Introduction to Geomicrobiology

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In memory of my mother

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Introduction toGeomicrobiology

Kurt Konhauser

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© 2007 Blackwell Science Ltda Blackwell Publishing company

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1 2007

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Konhauser, Kurt.Introduction to geomicrobiology / Kurt Konhauser.

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v

Contents

Preface, ix

1 Microbial properties and diversity, 11.1 Classification of life, 11.2 Physical properties of

microorganisms, 51.2.1 Prokaryotes, 51.2.2 Eukaryotes, 8

1.3 Requirements for growth, 101.3.1 Physical requirements, 101.3.2 Chemical requirements, 111.3.3 Growth rates, 17

1.4 Microbial diversity, 181.5 Life in extreme environments, 22

1.5.1 Hydrothermal systems, 231.5.2 Polar environments, 261.5.3 Acid environments, 281.5.4 Hypersaline and alkaline

environments, 291.5.5 Deep-subsurface

environments, 301.5.6 Life on other planets, 321.5.7 Panspermia, 34

1.6 Summary, 35

2 Microbial metabolism, 362.1 Bioenergetics, 36

2.1.1 Enzymes, 362.1.2 Oxidation-reduction, 372.1.3 ATP generation, 422.1.4 Chemiosmosis, 43

2.2 Photosynthesis, 472.2.1 Pigments, 472.2.2 The light reactions –

anoxygenic photosynthesis, 49

2.2.3 Classification of anoxygenic photosynthetic bacteria, 51

2.2.4 The light reactions – oxygenic photosynthesis, 54

2.2.5 The dark reactions, 562.2.6 Nitrogen fixation, 57

2.3 Catabolic processes, 582.3.1 Glycolysis and

fermentation, 592.3.2 Respiration, 61

2.4 Chemoheterotrophic pathways, 652.4.1 Aerobic respiration, 652.4.2 Dissimilatory nitrate

reduction, 662.4.3 Dissimilatory manganese

reduction, 672.4.4 Dissimilatory iron

reduction, 692.4.5 Trace metal and metalloid

reductions, 722.4.6 Dissimilatory sulfate

reduction, 742.4.7 Methanogenesis and

homoacetogenesis, 772.5 Chemolithoautotrophic

pathways, 792.5.1 Hydrogen oxidizers, 792.5.2 Homoacetogens and

methanogens, 812.5.3 Methylotrophs, 822.5.4 Sulfur oxidizers, 842.5.5 Iron oxidizers, 862.5.6 Manganese oxidizers, 892.5.7 Nitrogen oxidizers, 91

2.6 Summary, 92

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vi CONTENTS

3 Cell surface reactivity and metal sorption, 933.1 The cell envelope, 93

3.1.1 Bacterial cell walls, 933.1.2 Bacterial surface layers, 973.1.3 Archaeal cell walls, 1003.1.4 Eukaryotic cell walls, 100

3.2 Microbial surface charge, 1013.2.1 Acid–base chemistry of

microbial surfaces, 1013.2.2 Electrophoretic mobility, 1043.2.3 Chemical equilibrium

models, 1053.3 Passive metal adsorption, 108

3.3.1 Metal adsorption to bacteria, 108

3.3.2 Metal adsorption to eukaryotes, 111

3.3.3 Metal cation partitioning, 1123.3.4 Competition with

anions, 1143.4 Active metal adsorption, 114

3.4.1 Surface stability requirements, 115

3.4.2 Metal binding to microbial exudates, 116

3.5 Bacterial metal sorption models, 1193.5.1 Kd coefficients, 1193.5.2 Freundlich isotherms, 1203.5.3 Langmuir isotherms, 1213.5.4 Surface complexation

models (SCM), 1223.5.5 Does a generalized sorption

model exist?, 1243.6 The microbial role in contaminant

mobility, 1263.6.1 Microbial sorption to

solid surfaces, 1273.6.2 Microbial transport through

porous media, 1313.7 Industrial applications based on

microbial surface reactivity, 1333.7.1 Bioremediation, 1333.7.2 Biorecovery, 136

3.8 Summary, 138

4 Biomineralization, 1394.1 Biologically induced

mineralization, 1394.1.1 Mineral nucleation and

growth, 1394.1.2 Iron hydroxides, 1434.1.3 Magnetite, 1494.1.4 Manganese oxides, 1504.1.5 Clays, 1534.1.6 Amorphous silica, 1564.1.7 Carbonates, 1604.1.8 Phosphates, 1664.1.9 Sulfates, 1694.1.10 Sulfide minerals, 171

4.2 Biologically controlled mineralization, 1744.2.1 Magnetite, 1744.2.2 Greigite, 1784.2.3 Amorphous silica, 1794.2.4 Calcite, 183

