton and Claire Goulder FamilyProfessorship in Environmental Systems DeLong’s scientific interests focus primarily on central questions in marine microbialgenomics, biogeochemistry, ecology, and evolution A large part of DeLong’s efforts have been devoted to the study of microbes andmicrobial processes in the ocean, combining laboratory and field-based approaches Development and application of genomic,biochemical, and metabolic approaches to study and exploit microbial communities and processes is his another area of interest.DeLong is a fellow in the American Academy of Arts and Science, the U.S National Academy of Science, and the AmericanAssociation for the Advancement of Science

x About the Editors

Stephen LoryDepartment of Microbiology and ImmunologyHarvard Medical School

Boston, MAUSA

Stephen Lory received his Ph.D degree in Microbiology from the University of California in Los Angeles in 1980 The topic of hisdoctoral thesis was the structure-activity relationships of bacterial exotoxins He carried out his postdoctoral research on the basicmechanism of protein secretion by Gram-negative bacteria in the Bacterial Physiology Unit at Harvard Medical School In 1984, hewas appointed assistant professor in the Department of Microbiology at the University of Washington in Seattle, becoming fullprofessor in 1995 While at the University of Washington, he developed an active research program in host-pathogen interactionsincluding the role of bacterial adhesion to mammalian cells in virulence and regulation of gene expression by bacterial pathogens In

2000, he returned to Harvard Medical School where he is currently a professor of microbiology and immunobiology He is a regularreviewer of research projects on various scientific panels of governmental and private funding agencies and served for four years onthe Scientific Council of Institute Pasteur in Paris His current research interests include evolution of bacterial virulence, studies onpost-translational regulation of gene expression in Pseudomonas, and the development of novel antibiotics targeting multi-drug-resistant opportunistic pathogens

About the Editors xi

Erko StackebrandtLeibniz Institute DSMZ-German Collection of Microorganisms and Cell CulturesBraunschweig

Germany

Erko Stackebrandt holds a Ph.D in microbiology from the Ludwig-Maximilians University Munich (1974) During his postdoctoralresearch, he worked at the German Culture Collection in Munich (1972–1977), 1978 with Carl Woese at the University of Illinois,Urbana Champaign, and from 1979 to 1983 he was a member of Karl Schleifer’s research group at the Technical University, Munich

He habilitated in 1983 and was appointed head of the Departments of Microbiology at the University of Kiel (1984–1990), at theUniversity of Queensland, Brisbane, Australia (1990–1993), and at the Technical University Braunschweig, where he also was thedirector of the DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (1993–2009) He is involved in systematics,and molecular phylogeny and ecology of Archaea and Bacteria for more than 40 years He has been involved in many research projectsfunded by the German Science Foundation, German Ministry for Science and Technology, and the European Union, working on purecultures and microbial communities His projects include work in soil and peat, Mediterranean coastal waters, North Sea and BalticSea, Antarctic Lakes, Australian soil and artesian wells, formation of stromatolites, as well as on giant ants, holothurians, rumen ofcows, and the digestive tract of koalas He has been involved in the description and taxonomic revision of more than 650 bacteria taxa

of various ranks He received a Heisenberg stipend (1982–1983) and his work has been awarded by the Academy of Science atGo¨ttingen, Bergey’s Trust (Bergey’s Award and Bergey’s Medal), the Technical University Munich, the Australian Society forMicrobiology, and the American Society for Microbiology He held teaching positions in Kunming, China; Budapest, Hungary;and Florence, Italy He has published more than 600 papers in refereed journals and has written more than 80 book chapters He is theeditor of two Springer journals and served as an associate editor of several international journals and books as well as on national andinternational scientific and review panels of the German Research Council, European Science Foundation, European Space Agency,and the Organisation for Economic Co-Operation and Development

xii About the Editors

Fabiano ThompsonLaboratory of MicrobiologyInstitute of BiologyCenter for Health SciencesFederal University of Rio de Janeiro (UFRJ)Ilha do Funda˜o

Rio de JaneiroBrazil

Fabiano Thompson became the director of research at the Institute of Biology, Federal University of Rio de Janeiro (UFRJ), in 2012

He was an oceanographer at the Federal University of Rio Grande (Brazil) in 1997 He received his Ph.D in biochemistry from GhentUniversity (Belgium) in 2003, with emphasis on marine microbial taxonomy and biodiversity Thompson was an associate researcher

in the BCCM/LMG Bacteria Collection (Ghent University) in 2004; professor of genetics in 2006 at the Institute of Biology, UFRJ;and professor of marine biology in 2011 at the same university He has been a representative of UFRJ in the National Institute ofMetrology (INMETRO) since 2009 Thompson is the president of the subcommittee on the Systematics of Vibrionaceae–IUMS and

an associate editor of BMC Genomics and Microbial Ecology The Thompson Lab in Rio currently performs research on marinemicrobiology in the Blue Amazon, the realm in the southwestern Atlantic that encompasses a variety of systems, including deep sea,Cabo Frio upwelling area, Amazonia river-plume continuum, mesophotic reefs, Abrolhos coral reef bank, and Oceanic Islands(Fernando de Noronha, Saint Peter and Saint Paul, and Trindade)

About the Editors xiii

Table of Contents

Physiology and Biochemistry 1

1 Acetogenic Prokaryotes 3

Harold L Drake Kirsten Ku¨sel Carola Matthies

2 Virulence Strategies of Plant Pathogenic Bacteria 61

Maeli Melotto Barbara N Kunkel

3 Oxidation of Inorganic Nitrogen Compounds as an Energy Source 83

Eberhard Bock Michael Wagner

4 H2-Metabolizing Prokaryotes 119

Edward Schwartz Johannes Fritsch Ba¨rbel Friedrich

5 Hydrocarbon-Oxidizing Bacteria 201

Eugene Rosenberg

6 Lignocellulose-Decomposing Bacteria and Their Enzyme Systems 215

Edward A Bayer Yuval Shoham Raphael Lamed

7 Aerobic Methylotrophic Prokaryotes 267

Ludmila Chistoserdova Mary E Lidstrom

8 Dissimilatory Fe(III)- and Mn(IV)-Reducing Prokaryotes 287

Derek Lovley

9 Dissimilatory Sulfate- and Sulfur-Reducing Prokaryotes 309

Ralf Rabus Theo A Hansen Friedrich Widdel

Paul V Dunlap Henryk Urbanczyk

14 Halophilic and Haloalkaliphilic Sulfur-Oxidizing Bacteria 529

Dimitry Y Sorokin Horia Banciu Lesley A Robertson J Gijs Kuenen M S Muntyan Gerard Muyzer

15 Colorless Sulfur Bacteria 555

Gerard Muyzer J Gijs Kuenen Lesley A Robertson

16 Bacterial Stress Response 589

Eliora Z Ron

17 Anaerobic Biodegradation of Hydrocarbons Including Methane 605

Johann Heider Karola Schu¨hle

18 Physiology and Biochemistry of the Methane-Producing Archaea 635

Reiner Hedderich William B Whitman

xvi Table of Contents

Department of Biological Chemistry

The Weizmann Institute of Science

Rehovot

Israel

Dennis A Bazylinski

School of Life Sciences

University of Nevada at Las Vegas

zu BerlinBerlinGermany

Theo A HansenMicrobial Physiology (MICFYS)University of GroningenGroningen

The Netherlands

Reiner HedderichMax Planck Institute fu¨r TerrestricheMikrobiologie

MarburgGermany

Johann HeiderFachbereich BiologieLaboratorium fu¨r MikrobiologieMarburg

Germany

Mariangela HungriaEmbrapa SojaLondrinaBrazil

J Gijs KuenenDepartment of BiotechnologyDelft University of TechnologyDelft

The Netherlands

Barbara N KunkelDepartment of BiologyWashington University

St Louis, MOUSA

Kirsten Ku¨selFriedrich Schiller University JenaInstitute of Ecology

Limnology/Aquatic GeomicrobiologyJena

Germany

Raphael Lamed

Department of Molecular Microbiology and Biotechnology

George S Wise Faculty of Life Sciences

Tel Aviv University

Ramat Aviv

Israel

Christopher T Lefe`vre

CEA Cadarache/CNRS/Universite´ Aix-Marseille II, UMR7265

Service de Biologie Ve´ge´tale et de Microbiologie

Environnementale

Laboratoire de Bioe´nerge´tique Cellulaire

Saint Paul lez Durance

Belozersky Institute of Physico-Chemical Biology

Moscow State University

Moscow

Russia

Gerard MuyzerDepartment of BiotechnologyDelft University of TechnologyDelft

The Netherlandsand

Department of Aquatic MicrobiologyInstitute for Biodiversity and Ecosystem DynamicsUniversity of Amsterdam

AmsterdamThe Netherlands

Ernesto Ormen˜o-OrrilloGenomic Sciences Center, UNAMCuernavaca

Mexico

Ralf RabusInstitute for Chemistry and Biology of the MarineEnvironment (ICBM)

University of OldenburgOldenburg

Germany

Lesley A RobertsonDepartment of BiotechnologyDelft University of TechnologyDelft

The Netherlands

Eliora Z RonDepartment of Molecular Microbiology and BiotechnologyThe George S Wise Faculty of Life Sciences

Tel Aviv UniversityTel Aviv

Israel

Eugene RosenbergDepartment of Molecular Microbiology and BiotechnologyTel Aviv University

Tel AvivIsraelKarola Schu¨hleFachbereich BiologieLaboratorium fu¨r MikrobiologieMarburg

GermanyDirk Schu¨lerDepartment Biologie ILudwig-Maximilians-Universita¨t Mu¨nchenPlanegg-Martinsried

Germanyxviii List of Contributors

Department of Biotechnology and Food Engineering

Technion – Israel Institute of Technology

Haifa

Israel

Dimitry Y Sorokin

Winogradsky Institute of Microbiology

Russian Academy of Sciences

Michael WagnerDepartment of Microbial EcologyFaculty Center of EcologyFaculty of Life SciencesUniversity of ViennaVienna

Austria

William B WhitmanDepartment of MicrobiologyUniversity of GeorgiaAthens, GA

USA

Friedrich WiddelMax-Planck-Institut fu¨r Marine MikrobiologieBremen

Germany

List of Contributors xix

Physiology and Biochemistry

Discovery of Acetogenic Bacteria and Acetogenesis 6

Resolution of the Acetyl-CoA ‘‘Wood/Ljungdahl’’

The Acetyl-CoA Pathway and Bioenergetics 26

CO2as Terminal Electron Acceptor and the Concept of

Fermentation 26

Enzymology of the Acetyl-CoA Pathway 29

Conservation of Energy and Bioenergetics 31

Occurrence of the Acetyl-CoA Pathway in

Nonacetogenic Microorganisms 33

Diverse Physiological Talents of Acetogens 33

Diverse Electron Donors 33

Use of Diverse Terminal Electron Acceptors 36

Regulation of the Acetyl-CoA Pathway and Other

Metabolic Abilities 37

Tolerance to Oxic Conditions and Metabolism of O2 38

Ecology of Acetogens 39

Metabolic Interactions of Acetogens in Pure Cultures

and Complex Ecosystems 39

Diverse Habitats 41

Biotechnological Applications of Acetogens 47

Commercial Production of Acetic Acid from Sugars 47

Bioconversion of Synthesis Gas to Acetic Acid, Ethanol,

and Other Chemicals 48

Bioremediation, Bioreactors, and Landfills 48

Other Potential Applications 48

Summary and Conclusions 48

AbstractThis chapter circumscribes the acetogens, a physiologicallydefined group of the domain Bacteria that are anaerobes, usingthe acetyl-CoA pathway as a mechanism for the reductivesynthesis of acetyl-CoA from CO2, for a terminal-electron-accepting, energy-conserving process, and for mechanism forthe fixation (assimilation) of CO2in the synthesis of cell carbon.Three main metabolic features of these organisms were defined,such as the use of chemolithoautotrophic substrates (H2-CO2orCO-CO2) as sole sources of carbon and energy under anoxicconditions, the capacity to convert certain sugars stoichiometrically

to acetate, and the ability to O-demethylate methoxylated aromaticcompounds and metabolize the O-methyl group via the 420 acetyl-CoA pathway Acetogens have been assigned to more than 20different genera and they differ in their morphology, cytology,and physiology The most frequently isolated acetogenic species

to date are members of the genera Clostridium and bacterium The habitat, the morphological and physiological prop-erties, and the phylogenetic position of acetogenic species arepresented The electron flow of the “Wood/Ljungdahl” pathway