4.3 Fossilization, 1854.3.1 Silicification, 1864.3.2 Other authigenic

minerals, 1894.4 Summary, 191

5 Microbial weathering, 1925.1 Mineral dissolution, 192

5.1.1 Reactivity at mineral surfaces, 192

5.1.2 Microbial colonization and organic reactions, 195

5.1.3 Silicate weathering, 2005.1.4 Carbonate weathering, 2055.1.5 Soil formation, 2065.1.6 Weathering and global

climate, 2095.2 Sulfide oxidation, 211

5.2.1 Pyrite oxidation mechanisms, 211

5.2.2 Biological role in pyrite oxidation, 215

5.2.3 Bioleaching, 2235.2.4 Biooxidation of refractory

gold, 229

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CONTENTS vii

5.3 Microbial corrosion, 2305.3.1 Chemolithoautotrophs, 2315.3.2 Chemoheterotrophs, 2325.3.3 Fungi, 234

5.4 Summary, 234

6 Microbial zonation, 2356.1 Microbial mats, 235

6.1.1 Mat development, 2366.1.2 Photosynthetic mats, 2406.1.3 Chemolithoautotrophic

mats, 2466.1.4 Biosedimentary structures, 249

6.2 Marine sediments, 2596.2.1 Organic sedimentation, 2606.2.2 An overview of sediment

diagenesis, 2626.2.3 Oxic sediments, 2656.2.4 Suboxic sediments, 2666.2.5 Anoxic sediments, 2726.2.6 Preservation of organic

carbon into the sedimentary record, 280

6.2.7 Diagenetic mineralization, 2836.2.8 Sediment hydrogen

concentrations, 2876.2.9 Problems with the

biogeochemical zone scheme, 288

6.3 Summary, 292

7 Early microbial life, 2937.1 The prebiotic Earth, 293

7.1.1 The Hadean environment, 2947.1.2 Origins of life, 2967.1.3 Mineral templates, 301

7.2 The first cellular life forms, 3057.2.1 The chemolithoautotrophs, 3057.2.2 Deepest-branching Bacteria

and Archaea, 3097.2.3 The fermenters and initial

respirers, 3117.3 Evolution of photosynthesis, 312

7.3.1 Early phototrophs, 3127.3.2 Photosynthetic expansion, 3197.3.3 The cyanobacteria, 323

7.4 Metabolic diversification, 3277.4.1 Obligately anaerobic

respirers, 3277.4.2 Continental platforms as

habitats, 3317.4.3 Aerobic respiratory

pathways, 3347.5 Earth’s oxygenation, 340

7.5.1 The changing Proterozoic environment, 340

7.5.2 Eukaryote evolution, 3457.6 Summary, 349

References, 350Index, 406

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Preface

The past three decades have seen enormousadvances in our understanding of how microbiallife impacts the Earth. Microorganisms inhabitalmost every conceivable environment on theplanet’s surface, and extend the biosphere todepths of several kilometers into the crust. Signi-ficantly, the chemical reactivity and metabolicdiversity displayed by those communities makesthem integral components of global elementalcycles, from mineral dissolution and precipita-tion reactions, to aqueous redox processes. Inthis regard, microorganisms have helped shapeour planet over the past 4 billion years and madeit habitable for higher forms of life.

Introduction to Geomicrobiology was written inresponse to the need for a single, comprehensivebook that describes our current knowledge ofhow microbial communities have influenced biogeochemical and mineralogical processesthrough time. Though other books on geomicro-biology exist, they are either geared moretowards the physiological rather than the geo-logical aspects, or they are edited compilationswhere the individual papers are highly focusedon select topics. This has limited the usefulnessof those resources in teaching geomicrobiologyto my students. Consequently, Introduction toGeomicrobiology has taken a different approach.It is process-oriented, with one pervasive themethroughout, that being the geological conse-quences of microbial activity. I take an inter-disciplinary and “global” view on the issues, and although the chapters can stand alone, they are linked through a natural progression

of microorganism–environment interactions inmodern ecosystems, concluding with a synopsisof how the biosphere evolved during thePrecambrian. Designed primarily as a core textfor upper undergraduate/graduate students inearth and biological sciences, Introduction toGeomicrobiology is also rich in references, withthe aim being to provide the reader with a usefulstarting point for further study. In doing this, particular attention has been paid to making this book a useful resource for researchers from a number of other scientific backgrounds.

Introduction to Geomicrobiology covers the following topics:

n How microorganisms are classified, the physical constraints governing their growth, molecularapproaches to studying microbial diversity, and lifein extreme environments.

n Bioenergetics, microbial metabolic capabilities, andmajor biogeochemical pathways.

n Chemical reactivity of the cell surface, metal sorp-tion, and the microbial role in contaminant mobilityand bioremediation/biorecovery.

n Microbiological mineral formation and fossilization.

n The function of microorganisms in mineral dissolu-tion and oxidation, and the industrial and environ-mental ramifications of these processes.

n Elemental cycling in biofilms, formation of micro-bialites, and sediment diagenesis.

n The events that led to the emergence of life, evolutionof metabolic processes, and the diversification of thebiosphere.

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x PREFACE

One feature this book does not have is a methodssection. There are many techniques employed in such an interdisciplinary science, and it is myfeeling that since they are constantly evolving itis more appropriate for the reader to consult themost recent articles where the methodologies are fully explained.

The preparation of this book was greatly aided by discussions and reviews with a num-ber of colleagues. They include Jill Banfield,Roger Buick, Jeremy Fein, Bill Inskeep, AndreasKappler, Jon Lloyd, Anna-Louise Reysenbach,Sam Smith, Gordon Southam, and Nathan Yee.Special thanks go out to two of my students,Stefan Lalonde and Larry Amskold, for their help

with figures and proofreading. I am also indebtedto a number of other colleagues and publishers formaking available original photographs or allow-ing reproduction of previously published materialfor illustration. Lastly, but most importantly, I owemy wife and son many hours of undivided atten-tion for all the time missed during the writing ofthis book.