Aceto-as well Aceto-as properties and function of enzymes involved in the CoA pathway is shown in detail Several biotechnological applica-tions are described with the commercial production of acetic acidfrom sugars and the bioconversion of synthesis gas to acetic acid,ethanol, and other chemicals being the most important ones

acetyl-Introduction to Acetogenic Bacteria and the Process of Acetogenesis

This chapter presents an overview of the history, taxonomy, logenetics, biochemistry, physiology, ecology, and applied aspects

phy-of acetogens Acetogenic prokaryotes have only been found in thedomain bacteria These prokaryotes utilize a reductive one-carbon pathway for the synthesis of acetyl-CoA, a metabolic pre-cursor of both acetate and biomass This pathway fixes CO2and istermed ‘‘the acetyl-CoA pathway.’’ This pathway is often referred

to as ‘‘the Wood/Ljungdahl pathway’’ in recognition of the twoindividuals, Harland G Wood and Lars G Ljungdahl, who wereresponsible for elucidating most of its enzymological featuresfrom the model acetogen Moorella thermoacetica (>Fig 1.1; seethe section on >‘‘Historical Perspectives’’ in this chapter).Acetogenesis (i.e., the process by which acetogens synthesizeacetate) is often regarded as a fermentation process; however,

as outlined in the section on >‘‘CO2 as Terminal ElectronAcceptor and the Concept of Fermentation,’’ acetogenesis is

E Rosenberg et al (eds.), The Prokaryotes – Prokaryotic Physiology and Biochemistry, DOI 10.1007/978-3-642-30141-4_61,

# Springer-Verlag Berlin Heidelberg 2013

very dissimilar to classic fermentations Purinolytic bacteria that

synthesize acetate via the glycine pathway will not be considered

in this chapter However, certain features of this CO2-fixing,

glycine-reductase-dependent pathway are similar to those of

the acetyl-CoA pathway, and the reader is directed to the review

of Andreesen (1994) for a detailed assessment of this pathway

and organisms that use it

Acetogens Defined

Usage of the term ‘‘acetogen’’ has not been consistent in the

literature, and this inconsistent usage has caused a small amount

of confusion regarding which organisms utilize the acetyl-CoApathway for the synthesis of acetate The following definition forthe term acetogen has been previously proposed (Drake1994)and is applied in this chapter:

Acetogen: An anaerobe that can use the acetyl-CoA pathway

as a (1) mechanism for the reductive synthesis of acetyl-CoAfrom CO2, (2) terminal-electron-accepting, energy-conservingprocess, and (3) mechanism for the fixation (assimilation) of

CO2in the synthesis of cell carbon

Per this definition, the formation of acetate as an endproduct is unimportant, i.e., the fate of acetyl-CoA is lessimportant than the process by which it is formed For example,Eubacterium limosum, ‘‘Butyribacterium methylotrophicum,’’and Caloramator pfennigii (formerly Clostridium pfennigii),organisms that qualify as acetogens per the above definition,form butyrate from the acetyl-CoA that is formed via theacetyl-CoA pathway (Lynd and Zeikus 1983; Zeikus 1983;Krumholz and Bryant1985; Zeikus et al.1985; Loubiere et al

1992) Likewise, the acetogen Acetobacterium woodii formsethanol from acetyl-CoA under certain conditions (Buschhorn

et al.1989)

The term ‘‘acetogenic’’ is an adjective that could be used todescribe any organism that makes acetate or acetic acid How-ever, the metabolic processes by which acetate can be formedduring either the aerobic or anaerobic growth of diverse micro-organisms might not be equivalent The mechanism by whichacetate is formed via the oxidation of ethanol by Acetobacter aceti

is fundamentally different from that used by certain obligateanaerobes that synthesize acetate from CO2 via the reductiveacetyl-CoA pathway Thus, it is important that a differentialnomenclature be applied to distinguish between acetate-forming bacteria because failure to do so results in unnecessaryconfusion in the literature For example, Thermobacteroidesproteolyticus and the syntroph PA-1 have been referred to asacetogens because they form acetate from glucose (Ollivier

et al.1985b; Brulla and Bryant1989) However, these organismsuse protons, not CO2, as terminal electron acceptors and form

H2, not acetate, as their main reduced end product; in short, they

do not appear to use the acetyl-CoA pathway for the synthesis ofacetate Likewise, the butyrate-degrading syntrophSyntrophomonas wolfei has been described as an acetogen(Stams and Dong1995) However, this organism (1) convertsbutyrate to acetate and H2(which can subsequently be used toreduce CO2 to formate) by -oxidation via the crotonyl-CoApathway (Wofford et al 1986), (2) does not reduce CO2 toacetate, and (3) is not known to utilized the acetyl-CoA pathway

Usage of the Terms ‘‘Acetogenesis,’’

‘‘Homoacetogen,’’ and ‘‘Homoacetogenesis’’

The term ‘‘acetogenesis’’ could be used to describe the process bywhich any organism forms acetate For example, the termacetogenesis has been used to describe the (1) oxygen-dependentprocess by which Enterococcus RfL6 oxidizes lactate to acetate(Tholen et al.1997) and (2) the production of acetate during

(a) Electron micrograph of a sporulated cell of Clostridium

thermoaceticum , which was reclassified as Moorella thermoacetica

(Collins et al 1994 ) From Drake ( 1994 ), used with permission

from Kluwer Academic (b, c) The two biochemists who were

primarily responsible for resolving the enzymological features of

the acetyl-CoA ‘‘Wood/Ljungdahl’’ pathway in M thermoacetica.

From Drake and Daniel ( 2004 ), used with permission from Elsevier.

The dates of the photos for B and C are September 1977 (taken

after Harland Wood’s 70th birthday celebration/symposium at

Case Western Reserve University, during which Wood was

‘‘roasted’’ and given the honorary degree of Doctor of Mouse

Science [the image on the hood symbolizes the shape of

transcarboxylase as seen by electron microscopy]) and May 2000

(taken during the symposium honoring Harry D Peck, Jr., at the

University of Georgia), respectively

4 1 Acetogenic Prokaryotes

proteolysis by Treponema denticola (Mikx 1997) No evidence

suggests that these organisms utilize the acetyl-CoA pathway for

acetate synthesis Since such usage makes it difficult to

under-stand what process is being referred to, it has been suggested that

usage of the term acetogenesis be restricted to processes by

which two molecules of CO2are used to form one molecule of

acetate (Wood and Ljungdahl1991) Unfortunately, such usage

fails to adequately distinguish between the three known

meta-bolic processes by which acetate is formed from CO2: (1) the

acetyl-CoA pathway, (2) the glycine-synthase-dependent

path-way, and (3) the reductive citric acid cycle (Fuchs 1986, 1989;

Thauer1988; Wood and Ljungdahl1991)

The term ‘‘homoacetogen’’ is often used to distinguish

between organisms that use the acetyl-CoA pathway and those

that do not (Schink and Bomar1992) This term implies that

acetate is the sole product formed by a particular organism

However, organisms that use the acetyl-CoA pathway usually

do not form acetate as their sole end product Their capacity to

form any particular end product, including acetate, is dependent

upon cultivation conditions Butyrate (Lynd and Zeikus1983;

Krumholz and Bryant1985; Worden et al.1989; Grethlein et al

1991), ethanol (Buschhorn et al.1989), lactate (Lorowitz and

Bryant1984; Drake1993; Misoph and Drake 1996a), succinate

(Dorn et al 1978; Lorowitz and Bryant 1984; Matthies et al

1993; Misoph and Drake 1996a), reduced aromatic acrylates

(Tschech and Pfennig 1984; Parekh et al.1992; Misoph et al

1996b), reduced aromatic aldehydes (Lux et al 1990), CO

(Diekert et al 1986), H2 (Martin et al 1983; Lorowitz and

Bryant 1984; Savage et al 1987), CH4 (Savage et al 1987;

Buschhorn et al.1989), sulfide (Heijthuijsen and Hansen1989;

Beaty and Ljungdahl 1991), dimethylsulfide (Beaty and

Ljungdahl1991), nitrite (Seifritz et al.1993; Fro¨stl et al 1996),

and ammonium (Seifritz et al.1993,2003; Fro¨stl et al 1996) are

examples of reduced end products of so-called homoacetogens

Indeed, the production of such products can constitute the sole

energy-conserving, growth-supportive process of the cell (see

the section on>‘‘Use of Diverse Terminal Electron Acceptors’’

in this chapter) Thus, the conditions under which an acetogen

forms acetate should be qualified rather than merely referring to

the organism as a homoacetogen For example, Ruminococcus

productus (formerly Peptostreptococcus productus) is

homoacetogenic on pyruvate but forms acetate, lactate,

succi-nate, and formate when cultivated on fructose; this acetogen can

also form large amounts of ethanol during glycerol-dependent

growth (Misoph and Drake 1996a) Likewise, Moorella

thermoacetica is homoacetogenic when cultivated on H2/CO2

but does not form acetate when cultivated on H2/CO2in the

presence of nitrate; under this condition, the dissimilation of

nitrate is used preferentially to acetogenesis for the conservation

of energy (Fro¨stl et al 1996) Lastly, the term ‘‘homoacetate

production’’ has been used to describe the process by which

a genetically modified strain of Escherichia coli anaerobically

produces 2 moles of acetate per mole glucose fermented (Causey

et al.2003), yet this process is not homoacetogenic (i.e., does not

yield 3 moles of acetate per mole glucose) and the acetyl-CoA

pathway is not involved

Independent of these problems of usage, the production ofacetate as the sole end product from certain sugars, H2/CO2, orCO/CO2strongly suggests that the organism in question utilizesthe acetyl-CoA pathway per the definition for the term acetogen(see the section on>‘‘Acetogens Defined’’in this chapter)

Global Impact and Evolutionary Perspectives

Acetogens were initially viewed as obscure, poorly defined organisms For nearly five decades following the discovery ofacetogens in the 1930s, the major interest in them was restricted

micro-to resolving the biochemical features of the acetyl-CoA pathway(see the section on>‘‘Historical Perspectives’’in this chapter).The microbiology of acetogens drew little interest until the 1980swhen it started to become apparent that acetogens were a widelydistributed, phylogenetically diverse group of microorganisms.Added interest in the acetyl-CoA pathway occurred when it wasdiscovered that methanogens and sulfate-reducing bacteria usedmetabolic pathways that contained acetyl-CoA synthase, one ofthe key enzymes in the acetyl-CoA pathway (Fuchs 1986, 1989;Schauder et al 1986; Thauer et al 1989; see the section on

>‘‘Occurrence of the Acetyl-CoA Pathway in NonacetogenicMicroorganisms’’ in this chapter) Major bacterial groupsemploying this pathway in either the direction of acetate/bio-mass synthesis or acetate degradation include acetogens,methanogens, and sulfate-reducing bacteria

It is not possible to determine how much carbon is processedglobally via acetogens and pathways that make use of acetyl-CoAsynthase However, several facts are noteworthy:

1 The Calvin cycle, the reductive tricarboxylic acid cycle, thehydroxypropionate cycle, and the acetyl-CoAWood/Ljungdahlpathway facilitate the complete autotrophic fixation of CO2