I am grateful to all the individuals and pub-lishers who have given permission to reproducematerial in this book.

Kurt O. KonhauserUniversity of Alberta, Edmonton

For instructors, if you did not receive an artwork CD-ROM with your comp copy, please contact thisemail address: artworkcd@bos.blackwellpublishing.com

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Since their origin, perhaps some 4 billion yearsago, microorganisms have had a profound influ-ence on shaping our planet. From localized niches,that occur on the order of micrometers, to eco-systems as immense as the oceans, microorgan-isms are intimately involved in transforminginorganic and organic compounds to meet theirnutritional and energetic needs. Because themetabolic waste from one type of species nearlyalways provides substrates for another, there is a continuous recycling of elements throughoutthe biosphere. This interdependence can existbetween species growing in close proximity toone another, where any number of sorption, precipitation, and redox reactions inevitably create unique community-specific biogeochemicaland mineralogical signatures. Alternatively, thecommunities can be spatially separated, and elemental cycling may take on more complexand convoluted pathways, such as the transfer of metabolites across the sediment–water inter-face or from the ocean water column to theatmosphere. The latter examples are particularlyimportant for global-scale cycling of carbon,nitrogen, sulfur, and oxygen. Given sufficienttime, the collective metabolic activities of count-less microbial communities can even modify thedynamics of the entire Earth, controlling thecomposition of the air we breathe, the water wedrink, and the soils in which we grow plants. So intimate is this relationship that in order todiscover a part of the planet not fundamentallyaffected by microorganisms, it would be neces-sary to penetrate deep into the crust where temperatures are outside physiochemical limits

for life. From the early recognition that microbialactivity affects the environment, and vice versa,was born the field of Geomicrobiology. As this book is primarily concerned with the study ofmicrobially influenced biogeochemical and mineralogical reactions at present and throughtime, we begin by briefly examining some of thecharacteristic properties of microorganisms andtheir overall diversity in the biosphere.

1.1 Classification of life

Since the early eighteenth century, and the firstformal taxonomic classification of living organ-isms by Carolus Linnaeus, a number of attemptshave been made to categorize the different lifeforms according to morphological and nutritionalsimilarities. Traditionally, all organisms weregrouped into the kingdoms Animalia or Plantae.However, with the recognition of the immensediversity of microscopic organisms, Whittaker(1969) proposed a five-kingdom classification sys-tem that subsequently added Fungi, Protista, andMonera at the kingdom level. The Monera con-tained the bacteria that, based on their simplisticprokaryotic cell structure, were distinguished fromthe more complex eukaryotic organisms. TheProtista contained all eukaryotes that were neitheranimals, plants, or fungi – included algae, pro-tozoa, slime molds, and various other primitivemicroorganisms. According to Whittaker, theMonera were the first organisms on Earth, andthe Protista evolved directly from them. Fungi,Plantae, and Animalia evolved from the Protista

1Microbial properties and diversity

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2 CHAPTER 1

via three separate directions of evolution basedon differences in how the organisms met theirnutritional needs. Fungi evolved as the most com-plex multicellular organisms that still obtainedtheir nutrients by absorption; animals evolvedbased on their ability to ingest other organisms;while plants evolved a photosynthetic pathwayto synthesize their own organic compounds.

At around the same time as the Whittakerclassification scheme was being implemented,others recognized that comparative analyses ofspecific genes could be used to infer the evolu-tionary pathways of organisms (e.g., Zuckerkandland Pauling, 1965). This approach, termedmolecular phylogeny, is based on the number and location of differences in the nucleotidesequences of homologous genes from differentorganisms. This, in turn, provides an indicationof the evolutionary relatedness between thoseorganisms because the number of sequence differences identified is proportional to the stable mutational changes that accumulate in a sequence with time. Thus, two closely relatedspecies will have accumulated few differences in a sequence shared in common, whereas twomore distantly related organisms will have accumulated many more differences in the same sequence.

In order to determine evolutionary relation-ships it is essential that the gene chosen forsequencing studies: (i) be universally distributedbetween organisms; (ii) must be properly alignedso regions of homology can be identified; and (iii) have a sequence that changes at a rate com-mensurate with the evolutionary distance being measured (Olsen et al., 1986). There are severalevolutionary conserved genes that are present inmost organisms, one of which, however, is par-ticularly important because it encodes a specificcomponent of the protein-synthesizing ribo-somes, the ribosomal RNA (rRNA) molecule. In prokaryotic organisms this is known as the16S ribosomal RNA. In eukaryotes, where theequivalent molecule is slightly larger, it is called18S ribosomal RNA. Due to the fundamentalrole they play, rRNA molecules have remained

structurally and functionally conserved. However,some portions of the molecule are more import-ant to its function than others, and, as a result,these regions of the molecule are almost ident-ical in sequence from the smallest bacterium tothe largest animal. By contrast, regions that havea lesser role to play in the functionality of themolecule have very different sequences in dif-ferent organisms. This has produced a moleculethat consists of a mosaic of sequences that arehighly conserved in the rRNAs of all organismsand sequences that can be extremely variable.

When rRNA sequences from different organ-isms are compared with each other, the numberof changes in the sequence can be used to createa phylogenetic tree, with closely spaced “branches”symbolizing species that are genetically similar.There are a number of procedures used to con-struct phylogenetic trees, but the first stage inthese analyses is always the careful alignment of the rRNA nucleotide sequences. This is a relatively straightforward task for regions thathave a highly conserved sequence, yet it is con-siderably more problematic in regions of greatersequence variability. The mutations leading tosequence variability include additions, deletions,and reversions, further complicating the matter.Once the rRNA sequences have been aligned,phylogenetic trees of relatedness can be con-structed using a number of different approaches,including distance, parsimony, maximum likeli-hood, and Bayesian algorithms/methods (seeSchleifer and Ludwig, 1994 for details).

In their pioneering work, Woese and Fox(1977) and Fox et al. (1980) used single gene 16SrRNA sequences to determine the phylogeneticrelatedness between many different organisms.What they showed was that all life could begrouped into three major domains (a term thatsupplanted the use of kingdom), the Bacteria,Archaea, and Eukarya, and that these domainsdiverged from a “root,” a graphical representa-tion of a point in evolutionary time when allextant life on Earth had a common ancestor, alsoknown as the Last Universal Common Ancestor,or LUCA (Fig. 1.1). The comparative sequence

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MICROBIAL PROPERTIES AND DIVERSITY 3

analyses further allowed the definition of othermajor lineages (phyla, class, and order) withinthe three domains (Woese, 1987). Significantly,the new taxonomic system showed that life isdominated by microbial forms – Bacteria andArchaea are entirely prokaryotic, while the major-ity of the phyla in Eukarya are also microbial.The “higher” organisms, the plants and animals,are relegated to the peripheral branches of thetree. The degrees of variation in microbial diver-sity are similarly astounding, with the evolution-ary distances between the three domains beingmuch more profound than those that distinguishthe traditional kingdoms from each other(Woese et al., 1990). So, in terms of evolutionarydistance, humans are only slightly removed fromplants or fungi, while the distances betweenseemingly similar bacteria are considerablygreater – in this book the term bacteria writtenwith a lowercase “b” is synonymous with theterm prokaryote, not the domain.

Although it is presently impossible to cali-brate sequence changes against geological timebecause different lines of descent have evolved at different rates (i.e., sequence change is non-linear), the universal phylogenetic tree does give a relative timing for major evolutionarychange. For example, the Eukarya are more closelyrelated to Archaea than Bacteria (Iwabe et al.,1989). This suggests that Archaea and Eukaryahad a common history that excluded the descen-dants of the bacterial line. What is more, the majororganelles of eukaryotes – the mitochondria andchloroplasts – are derived from bacterial part-ners that had undergone specialization throughco-evolution within a host archaeal cell (see sec-tion 7.5.2 for details). Because Eukarya containboth bacterial and archaeal parts, their originswould appear to be very ancient (for an opposingview see Cavalier-Smith, 2002). This hypo-thesis, however, creates problems in trying toroot the universal phylogenetic tree because wecannot be sure whether the first divergences inthe tree separate: (i) Bacteria from a line that was to produce Archaea and Eukarya (as shown in Fig. 1.1); or (ii) a proto-eukaryotic lineage from

fully developed bacterial and archaeal lineages(Doolittle and Brown, 1994).

Despite the success of single gene taxonomy,phylogenetic reconstructions based on this tech-nique can lead to ambiguous evolutionary rela-tionships because single gene sequences lack sufficient information to resolve much of thedivergence pattern of their major lineages. Addi-tional complexity is also introduced by lateralgene transfer, a process whereby genes from onespecies are passed onto another, leading to theacquisition of physiological properties or meta-bolic traits that are not concordant with their16S rRNA phylogenies (Doolittle, 1999). Anexample we are all familiar with from the news ishow certain bacteria have suddenly developedresistance to antibiotics that used to destroythem. What we now suspect has happened is thatthey have acquired resistance by a donation ofgenes from those cells immune to the drug.Lateral gene transfer often results in a confusingpicture, where different phylogenies for the same organism are obtained when using differ-ent gene sequences because of an unusually high degree of similarity between the donor andrecipient genes of otherwise distantly relatedspecies. As a result of these incongruities, theentire premise of equating single gene phylo-geny with organismal phylogeny has been calledinto question. These problems can, in part, beaddressed by multi-gene- and whole-genome-based systematics (i.e., the study of genomics),with phylogenetic trees being built by either ana-lyzing a large number of conserved genes or byanalyzing for the presence or absence of genes in each genome, respectively (e.g., Fitz-Gibbonand House, 1999). Thus far, complete genomicsequences of more than 200 microorganismshave been made available. And, one of the mostexciting outcomes of these studies is that regard-less of domain, all of the microorganisms share a number of core genes, the so-called “house-keeping” genes that are responsible for criticalfunctions (Koonin, 2003). This suggests that lifemay indeed have had a common ancestor, withperhaps as few as 500–600 genes.