Of these pathways, the one-carbon acetyl-CoA pathway isbiochemically the most simple For example, the acetyl-CoApathway requires less ATP to fix a molecule CO2than doesthe Calvin cycle Furthermore, the acetyl-CoA pathway is

a linear process that does not depend on preformed, plex molecules to which CO2 is fixed in a cyclic process(e.g., the Calvin cycle, the reductive tricarboxylic acid cycle,the hydroxypropionate cycle are dependent upon ribulosebiphosphate, oxalacetate, and acetyl-CoA, respectively, forthe fixation of CO2) (see section on>‘‘The Acetyl-CoAPathway and Bioenergetics’’in this chapter) Methanogensutilize an acetyl-CoA-synthase-dependent pathway that isbiochemically very similar to the acetyl-CoA pathway uti-lized by acetogenic bacteria (see the section on>‘‘Occur-rence of the Acetyl-CoA Pathway in NonacetogenicMicroorganisms’’ in this chapter), and methanogens (orancestors of methanogens) may have been the first autotrophs(Schopf et al.1983; Brock1989) Thus, and since life origi-nated under anoxic conditions, the acetyl-CoA pathway or

com-a pcom-athwcom-ay closely relcom-ated to it mcom-ay hcom-ave been the first processused for the autotrophic fixation of CO2(Fuchs 1986, Woodand Ljungdahl1991; Lindahl and Chang2001)

Acetogenic Prokaryotes 1 5

2 Approximately half of the human population contains low

numbers of methanogens in their gastrointestinal systems

and produces relatively little CH4; the colon of these

indi-viduals, as well as of those who more actively emit CH4, is

heavily colonized by acetogens (Wolin and Miller 1983)

Indeed, the gastrointestinal systems of mammals, whether

they harbor methanogens or not, are heavily colonized with

acetogens (Prins and Lankhorst 1977; Breznak and Kane

1990; Mackie and Bryant 1994; Wolin and Miller 1994;

Leedle et al.1995)

3 Acetogens inhabit the human colon In this habitat, acetogens

produce 1010 kg of acetate per year from H2-CO2, and

acetogenesis is one of the dominant processes in the overall

metabolism of carbohydrate in the human colon (Lajoie

et al.1988; Wolin and Miller1994; Dore´ et al.1995; Bernalier

et al.1996a,b; Miller and Wolin1996; Wolin et al.1999)

4 Totally, 1012 kg of acetate are produced each year via the

reduction of CO2by acetogens in the hindgut of termites,

a number that is fivefold greater than the annual amount of

methane formed globally via the biogenic reduction of CO2

(Breznak and Kane1990) One-third of the energy

require-ments of the termite is provided by the acetate that is

synthesized by the reduction of CO2 by gut acetogens

(Breznak1994)

5 Totally 1013kg of acetate is formed and further metabolized

annually in terrestrial habitats such as soils and sediments,

and a minimum of 10 % of this acetate is likely formed by the

reduction of CO2via the acetyl-CoA pathway (Wood and

Ljungdahl1991)

6 Up to 25 % of the total organic carbon of soil can be turned

over through acetate under low temperature, anoxic

condi-tions (equivalent to nearly 40 g acetate per kg dry wt of soil;

Ku¨sel and Drake1994) The capacity to form acetate in soils

is concomitant with acetogenic activities and the occurrence

of H2-utilizing acetogens (Ku¨sel and Drake 1995; Wagner

et al.1996; Ku¨sel et al 1999c) Acetate is a dominant organic

compound in soil solution (Tani et al.1993), and

concen-trations can be in the mM range following a rainfall event

(Ku¨sel and Drake 1999a) Assuming a weight of 1017kg for

the first meter of the global terrestrial surface [based on

a surface area of 1014m2(Whitman et al.1998) and using

a weight conversion of 103kg per m3] and an acetate

con-centration of 0.1 mmol per kg of this material, it can be

estimated that 1012kg of acetate is present in the first meter

of the terrestrial surface at any one moment (i.e., per

‘‘snapshot’’) Even if only a small percentage of this acetate

were formed by acetogens, given the turnover dynamics

of acetate, the annual magnitude of the acetogen-derived

acetate in the terrestrial biosphere would be enormous The

number of prokaryotes in the terrestrial subsurface

might exceed that of the terrestrial surface by a factor of 10

(Whitman et al.1998) It can be projected that acetate and

acetogens are also involved in the cycling of carbon in

this poorly explored compartment of the terrestrial

ecosphere (see the section on>‘‘Diverse Habitats’’ in this

chapter)

7 The acetate formed by acetogenesis is an essential trophiclink during the turnover of carbon in diverse anoxic habitats(McInerney and Bryant1981)

Such observations not only illustrate that nature’s ability toform acetate is enormous, they also demonstrate that the acetyl-CoA Wood/Ljungdahl pathway is fundamental to the carboncycle of earth

organ-4H2þ 2CO2! CH2COOHþ 2H2O ð1:1ÞSuch a reaction had not been observed earlier With theexception of a small study on the nutritional requirements of

C aceticum (Karlsson et al 1948), no further work waspublished with this acetogen until it was reisolated in1980–1981 (Adamse 1980; Braun et al 1981; Gottschalk andBraun1981;>Fig 1.2)

Clostridium thermoaceticum was discovered a few years afterthe isolation of C aceticum (Fontaine et al 1942) and was theonly acetogen available for laboratory study for several decades(>Fig 1.1) This bacterium was reclassified as Moorellathermoacetica (Collins et al 1994) and will be referred to bythis name hereafter Moorella thermoacetica was isolated as anobligate heterotroph and was observed to convert glucose toacetate; the stoichiometry of this process approximated thefollowing reaction:

C6H12O6! 3CH3COOH ð1:2Þ

In the early 1940s, no known metabolic process couldexplain this reaction, and it was proposed that the CO2 pro-duced via oxidation was subsequently utilized in the synthesis ofacetate:

Since, in this fermentation, 2.5 moles of a two-carbon pound (acetic acid) are obtained from 1 mole of glucose, itseems probable that either there is some primary cleavage ofglucose other than the classical 3-3 split or that a one-carboncompound is being reabsorbed Of these two possibilities, therecent work on carbon dioxide uptake makes the latter seemmore likely (Fontaine et al 1942)

com-The latter statement was in reference to the discovery of CO2

fixation in heterotrophs (Wood and Werkman 1936, 1938;Wood et al.1941a,b) Subsequent proposals for the acetogenicconversion of glucose or pyruvate to acetate made it possible to

6 1 Acetogenic Prokaryotes

see that both the autotrophic and heterotrophic acetogenic

processes likely involved the reductive synthesis of acetate from

CO2(Barker1944)

Conversion of glucose to acetate:

ð1:3ÞReductive portion: 8Hþ 2CO2! CH3COOHþ 2H2O ð1:4Þ

Net reaction: C6H12O6! 3CH3COOH ð1:5Þ

Conversion of pyruvate to acetate:

ð1:6ÞReductive portion: 8Hþ 2CO2! CH3COOHþ 2H2O ð1:7Þ

Net reaction: 4C3H4O3þ 2H2O! 5CH3COOHþ 2CO2 ð1:8Þ

The overlap between reactions>1.1,>1.4, and>1.7cated that a unique reductive process was likely responsible foracetate synthesis from CO2

indi-Resolution of the Acetyl-CoA ‘‘Wood/Ljungdahl’’ Pathway

Barker and Kamen (1945) demonstrated in the first publishedbiological experiments with 14C (Kamen 1963) that

M thermoacetica incorporated14CO2equally into both carbonatoms of acetate This landmark experiment with14C demon-strated that the capacity of M thermoacetica to synthesize acetatefrom glucose was, in fact, similar to the capacity of C aceticum

to synthesize acetate from H2/CO2:

" It may be concluded that the acetic acid fermentation of glucose by C thermoaceticum involves a partial oxidation of the substrate to 2 moles each of acetic acid and carbon dioxide followed by a reduction and condensation of the carbon dioxide to a third mole of acetic acid (Barker and Kamen 1945 )

In 1952, Wood repeated the14C experiments of Barker andKamen with13CO2and confirmed that M thermoacetica syn-thesized acetate from two molecules of CO2(Wood1952a) Inthis work, mass spectrometry conclusively demonstrated that

CO2 was uniformly fixed into both the carboxyl and methylcarbons of the third molecule of acetate from glucose Utilizing[3,4-14C]-glucose, it was also shown that carbons 3 and 4 ofglucose were converted to CO2 (Wood 1952b) These earlystudies by Barker, Kamen, and Wood demonstrated that (1) glu-cose was subject to a classic 3-3 split between carbons 3 and 4and (2) CO2was fixed via an unknown CO2-fixing process intoacetate (>Fig 1.3)

It took decades of continued research before the enzymology

of this CO2-fixing process was fully resolved (see the section on

>‘‘The Acetyl-CoA Pathway and Bioenergetics’’in this ter) It is an irony of the history of acetogenesis that the modelorganism (i.e., M thermoacetica) used to resolve the biochem-istry of this autotrophic process was thought to be an obligateheterotroph during these decades of research Indeed, thechemolithoautotrophic nature of M thermoacetica (Daniel

chap-et al.1990) was resolved nearly five decades after its isolationand well after the enzymological details of the acetyl-CoApathway were firmly established The milestones of thenumerous studies that resolved both the enzymology of theacetyl-CoA pathway and the chemolithoautotrophic abilities ofthe model acetogen used in these studies can be found innumerous review articles (Ljungdahl and Wood 1969; Wood

1972, 1976, 1982, 1985, 1989, 1991; Ljungdahl 1986; Woodand Ljungdahl1991; Drake 1992, 1994; Ragsdale 1991, 1994,

1997) and are outlined in>Table 1.1 For additional insightsinto the early career years of Harland G Wood, see therecent excellent historical treatments by Singleton (Singleton

1997a,b,2000)

Fig 1.2

(a) Tube containing dried soil and spores of the first acetogen

to be isolated, Clostridium aceticum The tube was obtained from

H A Barker; the date on the tube is May 7, 1947 B Electron

micrograph of a peritrichously flagellated cell of C aceticum

[From Braun et al ( 1981 ), used with permission from Springer.

The photograph (panel A) was kindly provided by G Gottschalk]

Acetogenic Prokaryotes 1 7

Isolates to Date and Microbiological Methods

The number of known acetogens has increased significantly in

the last two decades, and approximately 100 different species

have been isolated to date from extremely diverse habitats

Acetogens can be found in almost all anoxic environments,

including some extreme habitats, as indicated by the isolation

of strain SS1 (Liu and Suflita 1993) and ‘‘Acetobacterium

psammolithicum’’ from deep subsurface sediment and

sand-stone, respectively (Krumholz et al.1999) Although most

iso-lates to date are mesophilic, thermophilic and psychrotolerant

species have also been isolated The occurrence and ecological

roles of acetogens in various habitats are discussed in the section

on acetogen ecology (see the section on >‘‘Ecology of

Acetogens’’in this chapter)

Homoacetogenic conversion of glucose to acetate Glucose is first

converted to two molecules of pyruvate via glycolysis (Box A);

glycolysis yields ATP by substrate-level phosphorylation (SLP).