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MICROBIAL PROPERTIES AND DIVERSITY 5

1.2 Physical properties ofmicroorganisms

1.2.1 Prokaryotes

Most Bacteria and Archaea are between 500nanometers (nm) and 2 micrometers (µm) indiameter, with a volume between 1 and 3 µm3

and a wet weight approximating 10 −12 grams.The only way to observe such small objects is by microscope, hence the term microorganisms or microbes. Individual cells most frequentlyoccur as rods (known as bacilli), spheres (knownas cocci), or helical shapes (known as either spirilla or vibrio) (Fig. 1.2). These differentshapes in turn affect the relationship betweenvolume and surface area: rods have a surface area to volume ratio of 10:1; spheres, 5.8:1; and helical-shaped, 16:1 (Beveridge, 1988).Individual cells also commonly form together in groups or clusters after division. When theygrow end on end, they form what are known as filaments.

The small size of Bacteria and Archaea has a number of ramifications. First and foremost,they are typically not large enough to differ-entiate the many cellular processes into com-partmentalized organelles. The lack of complexparts, however, benefits them because it meansthat there is little to fail, perhaps explaining why they are often found in “extreme” environ-ments that preclude the growth of more com-plex organisms (Nealson and Stahl, 1997).Given the lack of intracellular organelles, manyof the important metabolic and biosynthesisreactions take place instead at the cell’s peri-phery where the external sources of nutritionand energy are derived. Hence, establishing anappropriate shape with a high surface area to volume ratio is of considerable importance foroptimizing the diffusional properties of the cell(Fig. 1.3). If a cell grows too large in volume, thesurface area becomes small in comparison, andthe diffusion time becomes excessively long. On

the other hand, cells too small would suffer fromhaving insufficient surface area to accommodatethe numerous proteins required to transportnutrients into the cell, and not enough volumeto house all of the macromolecules and solutesnecessary to support normal cell growth(Beveridge, 1989a).

The physical makeup of Bacteria and Archaeais remarkably simple (see Madigan et al., 2003 for details). All possess a plasma membrane thatcompletely envelopes the cell and separates theinside of the microorganism from the externalenvironment. Its primary function is as a selec-tive and semipermeable barrier that regulates the flow of material into and out of the cell. The plasma membrane is 7–8 nm in thickness(Fig. 1.4). In Bacteria, it consists of a lipid bilayerwith a fatty acid portion sandwiched in the middle of two glycerol layers, while in Archaeathe fatty acid is replaced by repeating units of the hydrocarbon molecule isoprene. The place-ment of ionized phosphate compounds at theouter surface of the glycerol imparts a negativecharge that makes the phospholipid hydro-philic, that is, attracted to water (see Box 1.1).Therefore, the phosphate portions of one layerinteract with the water outside the cell and those of the other layer interact with the fluidswithin the cell. The fatty acids, on the otherhand, are made up of nonpolar hydrocarbonchains (with H–C bonds) that are hydrophobic.Accordingly, they cluster together and pointinwards towards each other in an attempt tominimize their exposure to the aqueous envir-onment. Despite the exclusion of water from their interior, most plasma membranes are actu-ally quite fluid, with a viscosity similar to lightgrade oil. As a result, the phospholipids have the capacity to move about with a certain degreeof flexibility.

The plasma membrane also houses a numberof different proteins, making up to 60% of themembrane weight. Some of these proteins arefound on the outer surfaces where they bind andprocess substrates for transport into the cell.Within the plasma membrane, various proteins

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6 CHAPTER 1

facilitate energy production in respiration andphotosynthesis (see Chapter 2). There are alsoproteins that completely span the membrane,having portions exposed to both the interior ofthe cell and the external environment. Some of

these proteins, called membrane transport pro-teins or permeases, provide passageways throughwhich specific molecules can pass.

Enclosed within the plasma membrane is thecell’s interior or cytoplasm, which is over 70%

A

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Figure 1.2 Basic shapes of bacteria as seen under the scanning electron microscope (SEM). (A) Rods,Escherichia coli (courtesy of Rocky Mountain Laboratories, NIAID and NIH). (B) Cocci, Staphylococcus aureus(courtesy of Janice Carr and CDC). (C) Spirilla, Leptospira sp. (courtesy of Janice Carr and CDC). (D) Filaments, Anabaena sp. (courtesy of James Ehrman).

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MICROBIAL PROPERTIES AND DIVERSITY 7

water and has a pH maintained near neutrality.Within the cytoplasm, raw materials from theexternal environment are enzymatically degradedand new organic macromolecules are biosyn-thesized. It contains a particulate fraction, con-sisting of: (i) its genome, which is in the form of a large double-stranded molecule (the bacterialchromosome) that aggregates to form a visiblemass called the nucleoid, as well as some extra-chromosomal DNA called plasmids that conferspecial properties on the cell and are amenable tolateral gene transfer; (ii) ribosomes, the machin-ery needed in the manufacture of proteins; (iii)

carboxysomes, polyhydroxybutyrate bodies, andvarious other inclusions and granules, that serveas specific storage sites; (iv) gas vacuoles, thatconfer buoyancy on the cells; and (v) magneto-somes, the magnetic particles found in somecells. The soluble portion (the cytosol) contains a variety of small organic molecules and dis-solved inorganic ions (Fig. 1.5).