Pyruvate is then oxidized and decarboxylated, yielding

acetyl-CoA, CO 2 , and reducing equivalents (Box B) The two

acetyl-CoA molecules that are produced from pyruvate are converted to

two molecules of acetate; this process yields additional ATP by

SLP The eight reducing equivalents that are produced via

glycolysis and pyruvate-ferredoxin oxidoreductase are utilized in

the acetyl-CoA pathway to reduce two molecules of CO 2 to an

additional molecule of acetate (Box C) The CO 2 that is reduced in

the acetyl-CoA pathway is likely derived primarily from

supplemental CO 2 rather than the CO 2 derived via the

decarboxylation of pyruvate (Modified from Drake 1994 )

Table 1.1

Milestones that led to resolving the acetyl-CoA pathway and chemolithoautotrophic abilities of Moorella thermoacetica

Year Event a

1932 H2-dependent conversion of CO2to acetate in

sewage sludge (Fischer et al 1932)

1936 Isolation of the first acetogen, Clostridium aceticum;

total synthesis of acetate from H2-CO2(Note: culture was lost, Wieringa 1936 , 1939–1940 )

1942 Discovery of the second acetogen, Moorella

thermoacetica (formerly Clostridium thermoaceticum); conversion of one glucose to three acetate molecules (Fontaine et al 1942)

1944 Acetogenic conversion of pyruvate to acetate

(Barker 1944 ) 1945–1952 Synthesis of acetate from 14 CO 2 (Barker and Kamen

1965 Autotrophic synthesis of cell-carbon precursors from

CO2(Ljungdahl and Wood 1965 ) 1966–1969 Proposal of one-carbon pathway for the

tetrahydrofolate/corrinoid-mediated synthesis of acetate from CO 2 (Ljungdahl and Wood 1966, 1969 ) 1973–1986 Resolution of the tetrahydrofolate pathway [reviewed

in Ljungdahl ( 1986 )]

1978–1980 Discovery of CO dehydrogenase as a

nickel-containing enzyme (Diekert and Thauer 1978 ; Drake et al 1980 )

1981 Resolution of enzymes required for synthesis of

acetyl-CoA from pyruvate and methyltetrahydrofolate (Drake et al 1981a ) 1981–1982 Demonstration that CO replaces the carboxyl

group of pyruvate and undergoes an exchange reaction with acetyl-CoA (Drake et al 1981b ;

Hu et al 1982 )

1982 Discovery of hydrogenase (Drake 1982 )

1983 Purification of CO dehydrogenase (Diekert and Ritter

1983 ; Ragsdale et al 1983 )

1983 Use of H 2 and CO under organotrophic conditions

(Kerby and Zeikus 1983 )

1984 Resolution of nutritional requirements (Lundie and

Drake 1984 )

1984 Enzyme system for H 2 -dependent synthesis of

acetyl-CoA (Pezacka and Wood 1984b ) 1984–1986 CO dehydrogenase is acetyl-CoA synthase (Pezacka

and Wood 1984a , b ; Regsdale and Wood 1985), and

CO is the carbonyl precursor in the acetyl-CoA pathway under growth conditions (Diekert et al.

1984 ; Martin et al 1985 ) 1985–1991 Catalytic mechanism of acetyl-CoA synthase

[reviewed in Ragsdale ( 1991 )]

8 1 Acetogenic Prokaryotes

Bacteria considered to be acetogens as defined above (see the

section on>‘‘Acetogens Defined’’in this chapter) are listed in

>Table 1.2 However, relatively few of these bacteria have been

examined in detail, and a good understanding of the metabolic

capabilities of most of the isolates is lacking In compiling the

list, the apparent acetogenic capability (see the section on

>‘‘Usage of the Terms ‘Acetogenesis,’ ‘Homoacetogen,’ and

‘Homoacetogenesis’’’ in this chapter) of each organism has

been taken into account In this regard, three main metabolic

features of these organisms (Drake 1994) are (1) the use of

chemolithoautotrophic substrates (H2-CO2 or CO-CO2) as

sole sources of carbon and energy under anoxic conditions,

(2) the capacity to convert certain sugars stoichiometrically to

acetate, and (3) the ability to O-demethylate methoxylated

aro-matic compounds and metabolize the O-methyl group via the

acetyl-CoA pathway Many acetogens display all three of these

metabolic capabilities

Most acetogenic isolates are rod-shaped, but coccoid forms

have also been observed (>Table 1.2) Staining properties vary,

sometimes within a genus, and both Gram-negative and

Gram-positive species have been reported (>Table 1.2) Some

acetogens have flagella and are motile Some form spores that

remain viable for long periods; the thermophilic sporeformers

are fairly resistant to high temperatures Indeed, spores of

M thermoacetica have a decimal reduction time (i.e., the time

required to decrease the population of viable spores by 90 %) of

111 min at 121 C (Byrer et al 2000) Cells of the acetogen

Clostridium glycolicum RD-1 are tethered by connecting

fila-ments, a morphological structure recently described for

Clos-tridium akagii and ClosClos-tridium uliginosum (Kuhner et al.2000;

Matthies et al 2001) Thus, the ultrastructural features of

acetogens are highly variable

Description of Species

Acetogens have been assigned to 21 different genera and differ in

their morphological, cytological, and physiological properties

(>Table 1.2) The genera Clostridium and Acetobacterium

harbor the most acetogenic species isolated to date The firstacetogen was classified as a clostridial species, C aceticum(Wieringa1936) The second acetogenic genus Acetobacteriumwas established when the first Gram-positive, nonsporeformingacetogen (Acetobacterium woodii; Balch et al.1977;>Fig 1.4)was isolated and could not be grouped with the acetogenicclostridia Conspicuously, all the heretofore isolatedpsychrotolerant acetogens and many N2-fixing acetogens belong

to the genus Acetobacterium (Schink and Bomar 1992;

>Table 1.2) About half of the genera that harbor acetogensonly contain one acetogenic species (e.g., Holophaga foetida,Acetohalobium arabaticum, Oxobacter pfennigii, Acetonemalongum;>Table 1.2) Recently, acetogenesis has been observed

in spirochetes (‘‘Treponema primitia’’) isolated from termite guts(Leadbetter et al.1999; Graber and Breznak 2004a; Graber et al.2004b;>Fig 1.5)

A brief overview of acetogenic species having validated names

is given in the following paragraphs The names of those isms not validated are in quotation marks Earlier compilationsinclude Breznak (1992), Diekert (1992), Hippe et al (1992),Schink and Bomar (1992), Mackie and Bryant (1994), andSchink (1994) Although relatively few of the acetogens listedbelow have been evaluated for their ability to tolerate O2, itshould be anticipated that many acetogens possess the ability

organ-to both organ-tolerate and consume small amounts of oxygen (Ku¨sel

et al.2001; Karnholz et al.2002; Boga and Brune2003)

Acetitomaculum ruminis This species was isolated from steerrumen fluid (Greening and Leedle1989) Cells are Gram-positive,nonsporeforming, motile, slightly curved rods Growth-supportive substrates include H2-CO2, CO, formate, cellobiose,glucose, ferulate, and syringate With all substrates, acetate is thesole reduced end product (Greening and Leedle1989)

Acetoanaerobium noterae This species was isolated fromsediment samples of the Notera oil exploration site in Israel(Sleat et al.1985) Cells are Gram-positive, nonsporeforming,motile, straight rods Acetoanaerobium noterae grows with

H2-CO2, glucose, and maltose and produces acetate as thesole product Propionate, butyrate, isobutyrate, and isovalerateare also formed when yeast extract serves as the growth-supportive substrate (Sleat et al.1985)

Acetoanaerobium romashkovii This organism was isolatedfrom the Romashkino oil field in Tatarstan (Davydova-Charakhch’yan et al 1992) Cells are Gram-positive,nonsporeforming, motile rods with rounded ends.Growth-supportive substrates include H2-CO2, formate,methanol, pyruvate, lactate, ethylene glycol, sugars, and aminoacids Acetate is the sole product from carbohydrates and

H2-CO2; propionate is also formed during growth on sucrose

‘‘Acetoanaerobium romashkovii’’ produces and excretespolysaccharides during growth on H2-CO2 or methanol(Davydova-Charakhch’yan et al.1992)

Acetobacterium bakii, Acetobacterium fimetarium, andAcetobacterium paludosum These species were isolated fromcold habitats (<6C) or from samples that were kept for morethan 1 year at 6C (Kotsyurbenko et al.1995) Cells of all three

Year Event a

1986–1990 H 2 - and CO-dependent electron transport system

coupled to the synthesis of ATP (Ivey and Ljungdahl

1986 ; Hugenholtz and Ljungdahl 1989 , 1990 ;

Das et al 1989 )

1990 Chemolithoautotrophic growth on H 2 -CO 2 and

CO-CO 2 (Daniel et al 1990 )

1991 Integrated model for catabolic, anabolic, and

bioenergetic features of the acetyl-CoA ‘‘Wood/

Ljungdahl’’ pathway (Wood and Ljungdahl 1991 )

a Events prior to the isolation of Moorella thermoacetica

Modified from Drake ( 1994 )

Acetogenic Prokaryotes 1 9

Table 1.2

Acetogenic bacteria isolated to date a

Acetogen Source ofisolate Gramtype b Cell

morphology Growthtemperature c G + C

(mol%) Deposited as References

Acetoanaerobium

ruminis

Rumen fluid, steer

+ Rod Mesophilic 34 ATCC 43876I Greening and Leedle

( 1989 ) Acetoanaerobium

+ Rod Mesophilic 38 DSM 2925 I Eichler and Schink ( 1984 )

Acetobacterium

dehalogenans

Sewage digester sludge

+ Coccus Mesophilic 48 DSM 11527 Traunecker et al ( 1991 )

Acetobacterium

fimetarium

Digested cattle manure

+ Rod Psychrotrophic 46 DSM 8238I Kotsyurbenko et al ( 1995 )

Acetobacterium

malicum

Freshwater sediment

+ Rod Mesophilic 44 DSM 4132I Tanaka and Pfennig

( 1988 ) Acetobacterium

 Rod Mesophilic n.r SMCC/W 751I Krumholz et al ( 1999 ) Acetobacterium tundrae Tundra soil + Rod Psychrotrophic 39 DSM 9173 I Simankova et al ( 2000 ) Acetobacterium tundrae Sewage

digester

+ Rod Mesophilic 43 DSM 1911I Braun and Gottschalk

( 1982 ) Acetobacterium woodii Marine

+ Rod Mesophilic 36 n.d Do¨rner and Schink ( 1991 ) Acetobacterium sp B10 Wastewater

pond

+ Rod Mesophilic n.r n.d Sembiring and Winter

( 1989 , 1990 ) Acetobacterium sp HA1 Sewage sludge + Rod Mesophilic n.r n.d Schramm and Schink

( 1991 ) Acetobacterium sp HP4 Lake sediment + Rod Psychrotrophic n.r n.d Conrad et al ( 1989 ) Acetobacterium

sp KoB58

Sewage sludge + Rod Mesophilic 44 n.d Wagener and Schink

( 1988 ) Acetobacterium sp.

+ Rod Mesophilic n.r n.d Emde and Schink ( 1987 ) Acetobacterium sp.

OyTac1

Freshwater sediment

+ Rod Mesophilic n.r n.d Emde and Schink ( 1987 )

Acetobacterium sp.

RMMac1

Marine sediment

 Rod Mesophilic 48 n.d Schuppert and Schink

( 1990 ) Acetobacterium sp 69 Sea sediment + Rod Mesophilic 48 n.d Inoue et al ( 1992 ) Acetobacterium sp Tundra

wetland soil

+ Rod Psychrotrophic 39 n.d Kotsyurbenko et al ( 1996 )

10 1 Acetogenic Prokaryotes

Table 1.2 (continued)

Acetogen Source ofisolate Gramtype b Cell

morphology Growthtemperature c G + C

(mol%) Deposited as References

Acetohalobium

arabaticum

Saline lagoon  Rod Mesophilic 34 DSM 5501I Zhilina and Zavarzin

( 1990 ) Acetonema longum Wood-eating

+ Rod Mesophilic 49 ATCC 33266I Zeikus et al ( 1980 )

Caloramator fervidus (?) Hot spring  Rod Thermophilic 39 ATCC 43204I Patel et al ( 1987 )

Clostridium aceticum Soil  Rod Mesophilic 33 DSM 1496I Wieringa ( 1936 )

Braun et al (1981)dClostridium

autoethanogenum (?)

Rabbit feces + Rod Mesophilic 26 DSM 10061 Abrini et al ( 1994 )

Clostridium coccoides Mouse feces,

human feces

+ Coccoid rod n.r 46 DSM 935I Kaneuchi et al ( 1976 )

Kamlage et al ( 1997 ) Clostridium difficile AA1 Rumen,

Tarmer et al (1993) Clostridium magnum Freshwater

digester

+ Rod Mesophilic 32 DSM 10521 I Schnu¨rer et al ( 1996 )

Clostridium sp CV-AA1 Sewage sludge  Rod Mesophilic 42 n.d Adamse and Velzeboer

( 1982 ) Clostridium sp M5a3 Human feces + Rod n.r n.r n.d Bernalier et al ( 1996a )

Leclerc et al ( 1997a , b ) Clostridium sp F5a15 Human feces + Rod n.r n.r n.d Bernalier et al ( 1996a )

Leclerc et al ( 1997a , b ) Clostridium sp Ag4f2 Human feces + Rod n.r n.r n.d Bernalier et al ( 1996a )

Clostridium sp TLN2 Human feces + Coccobacillus n.r n.r n.d Bernalier et al ( 1996a )

Eubacterium aggregans Olive oil mill

wastewater

+ Rod Mesophilic 55 DSM 12183I Mechichi et al ( 1998 )

Eubacterium limosum Rumen fluid,

sheep

+ Rod Mesophilic 48 ATCC 8486 I Sharak Genthner et al.