Most cells have internal solute concentrationsthat greatly exceed their external environment.As a result, there is a constant tendency through-out the life of the cell for external water to enterinto the cytoplasm to dilute the salt content.This creates an internal hydrostatic pressure thatmay reach up to 50 atmospheres. External hydro-static pressures are usually significantly lower.This pressure differential causes the delicate anddeformable plasma membrane to stretch near thebreaking point, and were it the sole structuralsupport, it would likely rupture and the cell woulddie (known as lysis). Bacteria and most Archaeahave, however, evolved an additional layer, thecell wall, which superimposes the plasma mem-brane, providing extra rigidity and support forthe cell, as well as governing cell morphogenesis(Beveridge, 1981). It also functions as an exter-ior armor that protects the cell from physical

Radius = 1 µmSurface Area (4πr2) = 12.6 µm2

Volume (4/3πr3) = 4.2 µm3

SA:V = 3

Radius = 2 µmSurface Area (4πr2) = 50.3 µm2

Volume (4/3πr3) = 33.5 µm3

SA:V = 1.5

7–8 nm

Phospholipids

Fatty acids

Glycerol withphosphate end

Substrate bindingproteins

Electron transportenzyme

HydrophobicgroupsHydrophilicgroups

Permease

Figure 1.4 Structure of a bacterial plasma membrane, showing the phospholipid molecules with theirhydrophilic groups pointing outwards and their hydrophobic groups pointing inwards. Embedded within themembrane are various proteins, each of which serves a specific cellular function.

Figure 1.3 The relationship between cell surfacearea and volume.

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8 CHAPTER 1

stresses, such as collision with other particles, andchemical perturbants, such as pH changes, dis-solved inorganic ions, and organic solvents (Koch,1983). Furthermore, the cell wall acts as a filtercontrolling the passage of dissolved molecules intothe cell (see Chapter 3). Without going into detailhere, it suffices to mention that there is variationin wall types based on morphology and composi-tion. Bacteria are typically classified according tohow they react with what is known as the Gramstain. Gram-positive cells stain purple, and thisreflects their thick, single-layer walls made up of amaterial called peptidoglycan. Gram-negative cellsstain pink, a reflection of their more complexstructure, with a thin peptidoglycan layer, overlainby gel-like space known as the periplasm, on topof which lies the outer membrane. Archaea aremuch more diverse, and have several wall types.

1.2.2 Eukaryotes

Eukaryotes include microorganisms, such as protists and fungi, as well as multicellular life,such as the plants and animals. Of the variousprotists, two will be covered in the followingchapters, the algae and protozoans. Algae are adiverse group of photosynthetic organisms thatproduce O2 as a byproduct of their metabolism.

They are typically found in fresh and sea water,but they can also grow on rocks and trees, or insoils when water is available to them. Algae arebroadly classified into green, brown, golden, andred based on the biochemical characteristics of their chlorophyll molecules and accessory pigments. Collectively they display a wide varietyof morphologies, ranging from unicellular formsto macroscopic seaweeds that can grow tens of meters in length (see van den Hoek et al.,1996 for details). Others can grow symbiotic-ally with animals, such as the dinoflagellatesassociated with coral reefs. Despite being rela-tively primitive, green algae are classified in thesame clade as the plants because of their shareduse of photosynthetic pigments. Some algae even form mineralized shells (or exoskeletons),including the silica-precipitating diatoms andthe calcite-precipitating coccolithophores (seeChapter 4). By contrast, protozoans are color-less and unicellular animal-like organisms thatobtain food by ingesting other organisms or particulate organic matter (see Sleigh, 1992 fordetails). They are classified according to theirability to move in a concerted manner (known as motility); some use flagella, which are whip-like appendages that arise from the cell surface,others have shorter appendages called cilia that

Flagella

Pili

1 µm

Plasmamembrane

Cell wallVacuoleMagnetosome

Storage granulesRibosomes

CytoplasmNucleoid

Figure 1.5 Idealized drawing of a prokaryotic cell, showing some of its internal structure.

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MICROBIAL PROPERTIES AND DIVERSITY 9

move in a wave-like manner, while some useextensions of their cytoplasm called pseudopods.Many protozoans live as parasites, absorbing or ingesting organic compounds from their hostcell; one well known example is Plasmodium sp., a parasite that causes malaria. Some protozoansform mineralized shells as well, such as the calcite-forming foraminifera and the silica-forming radiolarians.

Fungi also have a range of morphologies, from unicellular yeasts, to filamentous molds that clump together to form what is known asmycelia, to macroscopic forms such as mush-rooms. Their most important roles are: (i) asdecomposers of organic material, leading tonutrient cycling and spoilage of natural and synthetic materials, such as wood or food; (ii) as symbionts of algae and cyanobacteria (e.g., as lichens); and (iii) as producers of economic-ally important substances, such as ethanol, citric acid, antibiotics, and vitamins. They areoften dominant in acidic conditions and, in soil, represent the largest fraction of biomass (see Moore-Landecker, 1996 for details).