( 1981 )rAcetogenic Prokaryotes 1 11

Table 1.2 (continued)

Acetogen Source ofisolate Gramtype b Cell

morphology Growthtemperature c G + C

(mol%) Deposited as References

Holophaga foetida Freshwater

ditch mud

 Rod Mesophilic 62 DSM 6591I Bak et al ( 1992 )

Liesack et al ( 1994 ) Moorella glycerini Hot spring

sediment

+ Rod Thermophilic 54 DSM 11254 I Slobodkin et al ( 1997 )

Moorella mulderi Bioreactor + Rod Thermophilic 53 DSM 14980I Balk et al ( 2003 ) Moorella thermoacetica Horse manure +/  Rod Thermophilic 54 ATCC 35608 I Fontaine et al (1942) Moorella

thermoautotrophica

Hot spring +/  Rod Thermophilic 54 ATCC 33924 I Wiegel et al ( 1981 ) Moorella sp F21g Soil + Rod Thermophilic n.r n.d Karita et al ( 2003 ) Natroniella acengena Soda lake

+ Rod Mesophilic 32 DSM 11416I Zhilina et al ( 1998 )

Oxobacter pfennigii Rumen fluid,

steer

+ Rod Mesophilic 38 DSM 3222 I Krumholz and Bryant

( 1985 ) Ruminococcus

+ Coccus Mesophilic 45 ATCC 35244 Lorowilz and Bryant

(1984) Ruminococcus

productus Marburg

Sewage digester

+ Coccus Mesophilic 46 ATCC 43917 Geerligs et al ( 1987 )

Ruminococcus schinkii Rumen,

3-day-old lamb

+ Coccoid rod Mesophilic 46 DSM 10518I Rieu-Lesme et al ( 1996b )

Ruminococcus sp TLF1 Human feces + Coccobacillus n.r n.r n.d Bernalier et al ( 1996a ) Sporomusa acidovorans Distillation

soil

+ Rod Mesophilic 43 DSM 10669I Kubner et al (1997)

Sporomusa sphaeroides River mud  Rod Mesophilic 47 DSM 2875 I Mo¨ller et al ( 1984 ) Sporomusa termitida Wood-eating

termite, gut

 Rod Mesophilic 49 DSM 4440I Breznak et al ( 1988 ) Sporomusa sp DR6 h Rice field soil + Rod n.r n.r n.d Rosencrantz et al ( 1999 ) Sporomusa sp DR1/8 Rice field soil + Rod n.r n.r n.d Rosencrantz et al ( 1999 ) Syntrophococcus

sucromutans

Rumen fluid, steer

 Coccus Mesophilic 52 DSM 3224I Krumholz and Bryant

( 1986 ) Thermoacetogenium

phaeum

Pulp waste water reactor

+ Rod Thermophilic 54 DSM 12270 I Hattori et al ( 2000 )

n.r Spirochete Mesophilic 51 DSM 1247 Graber et al ( 2004 )

12 1 Acetogenic Prokaryotes

Table 1.2 (continued)

Acetogen Source ofisolate Gramtype b Cell

morphology Growthtemperature c G + C

(mol%) Deposited as References

digester

+ Rod Thermophilic 47 n.d Lee and Zinder ( 1988 ) CS1 Van Human feces + Rod Mesophilic n.r n.d Wolin and Miller ( 1993 )

CS3Glu Human feces + Coccoid rod Mesophilic n.r n.d Wolin and Miller ( 1993 )

CS7H Human feces + Rod Mesophilic n.r n.d Wolin and Miller ( 1993 )

+ Rod n.r n.r n.d Plugge et al ( 1990 )

HA Horse feces  Coccobacillus n.r n.r n.d Miller and Wolin ( 1995 )

I52 Human feces  Coccoid rod Mesophilic n.r n.d Wolin and Miller ( 1994 )

S5a2h Human feces + Coccus n.r n.r n.d Bernalier et al ( 1996a )

Leclerc et al ( 1997a , b )

 Rod Mesophilic n.r n.d Samain et al ( 1982 )

ZJ j Tundra soil + Rod Psychrophilic n.r n.d Kotsyurbenko et al ( 1992 )

Nozhevnikova et al.

( 1994 ) 417/2 Oil field  Rod Mesophilic 43 n.d Davydova-Charakhch’yan

et al ( 1992 ) 417/5 Oil field  Rod Mesophilic 43 n.d Davydova-Charakhch’yan

et al ( 1992 ) New acetogenic

bacterium

Rumen, old lamb

15-h-+ Coccoid rod Mesophilic 46 DSM 12568 Rieu-Lesme et al ( 1996a )

Symbols and abbreviations: + positive,  negative, +/ variable, n.d not deposited, n.r not reported; I

, type strain, ATCC American Type Culture Collection, DSM Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, SMCC and Subsurface Microbial Culture Collection

a Bacteria listed appear to use the acetyl-CoA pathway for the synthesis of acetate and growth [modified from Drake (1992, 1994)] If the acetogenic nature of an organism is uncertain, a question mark occurs after the name of the organism (see text) Organisms not having validated names are enclosed in quotation marks Unless otherwise indicated, type strains (marked with a ‘‘T’’ adjacent to the deposition number) are available from American Type Culture Collection (ATCC),

10801 University Boulevard, Manassas, Virginia, U.S.A., and Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) Mascheroder Weg 1b, Braunschweig, Germany

b Gram type is based on electron microscopic analyses of the cell wall structure if reported Otherwise, Gram type is based on the Gram-stain reaction (Note: results

of the Gram-stain reaction are not always in agreement with the electron microscopic analysis of the cell wall)

c General temperature preference: psychrophilic (5–10C), psychrotolerant (16–18C), mesophilic (31–34C), and thermophilic (58–62C)

d See also Adamse ( 1980 )

e See also E1 Ghazzawi ( 1967 )

f See also Moore and Cato ( 1965 )

species are Gram-positive, oval-shaped, and motile rods.

Although these species were isolated at 6C, their optimum

growth temperatures are 20C (A bakii and A paludosum) and

30C (A fimetarium) However, their ability to grow at a

tem-perature as low as 1C qualifies them as psychrotolerant

bacte-ria Acetobacterium bakii, A fimetarium, and A paludosum grow

with H2-CO2, CO, formate, and certain sugars and

stoichiomet-rically convert these substrates to acetate as the sole product

(Kotsyurbenko et al.1995) Although these three species are verysimilar, DNA-DNA hybridization supported the designation ofthree different species

Acetobacterium carbolinicum This species was isolated fromfreshwater sediments (Eichler and Schink1984) Cells are Gram-positive, nonsporeforming, rods with slightly pointed ends;some strains are motile The type strain grows with ethanol,propanol, butanol, 1,2-propanediol, and 2,3-butanediol; oxi-dizes these substrates incompletely to the corresponding fattyacids; and uses the reducing equivalents to reduce CO2to acetatevia the acetyl-CoA pathway (Eichler and Schink 1984) Othergrowth-supportive substrates include H2-CO2, formate, pyru-vate, lactate, methanol, hexoses, ethylene glycol, andmethoxylated aromatic acids Strain A carbinolicum KoMac1(DSM 5193) was isolated with the methylalkylethermethoxyacetate and can also grow on glycol ethers The etherbond of these compounds is cleaved, and acetate is formed as thesole product (Schuppert and Schink1990) Strain KoMac1 alsoutilizes the O-methyl group of methoxylated aromatics andbetaine (Schuppert and Schink1990)

Acetobacterium dehalogenans This organism (also termed

‘‘strain MC’’) was isolated from sewage sludge (Traunecker

et al 1991) Cells are Gram-positive, nonsporeforming,nonmotile, elongated cocci ‘‘Acetobacterium dehalogenans’’ isthe only known acetogen able to utilize and grow with methylchloride Methyl chloride is dehalogenated via a methyl chloridedehalogenase (Meßmer et al.1996) and is further metabolized toacetate via the acetyl-CoA pathway Other substrates supportinggrowth of the organism include H2-CO2, CO, glucose, fumarate,methanol, and methoxylated aromatic compounds

Acetobacterium malicum This species was enriched and lated with 2-methoxyethanol as growth substrate from

iso-a freshwiso-ater sediment (Tiso-aniso-akiso-a iso-and Pfennig 1988) Cells areGram-positive, nonsporeforming, motile rods with slightlypointed ends Similar to a few other species of Acetobacterium,

A malicum can grow at the expense of the ether compounds2-methoxymethanol and 2-ethoxyethanol, which are metabo-lized to acetate and the corresponding alcohols Othergrowth-supportive substrates include malate, H2-CO2, formate,pyruvate, fructose, betaine, and the O-methyl groups ofmethoxylated aromatic compounds (Tanaka and Pfennig

1988) Acetate is the sole product of these substrates

Acetobacterium psammolithicum This species was isolatedfrom subsurface sandstone and represents the second acetogenisolated from a subsurface ecosystem (Krumholz et al 1999).[The first acetogenic isolate from a subsurface habitat was theunclassified strain SS1 (Liu and Suflita1993).] Cells are Gram-negative, nonsporeforming, nonmotile rods Growth is very slow

in mineral medium In medium supplemented with yeast extract,growth is very good on H2-CO2, methanol, formate, glucose,syringate, alcohols, and organic acids (Krumholz et al 1999).Acetate is the product of H2-CO2-dependent growth; products

of other growth-supportive substrates are not reported.Acetobacterium tundrae This organism is psychrotolerantand was isolated from a tundra wetland soil (Simankova et al

2000) Cells are Gram-positive, nonsporeforming, motile rods

P

F

Fig 1.4

Electron micrograph of cells of Acetobacterium woodii (ATCC

29683 T , DSM 1030 T ) with a single subterminal flagellum (F) and

pili-like structures (P) [From Balch et al ( 1977 ), used with

permission from International Union of Microbiological Societies.