Eukarya are larger and much more complex instructure than either Bacteria or Archaea, typically5–100 µm in diameter. However, this demarca-

tion has been clouded by the discovery of marinebacteria on the seafloor that can reach sizes aslarge as 750 µm (Schulz et al., 1999) and therecent recognition that a diversity of 0.5–5 µmsized “picoeukaryotes,” with compact organiza-tion and limited organelles, exist throughout theocean waters (Moreira and López-García, 2002).Most eukaryotes have a rigid cytoskeleton thatvaries in type amongst the different phyla. Theyalso have a plasma membrane that contains complex lipids known as sterols. These mole-cules stabilize the cell structure, making themless flexible than their prokaryotic counterparts,and, depending on the type of cell, sterols cancomprise up to 25% of the total lipids in themembrane (Madigan et al., 2003). Eukaryotesalso contain a membrane-enclosed nucleus thataccommodates its genome in the form of DNA-containing chromosomes, as well as a number of membrane-bound organelles that are used tosegregate the various cellular functions from one another. These include, amongst many, themitochondria for energy production and thechloroplasts, the chlorophyll-containing sitesused by algae (and plants) for photosynthesis(Fig. 1.6). This compartmentalization is necessaryfor localizing the various metabolic processes,

Chloroplast

Cytoplasm

Cell wall

Nuclearmembrane

Golgi complex

Nucleus

Ribosomes

Vacuole

10 µm

MitochondriaEndoplasmicreticulum Granules

Nucleolus

Figure 1.6 Idealized drawing of an eukaryotic cell, showing some of its internal structure.

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10 CHAPTER 1

and thereby minimizing the inherent problemsthat their large size would face in obtainingnutrients and excreting waste products simply by diffusional processes (Beveridge, 1988).

1.3 Requirements for growth

Although microorganisms are ubiquitous in thesurface environment, each particular species issubject to a number of variables that affect theirrates of growth. We can divide these requirementsinto two categories, physical and chemical. Phys-ical aspects include temperature, pH, and osmoticpressure, while the chemical aspects includesources of nutrition and energy.

1.3.1 Physical requirements

(a) Temperature

Each microorganism has a temperature rangeover which it can grow. Psychrophiles are cap-able of growth up to 20°C. In nature, they arecommonly found in deep ocean waters or in polar regions, but they also include those thatgrow within our refrigerators. Mesophiles growbetween 15 and 45°C. These are the most common types of microorganisms in nature, and include most of the pathogenic species.Thermophiles grow between 45 and 80°C andhyperthermophiles grow above 80°C. The lasttwo groups of microorganisms are associated with terrestrial geothermal regions or seafloor hydro-thermal vent systems.

The temperature limits of life are constrainedin one of two ways. At high temperatures, subtlechanges occur in the configuration of the pro-teins and nucleic acids causing them to becomeirreversibly altered. As temperatures approachthe surface boiling point of water, the monomersthat make up the cells also begin to hydrolyse(bonds break by reacting with water); the half-life of adenosine triphosphate (ATP) is less than30 minutes at 100°C (Miller and Bada, 1988).Also, as reactions generally proceed faster at

higher temperatures, solute transfer across theplasma membrane may become excessivelyquick, thereby inhibiting the cells’ ability to generate energy through what is known as theproton motive force (see section 2.1.4(b)). To date, the uppermost survival temperaturemeasured is 121°C for an archaeal species mostsimilar to Pyrodictium occultum (Kashefi andLovley, 2003). Hyperthermophiles compen-sate for the high temperatures in a number ofways, including altering some of their proteinstructures and by possessing plasma membranescomposed of hydrocarbons with repeating unitsof isoprene (instead of fatty acids) that are nearly impermeable to ions and protons (van de Vossenberg et al., 1998).

One principal factor that governs a micro-organism’s minimum temperatures is whetherthe cell membrane retains its fluid state, so that its capability for nutrient transport andenergy generation still exists. Experiments withpsychrophiles have shown that as temperature is decreased, lipids in their plasma membranechange composition by adding an increasing proportion of unsaturated fatty acids that helpmaintain an optimal degree of fluidity (Gounot,1986). Other adaptations include producing cold-acclimation enzymes that allow the cells to meta-bolize at rates comparable to mesophiles (Fellerand Gerday, 2003) and formation of extracellularlayers (i.e., outside the cell wall) containingcompounds that increase the viscosity of theimmediate fluid phase, in essence acting as a natural antifreeze agent (Raymond and Fritsen,2001). Thus far, the minimum temperaturerecorded for actively growing bacteria is −20°Cin Siberian permafrost (Rivkina et al., 2000).

(b) pH

Each microorganism has an external pH rangewithin which growth is possible. For most micro-organisms (e.g., the neutrophiles), this is withinpH 5–9, although the intracellular – within thecell – pH must remain neutral in order to preventdestruction of its cytoplasmic macromolecules.

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MICROBIAL PROPERTIES AND DIVERSITY 11

Only a few species are tolerant of pH values below2, these are the so called acidophiles. They aregenerally restricted to mine drainage and geo-thermal environments, where acid is generatedfrom the oxidation of reduced sulfur-containingcompounds. Other microorganisms, the alka-liphiles, can grow at pH 10–11. Such extremelyalkaline environments are often associated withsoda lakes and carbonate-rich soils.