The micrograph was kindly provided by R S Wolfe]

Fig 1.5

Phase-contrast micrograph of spirochete Treponema sp., strain

ZAS-2 The length of cells varies from 3 to 7 mm (The micrograph

was kindly provided by J Breznak)

14 1 Acetogenic Prokaryotes

Growth-supportive substrates include H2-CO2, CO, formate,

methanol, and sugars; acetate is the sole reduced end product

As with the psychrotrophic acetogens A bakii and A paludosum,

A tundrae has a minimum growth temperature of 1C and an

optimal growth temperature of 20C

Acetobacterium wieringae This species was isolated from

a sewage digester (Braun and Gottschalk 1982) Cells are

Gram-positive, nonsporeforming, motile rods H2-CO2,

fruc-tose, and lactate are growth-supportive substrates; acetate is the

sole reduced end product (Braun and Gottschalk 1982)

Acetobacterium wieringae tolerates 300 mM acetate (Menzel

and Gottschalk1985)

Acetobacterium woodii The type strain of A woodii was

enriched and isolated from black sediment of a marine estuary

with H2-CO2 as substrate (Balch et al.1977) Cells are

Gram-positive, nonsporeforming, motile rods with slightly pointed ends

(>Fig 1.4) Growth-supportive substrates include H2-CO2, CO,

formate, methanol, 2,3-butandiol, ethylene glycol, acetoin,

glycerol, sugars, betaine, and several methoxylated aromatic

acids (Balch et al.1977; Bache and Pfennig 1981; Eichler and

Schink 1984; Sharak Genthner and Bryant 1987; Schink and

Bomar1992) Cultures demethylate the osmolytes

dimethylsulfo-niopropionate and glycine-betaine to methylthiopropionate and

dimethylglycine, respectively; however, only the demethylation of

glycine-betaine supported growth of the organism (Jansen and

Hansen2001) Acetobacterium woodii growths mixotrophically

on (i.e., can simultaneously utilize) H2-CO2and organic

com-pounds (e.g., fructose; Braun and Gottschalk1981) and can use

aromatic acrylates as energy-conserving, growth-supportive

ter-minal electron acceptors (Bache and Pfennig1981; Tschech and

Pfennig 1984) Growth, motility, and acetate formation from

H2-CO2are strictly dependent on sodium ions (Heise et al.1989;

Mu¨ller and Bowien 1995; Aufurth et al 1998) Several

Na+-dependent reactions in the metabolism of A woodii have

been identified, and associated enzymes have been purified

and characterized (Heise et al 1989, 1991, 1992, 1993;

Mu¨ller and Gottschalk 1994; Reidlinger and Mu¨ller 1994a;

Reidlinger et al 1994b; Mu¨ller et al 2001) Cells reductively

dechlorinate carbon tetrachloride (Egli et al.1988; Stromeyer

et al 1992); dechlorination is enhanced by the addition

of hydroxocobalamin (Hashsham and Freedman 1999)

Cells also tolerate and consume small amounts of oxygen

(Karnholz et al.2002)

Acetohalobium arabaticum This organism was isolated

from a cyanobacterial mat in a saline lagoon and was the

first obligately halophilic acetogen to be described (Zhilina

and Zavarzin1990) Sodium chloride (10–25 %) is necessary

for growth Cells are motile, straight rods often aggregated in

palisades H2-CO2, CO, trimethylamine, formate, betaine,

lac-tate, pyruvate, and histidine are growth-supportive

substrates Acetate is the main product during growth on

trimethylamine and betaine and is accompanied by minor

amounts of methylamines (Zhilina and Zavarzin1990; Zavarzin

et al.1994) Cell extracts have CO dehydrogenase and

hydroge-nase activities, which are stimulated by increased salt

concentrations

Acetonema longum This organism was isolated from the gutcontents of the wood-feeding termite Pterotermes occidentis(Kane and Breznak 1991a) Cells are sporeforming, motile rods

of unusually large size; cells can be up to 60 mm in length.Growth-supportive substrates include H2-CO2, pyruvate, fuma-rate, glucose, mannitol, and ribose; poor growth occurs oncitrate, propanol, ethylene glycol, and 3,4,5-trimethoxy-benzoate Homoacetogenesis only occurs with H2-CO2.Butyrate and acetate are the main products from carbohydratesand pyruvate; fumarate is metabolized to propionate and ace-tate, and rhamnose yields 1,2-propanediol as the major product(Kane and Breznak 1991a)

Bryantella formatexigens This species was isolated fromhuman feces (Wolin et al 2003) Cells are Gram-positive,nonmotile short rods (approx 1.2  0.7 mm) Single cells,and pairs and short chains of cells, are apparent Upon isolation,the type strain (I-52; Wolin and Miller1994) fermented vegeta-ble cellulose and carboxymethylcellulose but lost this abilityafter storage under frozen conditions No growth occurs on

H2-CO2 or formate, and formate is required for optimalhomoacetogenic conversion of glucose The lack of supplemen-tal formate yields succinate, lactate, and acetate as productsfrom glucose These characteristics indicate that the formatedehydrogenase is negligible Growth is supported by stachyose,sucrose, lactose, maltose, galactose, mannose, and xylose.Cells are catalase and oxidase negative, and nitrate is notreduced

Butyribacterium methylotrophicum This organism was lated from a sewage digestor (Zeikus et al.1980) Cells are Gram-positive, sporeforming, nonmotile rods Growth is supported by

iso-H2-CO2, formate, methanol, glucose, fructose, sucrose, vate, lactate, and glycerol (Zeikus et al.1980; Kerby and Zeikus

pyru-1987) Homoacetogenic utilization of substrates only occurswith H2-CO2and formate With other substrates, butyrate and

H2are also produced (Zeikus et al.1980; Lynd and Zeikus1983).After prolonged incubation in medium with CO in the gasphase, the type strain grew on and utilized CO; acetate was thesole product from CO (Lynd et al.1982) There is substantialevidence that ‘‘B methylotrophicum’’ and Eubacterium limosumare the same species: (1) the metabolic properties of the twoorganisms are nearly identical and (2) the 16S rRNA genesequences of the two organisms are very similar (99.4% sequencesimilarity; Moore and Cato1965; Sharak Genthner et al.1981;Tanner et al.1981; Sharak Genthner and Bryant1982; Tannerand Woese1994; Jansen and Hansen2001)

Caloramator fervidus This species was isolated from a hotspring in New Zealand and was first described as Clostridiumfervidus (Patel et al 1987) Cells are Gram-negative,sporeforming, motile rods Carbohydrates support growth,and acetate is the major end product However, growth onone-carbon compounds (e.g., formate) or other typicalacetogenic substrates (e.g., H2-CO2) has not been reported,and substrate/product stoichiometries of carbohydrate utiliza-tion are not available Thus, the true acetogenic nature of thisorganism has not been established Until otherwise proven, oneshould assume that the organism might not be an acetogen

Acetogenic Prokaryotes 1 15

Clostridium aceticum This species was the first acetogen to

be isolated (>Fig 1.2) It was isolated from soil and described by

Wieringa (Wieringa1936,1939–1940) After early studies with

the organism (Karlsson et al.1948), it was lost for about 30 years

However, C aceticum was reisolated from soil using Wieringa’s

enrichment procedure, and almost at the same time, spores of

the original Wieringa strain in sterile dried soil were found in

Barker’s laboratory and revived (Adamse 1980; Braun et al

1981) Cells are Gram-negative, sporeforming, motile rods

(Wieringa1939–1940; Braun et al.1981) Growth-supportive

substrates include H2-CO2, CO, fructose, glutamate fumarate,

pyruvate, aldehyde groups of aromatic compounds, and

methoxylated aromatic compounds (Wieringa 1939–1940;

Braun et al.1981; Lux and Drake1992; Matthies et al.1993;

Go¨ßner et al.1994) As with C formicoaceticum (see below),

fumarate is dismutated by C aceticum to acetate and succinate

and is metabolized independent of the acetyl-CoA pathway;

fumarate also serves as an alternative electron acceptor and

is reduced to succinate (Matthies et al 1993) N2 is fixed

(Cato et al.1986)

Clostridium autoethanogenum This organism was isolated

from rabbit feces (Abrini et al.1994) Cells are Gram-positive,

sporeforming, motile rods The range of substrates includes

H2-CO2, CO, pyruvate, hexoses, pentoses, and glutamate and

is similar to the range of substrates used by Clostridium

ljungdahlii (Abrini et al 1994; Tanner et al 1993) CO is

converted to acetate and ethanol (Abrini et al.1994) Ethanol

production from CO was also reported for C ljungdahlii;

how-ever, this metabolic potential is not necessarily stable (Barik et al

1988; Tanner et al 1993) The 16S rRNA gene sequences of

‘‘C autoethanogenum’’ and C ljungdahlii are essentially identical

(Stackebrandt et al.1999)

Clostridium coccoides Two acetogenic strains of C coccoides

(strains 1410 and 3110) were isolated from the human intestinal

tract (Kamlage et al 1997) The type strain of C coccoides

isolated from mouse feces was not initially described as an

acetogen; however, it has recently been shown to contain all

the enzymes of the acetyl-CoA pathway when grown on

H2-CO2-formate (Kaneuchi et al.1976; Kamlage et al 1997)

Cells of C coccoides strain 1410 (which is probably identical to

strain 3110) are Gram-variable, coccoid rods Clostridium

coccoides strain 1410 grows on a variety of hexoses, pentoses,

sugar alcohols, H2-CO2-formate, and H2-CO2-vanillate

Prod-ucts from growth have not been reported However, resting cells

convert formate, H2-CO2, and O-methyl groups of vanillate to

acetate at stoichiometries indicative of acetogenesis; the

aro-matic ring of vanillate remains intact (Kamlage et al 1997)

Resting cells of C coccoides strain 1410 convert glucose to

ace-tate, succinate, andD-lactate

Clostridium difficile Five acetogenic strains of C difficile

were isolated from the rumen of newborn lambs; strain AA1 is

considered as a representative strain (Rieu-Lesme et al.1998in

this chapter) No acetogenic potentials have been documented

for the type strain of C difficile Cells of C difficile strain AA1 are

Gram-positive, sporeforming, giant filamentous rods Growth

of strain AA1 is supported by H-CO, fructose, glucose,

cellobiose, maltose, mannose, and syringate Acetate is the soleproduct from H2-CO2, and the substrate/product stoichiometry

is indicative of acetogenesis; however, glucose and fructose aremetabolized to almost equal amounts of acetate and butyrate,and small amounts of ethanol and isovalerate (Rieu-Lesme et al

1998)

Clostridium formicoaceticum The first strain of

C formicoaceticum was probably isolated from pond sediment

by El Ghazzawi (1967) Although the organism was called tridium aceticum in the title of the German publication, ElGhazzawi stated that his isolate differed from C aceticum andtentatively named his organism ‘‘Clostridium formicoaceticum’’because it produced both formate and acetate (El Ghazzawi

Clos-1967) The type strain of C formicoaceticum was isolated fromsewage sludge (Andreesen et al.1970) Cells are Gram-negative,sporeforming, motile, straight or slightly curved rods The range

of substrates is very similar to that of C aceticum (see above) butalso includes glycerol, gluconate, glucuronate, and glycerate(Andreesen et al 1970) Clostridium formicoaceticum can bedifferentiated from C aceticum by its inability to grow with

H2-CO2 and its ability to grow with methanol and lactate(Andreesen et al 1970; Lux and Drake 1992) As with

C aceticum, the utilization of fumarate by C formicoaceticumdoes not involve the acetyl-CoA pathway; fumarate isdismutated to acetate and succinate (Dorn et al.1978) Fuma-rate can also serve as an alternative electron acceptor (Matthies

et al 1993), and N2is fixed (Bogdahn et al.1983) Reductantderived from the oxidation of the aldehyde groups of certainaromatic compounds (e.g., 4-hydroxybenzaldehyde) is growthsupportive (Go¨ßner et al.1994), preferentially used in the acetyl-CoA pathway, and inhibits the use of fructose (Frank et al 1998).Clostridium glycolicum Two acetogenic strains of

C glycolicum have been isolated Strain 22 was isolated fromsewage sludge, grows on H2-CO2, and produces mainly acetate;cells are Gram-positive rods that form oval, subterminal spores(Ohwaki and Hungate1977) Strain 22 has been deposited at theAmerican Type Culture Collection (ATCC) and has been iden-tified as a strain of Clostridium glycolicum; however, the 16SrRNA gene sequence is not available (per information from theATCC Bacteriology Program) Strain RD-1 was isolated from seagrass roots and was identified as an acetogenic strain of

C glycolicum by analysis of the 16S rRNA gene sequence (Ku¨sel

et al 2001) Cells of strain RD-1 are Gram-positive,sporeforming, motile rods that can be linked by connectingfilaments Growth-supportive substrates of strain RD-1 include

H2-CO2, formate, pyruvate, lactate, ethylene glycol, and certainsugars Except for growth on sugars and ethylene glycol, acetate

is the sole reduced end product Strain RD-1 is aerotolerant andgrows at O2concentrations of up to 6 % in the headspace ofstatic liquid cultures and up to 4 % in the headspace of shakenliquid cultures; ethanol, lactate, and H2 are the reducedend products under oxic conditions (Ku¨sel et al.2001; see thesection on >‘‘Tolerance to Oxic Conditions and Metabolism

of O2’’) No acetogenic potentials have been found for thetype strain of C glycolicum (Gaston and Stadtman 1963;Ku¨sel et al.2001)

16 1 Acetogenic Prokaryotes

Clostridium ljungdahlii This organism was isolated from

chicken manure/waste (Barik et al 1988; Tanner et al 1993)

Cells are Gram-positive, sporeforming, motile rods (>Fig 1.6)

The organism grows autotrophically on H2-CO2and CO;

het-erotrophic growth occurs on formate, ethanol, pyruvate,

fuma-rate, and sugars (including fructose and xylose; Tanner et al

1993) The sole product from H2-CO2and fructose is acetate;

however, from synthesis gas (a mixture of H2, CO, and CO2),

acetate and ethanol are produced (Tanner et al.1993; Phillips

et al.1994) Nitrate is reduced to ammonium; however, unlike

the dissimilation of nitrate by M thermoacetica (see the sections

on >‘‘Use of Diverse Terminal Electron Acceptors’’ and

>‘‘Regulation of the Acetyl-CoA Pathway and Other Metabolic

Abilities’’in this chapter), the reduction of nitrate does not have

a regulatory effect on acetogenesis and likewise does not enhance

the growth of the organism (Seifritz et al.1993; Fro¨stl et al 1996;

Laopaiboon and Tanner1999)

Clostridium magnum This species was isolated from

pas-teurized freshwater sediment (Schink 1984) Cells are

Gram-positive, sporeforming, motile, large straight rods H2-CO2,

formate, methanol, 2,3-butandiol, acetoin, malate, citrate, and

a few sugars are substrates, and acetate is the sole reduced end

product N2is fixed (Bomar et al.1991), and small amounts of

O2are tolerated and consumed (Karnholz et al.2002)

Clostridium mayombei This organism was isolated from the

gut of a soil-feeding termite (Kane et al 1991b) Cells are

Gram-positive, sporeforming, motile, straight rods Growth occurs on

H2-CO2, sugars, sugar alcohols, organic acids, and amino acids

The main reduced end product is acetate; however, succinate is

metabolized to CO2and propionate (Kane et al 1991b)

Clostridium methoxybenzovorans This species was isolated

from an olive mill wastewater digester (Mechichi et al 1999)

Cells of C methoxybenzovorans are Gram-positive,

sporeforming, nonmotile rods Growth occurs on H-CO,

methanol, lactate, sugars, methoxylated aromatic compounds,betaine, dimethylglycine, dimethylsulfide, casaminoacids, andpeptone H2-CO2is metabolized to acetate and formate Metab-olism of betaine, dimethylglycine, and dimethylsulfide yieldsacetate, and sugars are metabolized to acetate, formate, ethanol,

H2, and CO2 O-methyl groups, methanol, and lactate aremetabolized to acetate and butyrate (Mechichi et al 1999).Since no substrate/product stoichiometries have been reportedfor the organism, the acetogenic utilization of most substrates isuncertain

Clostridium scatologenes An acetogenic strain of

C scatologenes (SL1) was isolated from sediment of an acidiccoal mine pond (Ku¨sel et al 2000) The type strain of

C scatologenes was isolated from soil and was not originallydescribed as an acetogen (Holdeman et al 1977) However,both strain SL1 and the type strain utilize H2and CO with theconcomitant production of acetate, and cell extracts of bothorganisms have CO dehydrogenase, hydrogenase, and formatedehydrogenase activities (Ku¨sel et al 2000) Cells are Gram-positive, sporeforming, motile, long rods and produce skatole,

a dung odor component (Holdeman et al 1977; Ku¨sel et al

2000) Substrates include fructose, arabinose, ethanol, formate,vanillate, H2-CO2, and CO The major reduced end product isacetate However, in addition to acetate, butyrate and traces of

H2are also produced from sugars (Ku¨sel et al.2000)

Clostridium ultunense This species was isolated from ananaerobic acetate-oxidizing triculture that was enriched from

a digester fed with swine manure (Schnu¨rer et al.1994,1996).Cells are Gram-positive, sporeforming rods that change cell size,cell form, and motility during growth The only known growth-supportive substrates are formate, betaine, glucose, pyruvate,ethylene glycol, and cysteine The main end products are acetate,formate, and traces of H2 H2-CO2does not support growth;however, H2-CO2 is converted to acetate by resting cells(Schnu¨rer et al 1996) Acetate is oxidized in coculture with

a methanogen, and the oxidation of acetate appears to occurvia a reversal of the acetyl-CoA pathway (Schnu¨rer et al.1997)

An acetogen (strain AOR) that also oxidized acetate in coculturewith a methanogen was previously isolated (Lee and Zinder

1988); however, this strain has been lost (S.H Zinder, personalcommunication)

Eubacterium aggregans This organism was isolated from anolive mill wastewater digestor (Mechichi et al.1998) Cells areGram-positive, nonsporeforming, nonmotile rods that formaggregates Substrates include H2-CO2, glucose, fructose,sucrose, lactate, formate, methanol, betaine, and numerousmethoxylated aromatic compounds Although E aggregans isdescribed as homoacetogenic, H2, formate, acetate, and butyrateare produced from sugars (Mechichi et al.1998) Acetate is thesole reduced end product with formate and methanol.Methoxylated aromatic compounds are O-demethylated, andacetate, butyrate, and the corresponding hydroxylated aromaticcompounds are formed Aldehyde groups of methoxylated aro-matic compounds are oxidized to carboxylate groups

Eubacterium limosum This species was isolated from sheeprumen and digester sludge (Sharak Genthner et al.1981) Cells

Fig 1.6

Electron micrograph of cells from a young culture (16 h, fructose

grown) of Clostridium ljungdahlii (ATCC 55383 T ) with

peritrichously inserted flagella Bar equals 1 mm (The micrograph

was kindly provided by R.S Tanner)

Acetogenic Prokaryotes 1 17

are Gram-positive, nonsporeforming, nonmotile straight rods

that become more pleomorphic after prolonged incubation

Eubacterium limosum is metabolically very versatile; its substrate

range includes sugars, amino acids, methoxylated aromatic

compounds, glycine, betaine, lactate, methanol, H2-CO2, and

CO (Sharak Genthner et al.1981; Sharak Genthner and Bryant

1982,1987; Jansen and Hansen2001) Both acetate and butyrate

are produced from one-carbon compounds (Sharak Genthner

et al 1981; Pacaud et al 1985; see ‘‘Butyribacterium

methylotrophicum,’’ above) Cultures demethylate the osmolytes

dimethylsulfoniopropionate and glycine-betaine to

methylthio-propionate and dimethylglycine, respectively; however, only

the demethylation of glycine-betaine supports growth of the

organism (Jansen and Hansen2001)

Holophaga foetida This organism (strain TMBS4) was

isolated from freshwater sediment (Bak et al 1992;

Liesack et al.1994) Cells are Gram-negative, nonsporeforming,

nonmotile rods The substrate range is rather small and mainly

consists of pyruvate and aromatic compounds, especially

meth-ylated and nonmethmeth-ylated trihydroxybenzenes Acetate is the

main reduced end product In contrast to other acetogens,

H foetida degrades aromatic rings to acetate (Bak et al.1992;

Kreft and Schink1993) Dimethylsulfide and methanediol are

produced from methoxylated aromatic compounds when cells

are cultured in sulfide-containing media, indicating that sulfide

can serve as a methyl acceptor (Bak et al.1992) CO2and CO can

also be used as methyl acceptors with the subsequent formation

of acetate CO dehydrogenase activity is present in cells grown

on methoxylated aromatic compounds (Kreft and Schink1993)

Holophaga foetida occupies a fairly isolated position in the

phylogenetic tree of the bacteria (Liesack et al.1994,1997; see

the section on>‘‘Taxonomy and Phylogeny’’)

Moorella glycerini This species is a thermophilic acetogen

and was isolated from the sediment of a hot spring at

Yellowstone National Park (Slobodkin et al 1997) The cells

are Gram-positive, sporeforming, motile, straight rods Growth

is supported by glycerol, sugars, lactate, glycerate, pyruvate, and

yeast extract; however, H2-CO2 is not growth supportive

Acetate is the only product from glycerol and glucose Fumarate

is reduced to succinate, and the reduction of thiosulfate yields

elemental sulfur Nitrate is not dissimilated Optimum growth

occurs at 58C

Moorella mulderi This organism is a thermophilic acetogen

and was isolated from a high-temperature bioreactor (Balk et al

2003) The cells are Gram-positive, sporeforming rods Growth

is supported by H2-CO2, formate, methanol, hexoses, cellobiose,

lactate, and pyruvate The reduction of thiosulfate yields sulfide

Nitrate is not dissimilated

Moorella thermoacetica This organism is a thermophilic

acetogen that was isolated from horse manure and was first

described as Clostridium thermoaceticum (Fontaine et al 1942)

On the basis of phylogenetic analysis of the 16S rRNA gene

sequence, C thermoaceticum was reclassified as M thermoacetica

(Collins et al 1994) Although the organism was originally

isolated from horse manure, the organism is a common

inhab-itant of soils (Go¨ßner and Drake1997; Go¨ßner et al.1998,1999;

Karita et al.2003) Cells are Gram-variable, sporeforming, iably motile, straight rods (>Fig 1.1) The optimum tempera-ture of growth is 55–60 C (Fontaine et al 1942), and thevitamin nicotinic acid is required for growth (Lundie andDrake 1984) Moorella thermoacetica was the first bacteriumthat was shown to produce 3 moles of acetate from 1 mole ofhexose (Fontaine et al 1942) and is one of the most metaboli-cally robust acetogens characterized to date Moorellathermoacetica was originally isolated as an obligate heterotroph(Fontaine et al 1942), but nearly five decades later, it was shown

var-to be capable of auvar-totrophic growth (Daniel et al.1990) Thisbacterium displays very diverse physiological capabilities (Drakeand Daniel 2004) Growth-supportive substrates include CO,

H2-CO2, formate, methanol, hexoses, pentoses, methoxylatedbenzoic acids, and several two-carbon compounds (e.g., oxalate,glycolate, and glyoxylate; Fontaine et al 1942; Daniel et al.1990,

2004; Daniel and Drake1993; Drake et al 1997; Seifritz et al

1999; Kim et al.2002) Carboxyl groups of aromatic compoundscan serve as CO2equivalents in the acetyl-CoA pathway (Hsu

et al.1990a,b) Thiosulfate (Beaty and Ljungdahl1990,1991),nitrate (Seifritz et al.1993), and nitrite (Seifritz et al.2003) serve

as alternative electron acceptors Nitrate is dissimilated to bothnitrite and ammonium, and nitrite is dissimilated to ammo-nium Ethanol and n-propanol are oxidized and aregrowth-supportive substrates when nitrate is dissimilated; nei-ther ethanol nor n-propanol is utilized as an acetogenic substrate(Fro¨stl et al 1996) Reductively dechlorinates carbon tetrachlo-ride (Egli et al.1988) Tolerates and consumes small amounts ofoxygen (Karnholz et al.2002) A recent isolate that is phyloge-netically nearly identical to M thermoacetica is cellulolytic(Karita et al.2003) Moorella thermoacetica is the most studiedacetogen, and the enzymology of the acetyl-CoA pathway wasresolved with this organism (see the section on >‘‘HistoricalPerspectives’’and>Table 1.1in this chapter)

Moorella thermoautotrophica This organism is

a thermophilic acetogen that was isolated from a hot spring atYellowstone National Park and was first described as Clostridiumthermoautotrophicum (Wiegel et al 1981) On the basis ofphylogenetic analysis of the 16S rRNA gene sequence,

C thermoautotrophicum was reclassified as M thermoautotrophica(Collins et al 1994) Cells are Gram-variable, sporeforming,motile rods (Wiegel et al.1981) Moorella thermoautotrophicawas initially described as being metabolically distinct from theclosely related M thermoacetica (Collins et al.1994); this dis-tinction was primarily based on the H2-dependent acetogenicabilities of the former bacterium (Wiegel et al.1981) However,later studies demonstrated that M thermoacetica growschemolithoautotrophically on H2-CO2 (Daniel et al 1990).Both of these species of Moorella display a similar substraterange Both species also require the vitamin nicotinic acid forgrowth (Lundie and Drake1984; Savage and Drake1986) Thesubstrate range of M thermoautotrophica includes H2-CO2, CO,formate, methanol, glucose, fructose, glycerate, glycolate, andmethoxylated aromatic compounds (Wiegel et al 1981; Fro¨stl

et al 1996; Seifritz et al.1999) Nitrate is utilized as an alternativeelectron acceptor and is dissimilated to nitrite and ammonium;

18 1 Acetogenic Prokaryotes

ethanol and n-propanol are growth-supportive substrates only

when nitrate is available for dissimilation (Fro¨stl et al 1996)

Natroniella acetigena This organism is a haloalkaliphilic

acetogen and was isolated from the soda deposits at Lake

Magadi, Kenya (Zhilina et al.1996) Cells are Gram-negative,

sporeforming, motile, large rods The substrate range is limited

and includes lactate, pyruvate, ethanol, glutamate, and

propanol Growth does not occur on H2-CO2 or CO-CO2

Acetate is the sole reduced end product Propionate is formed

during growth on propanol The optimal pH is 10, and the

optimal salinity for growth is 12 % NaCl (w/v)

Natronincola histidinovorans This species is a moderately

haloalkaliphilic acetogen and was isolated from soda deposits

at Lake Magadi, Kenya (Zhilina et al 1998) Cells are

Gram-positive, motile rods; sporeforming and nonsporeforming strains

have been isolated Natronincola histidinovorans is specialized in

using amino acids (histidine, glutamate, and casaminoacids) as

sources of energy Neither H2-CO2 nor CO-CO2 support

growth Optimal growth occurs at pH 9 and a salinity of 9 %

NaCl Acetate and ammonium are the main end products

Oxobacter pfennigii This organism was isolated from the

rumen fluid of a steer and was first described as Clostridium

pfennigii (Krumholz and Bryant1985) On the basis of

phyloge-netic analysis of the 16S rRNA gene sequence, C pfennigii was

reclassified as O pfennigii (Collins et al.1994) Cells are

Gram-positive, motile, sporeforming, slightly curved rods Substrates

include CO, pyruvate, vanillate, vanillin, ferulate, syringate, and

trimethoxybenzoate In contrast to most other acetogens, acetate

is not produced from methoxybenzenoids (O-methyl groups

are utilized, and butyrate and the respective hydroxybenzenoids

are formed; Krumholz and Bryant1985) During growth on CO

or pyruvate, acetate is formed in addition to butyrate or is the

sole product, respectively

Ruminococcus hydrogenotrophicus This species is

a nonsporeforming coccobacillus that was isolated from

human feces (Bernalier et al.1996c) Ruminococcus

hydrogeno-trophicus grows on H2-CO2, formate, pyruvate, and several

sugars Acetate is the sole product from H2-CO2-dependent

growth; however, glucose and fructose are metabolized to

ace-tate, lacace-tate, ethanol, and small amounts of isobutyrate and

isovalerate (Bernalier et al 1996c) Thus, the metabolism of

sugars involves several fermentative processes

Ruminococcus productus This organism was originally

iso-lated from various mammalian gastrointestinal tracts and was

described as Peptostreptococcus productus; the original isolates

were not described as acetogens (Moore and Holdeman1974;

Varel et al.1974; Holdeman-Moore et al 1986) On the basis

of phylogenetic analysis of the 16S rRNA gene sequence,

P productus was reclassified as R productus (Ezaki et al.1994)

Two acetogenic strains (strain U-1 [ATCC 35244] and strain

Marburg [ATCC 43917]) of R productus have been isolated

from sewage sludge (Lorowitz and Bryant1984; Geerligs et al

1987) Cells are Gram-positive, nonsporeforming, nonmotile

elongated cocci occuring often in pairs or chains (Lorowitz

and Bryant1984; Holdeman-Moore et al.1986; Geerligs et al

1987) Growth-supportive substrates of the acetogenic strains

include CO, H2-CO2, monomeric and dimeric sugars, andmethoxylated aromatic compounds; growth is particularlygood on CO (Lorowitz and Bryant 1984; Geerligs et al.1987;Parekh et al.1992) The acrylate side chain of methoxylated andnonmethoxylated phenylacrylates can be used as alternativeelectron acceptor (Parekh et al 1992; Misoph et al 1996b).The major reduced end product is acetate; however, under

CO2-limited conditions or when substrate concentrations arehigh (e.g., 10 mM fructose), lactate, succinate, and formate arealso formed (Misoph and Drake 1996a)

Ruminococcus schinkii This organism was isolated fromrumen content of 1–3-day-old lambs (Rieu-Lesme et al

1996b) Cells are Gram-positive, nonsporeforming, nonmotilecocci Substrates include H2-CO2, various sugars, glycerol,syringate, and ferulate Acetate is the sole reduced end product.Sporomusa acidovorans This species was isolated from

a distillation wastewater fermentor (Ollivier et al.1985a) Cellsare Gram-negative, sporeforming, motile, curved rods Growth-supportive substrates mainly include organic acids, H2-CO2,methanol, glycerol, and a few sugars; acetate is the sole reducedend product with all substrates

Sporomusa aerivorans This organism was isolated from

a soil-feeding termite (Boga et al.2003) Cells are tive, sporeforming, motile, curved rods Growth-supportivesubstrates include H2-CO2, formate, methanol, ethanol, lactate,pyruvate, mannitol, citrate, and various methoxylated aromaticcompounds; hexoses are not utilized Cells tolerate and consumesmall amounts of oxygen and are catalase positive (Boga andBrune2003)

Gram-nega-Sporomusa malonica This species was isolated fromfreshwater sediment (Dehning et al.1989) Cells are Gram-neg-ative, sporeforming, motile, curved rods The organism exhibits

a very versatile metabolism and utilizes H2-CO2and numerousorganic compounds, including formate, pyruvate, alcohols,dicarboxylic acids, fructose, and trimethoxycinnamate Acetate

is the reduced end product when typical acetogenic substratessuch as H2-CO2, formate, methanol, fructose, pyruvate, or theO-methyl groups of trimethoxycinnamate are metabolized(Dehning et al.1989) Alcohols yield acetate and the respectivefatty acids, and crotonate and 3-hydroxybutyrate yield acetateand butyrate As with relatively few anaerobes, S malonicametabolizes simple dicarboxylic acids (e.g., malonate and succi-nate) by decarboxylation to the respective fatty acids

Sporomusa ovata This organism was isolated from sugarbeet leaf silage (Mo¨ller et al.1984) Cells are Gram-negative,sporeforming, motile, curved rods Growth is supported by

a variety of substrates including H2-CO2, pyruvate, lactate, hols, fructose, betaine, dimethylglycine, and sarcosine Acetate isthe sole reduced end product; methylamines are formed fromN-methyl compounds Reductively dechlorinates tetrachlor-oethylene to trichloroethylene (Terzenbach and Blaut 1994).Cultures demethylate the osmolytes dimethylsulfonio-propionate and glycine-betaine to methylthiopropionate anddimethylglycine, respectively; however, only the demethylation

alco-of glycine-betaine supports growth alco-of the organism (Jansen andHansen2001)

Acetogenic Prokaryotes 1 19

Sporomusa paucivorans This species was isolated from lake

sediment (Hermann et al 1987) Cells are Gram-negative,

nonsporeforming, motile, slightly curved rods H2-CO2,

for-mate, methanol, pyruvate, serine, betaine, alcohols, and ethylene

glycol support growth Acetate is the sole reduced end product

Oxidation of alcohols yields the corresponding fatty acids

Sugars are not utilized

Sporomusa silvacetica This organism was isolated from

for-est soil (Kuhner et al 1997) Cells are Gram-negative,

sporeforming, motile, slightly curved rods (>Fig 1.7) Growth

occurs on H2-CO2, formate, methanol, pyruvate, vanillate,

ferulate, fructose, betaine, fumarate, 2,3-butanediol, ethanol,

lactate, and glycerol With most substrates, acetate is the main

reduced end product Fumarate is dismutated to acetate and

succinate Vanillate and ferulate are O-demethylated and

reduced, respectively Cells tolerate and consume small amounts

of oxygen (Karnholz et al.2002)

Sporomusa sphaeroides This species was isolated from river

mud (Mo¨ller et al 1984) Cells are Gram-negative,

sporeforming, motile, curved rods Growth occurs on H2-CO2,

pyruvate, lactate, alcohols, glycerol, serine, ethyleneglycol,

beta-ine, and other N-methyl compounds Acetate is the sole reduced

end product; methylamines are formed from N-methyl

com-pounds Cultures demethylate the osmolytes

dimethylsulfonio-propionate and glycine-betaine to methylthiodimethylsulfonio-propionate and

dimethylglycine, respectively; however, only the demethylation

of glycine-betaine supports growth of the organism (Jansen andHansen2001)

Sporomusa termitida This organism was isolated from thegut of a wood-feeding termite (Breznak et al 1988) Cells of

S termitida are Gram-negative, sporeforming, motile, straight

to slightly curved rods Substrates include H2-CO2, CO, formate,methanol, ethanol, betaine, sarcosine, lactate, pyruvate, oxalo-acetate, citrate, malonate, succinate, mannitol, and trimethox-ybenzoate Acetate is the main reduced end product As with

S malonica, S termitida decarboxylates succinate to propionate(Breznak et al.1988; Dehning et al.1989) Sporomusa termitidagrows mixotrophically, e.g., by utilizing H2 and methanol orlactate at the same time (Breznak and Switzer Blum1991).Syntrophococcus sucromutans This organism is a Gram-negative, nonsporeforming, nonmotile, coccoid bacteriumthat was isolated as a dominant methoxybenzenoids utilizerfrom the rumen contents of a steer (Krumholz and Bryant

1986) Syntrophococcus sucromutans has a unique metabolism:growth with carbohydrates or pyruvate is only possible in thepresence of electron acceptors such as formate, O-methyl groups,

or a hydrogenotrophic methanogen (Krumholz and Bryant

1986) Formate and O-methyl groups are metabolized via anacetyl-CoA pathway that lacks formate dehydrogenase and istherefore incomplete (Dore´ and Bryant1990)

Thermoacetogenium phaeum This species is

a thermophilic acetogen that was isolated from an anoxicpulp wastewater reactor (Hattori et al 2000) Cells areGram-positive, sporeforming, nonmotile, straight or slightlycurved rods Substrates include H2-CO2, formate, methanol,n-propanol, methoxylated benzoic acids, glycine, andcysteine Acetate is the sole reduced end product Acetate isoxidized in the presence of hydrogenotrophic methanogens or

an alternative electron acceptor (e.g., sulfate or thiosulfate);concomitantly, methane is produced by the syntrophicmethanogen or the alternative electron acceptor is reduced Itsability to oxidize acetate in syntrophic association withhydrogenotrophic methanogens is similar to that of two otheranaerobic acetate oxidizers, strain AOR and Clostridiumultunense (Zinder and Koch 1984; Lee and Zinder 1988;Schnu¨rer et al.1996)

Thermoanaerobacter kivui This species is a thermophilicacetogen that was isolated from lake sediments of Lake Kivu,Africa, and was first described as Acetogenium kivui (Leigh et al

1981) On the basis of phylogenetic analysis of the 16S rRNAgene sequence, A kivui was reclassified as T kivui (Rainey et al

1993; Collins et al.1994) Cells are nonmotile, nonsporeformingrods often occurring in pairs or chains (Leigh et al 1981;

>Fig 1.8) The cell wall is covered by a hexagonally structuredS-layer consisting of an 80-kDa protein (Rasch et al.1984; Lupas

et al 1994) The temperature optimum is 66C Autotrophicgrowth occurs on H2-CO2, and heterotrophic growth occurs onglucose, mannose, fructose, pyruvate, and formate; acetate is themain reduced end product (Leigh et al.1981) Growth does notoccur on CO-CO2 (Daniel et al 1990) Thermoanaerobacterkivui grows robustly on H -CO, a substrate that yields very

Fig 1.7

Electron micrograph of a vegetative cell of Sporomusa silvacetica

(DSM 10669 T ) showing flagella inserting at the concave side of

the cell [From Kuhner et al ( 1997 ), used with permission from

International Union of Microbiological Societies]

20 1 Acetogenic Prokaryotes