(c) Water availability

The primary impediment for microbial life is the availability of water. Water is crucial becauseit facilitates biochemical reactions by serving asthe transport agent for reactants and products.When a substance cannot move across the plasmamembrane in response to a chemical gradient,water will move across instead. This occursbecause the presence of solutes changes the con-centration of water. Such movement of water iscalled osmosis, and the osmotic pressure is theforce with which water moves from low to highsolute concentrations when the solutions areseparated by a semipermeable barrier. In a hypo-tonic medium, the solute concentration is higherin the cell. As a consequence, water will moveinto the cell to attain positive water balance. Ifthe flow into the cell was unrestricted, the plasmamembrane would burst. The reverse occurs in ahypertonic medium, where the solute concentra-tion is higher outside the cell than inside. Inbrines, a cell would inadvertently shrink throughplasmolysis if it did not naturally possess themeans to deal with osmotic stresses. Thus, micro-organisms living in evaporitic environments gener-ally have some specific requirements for sodiumions (Na+) to maintain osmotic equilibrium.Such organisms are called halophiles.

1.3.2 Chemical requirements

Life is made up of a few basic organic ingredients.Besides water, carbon is the primary requirementfor growth, making up 50% of an organism’s dryweight. It serves as the structural backbone of

living matter, to which carbon and a number of other elements, such as hydrogen, oxygen,nitrogen, sulfur, and phosphorous, can bind.Covalently linked carbon atoms can form linearchains, branched chains (aliphatics), benzenerings (aromatics), or ring structures containing oneor more noncarbon atoms (heterocyclic). Theother elements usually take the form of anions orneutral species that have a definite geometricalarrangement around the central carbon atom.The resultant molecules form what are known as functional groups, each of which possessescharacteristic chemical and physical propertiesthat can be observed in a range of organic compounds. Many organic macromolecules are polyfunctional, containing two or more differ-ent kinds of functional groups. Figure 1.7 shows the common functional groups associated withmicrobial cell surfaces, some of which play animportant role in metal sorption processes (seeChapter 3).

The various functional groups are bondedtogether to form low molecular weight com-pounds called monomers, which in turn combineto form much larger macromolecules (Box 1.1).Monomers are covalently bonded together, oftenthrough reaction of a hydrogen atom from onemonomer with the hydroxyl group from another,liberating a molecule of water. These are referredto as dehydration or condensation reactions. Bycontrast, macromolecules can be broken downinto their precursor monomers through the addition of water. These chemical reactions areknown as digestion or hydrolysis. When severalof the same monomers are repetitively bondedtogether, the macromolecule is called a polymer.The important macromolecules in living systems,in order of their dry cell percentage, are pro-teins (55%), nucleic acids (24%), lipids (9%),and carbohydrates (10%).

(a) Nutrition

Organisms obtain their carbon in one of two ways.Those that convert CO2 to organic carbon, suchas glucose (C6H12O6), function autotrophically,

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12 CHAPTER 1

whereas those that consume pre-existing organicmaterials act heterotrophically.

A living cell further requires a number of trace metals to fulfill essential biochemical rolesin cell metabolism. For example, iron, nickel,copper, manganese, cobalt, molybdenum, andzinc are all important components of differentmetalloenzymes. Other trace metals, however,are toxic to the cell (e.g., arsenic, cadmium, copper, and mercury), and in order for a cell to survive when they are present in excess, theymust actively prevent their intracellular trans-port. As such, microorganisms possess a suite of genes that serve to activate specific proteins

designed either to facilitate metal transport into the cell or, alternatively, help rid the cells of them through their efflux, biomethylation,volatilization, or immobilization (e.g., Nies,2000).

Because of the unique physicochemical prop-erties of each individual ion or compound, a cell requires more than one transport mechan-isms to obtain the full spectrum of solutes andcompounds required. There are a wide variety of mechanisms utilized by microorganisms, ofwhich four will be covered here. These include:(i) passive diffusion; (ii) facilitated diffusion;(iii) active transport; and (iv) cytosis.

R

O

C

R OH

R1 C R2

H

R

H

H

C H

R

H

H

C H

O

R C OH

O

R1

R1

R1 R2

R2

C O

O

C

O

OC

O

C R2

O

C

Hydroxyl (alcohol)

Carbonyl (aldehyde)

Carbonyl (ketone)

Carboxyl (carboxylic acid)

Anhydride (acid anhydride)

Acetyl

Methyl (alkane)

Ether (ether)

Ester (ester)

R S H

O

CR1 S R2

SR1

R1

S R2

R2

R

O

OH

OH

C

O

R

OH

RH

H

O P O

O

R

OH

O P OH

O

N

CH

HN

R C

H

CN

CH N

H

Thioester

Disulfide

Sulfhydryl (thiol)

Phosphodiester

Phosphate (organic phosphate)

Amino (amine)

Phosphoryl (organic phosphate)

Amide (amide)

Imidazole (amide)

Figure 1.7 Some organic functional groups, shown in their protonated state. The symbol “R” is used torepresent any substituents, but typically it is a carbon-containing moiety. When two substituents are shown in amolecule, they are designated “R1” and “R2”.

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MICROBIAL PROPERTIES AND DIVERSITY 13

Box 1.1 Organic macromolecules

Proteins

The importance of proteins to living organismsstems from their roles as enzymes, as well as their involvement in solute transport and cellstructure. A single cell of Escherichia coli, a typical bacterium, contains about 1900 differ-ent kinds of proteins and 2.4 million total pro-tein molecules (Madigan et al., 2003). The basicbuilding blocks of proteins are amino acids,which in themselves comprise both amino andcarboxyl functional groups, as well as an addi-tional side group that distinguishes the variousamino acids (there are 20 different kinds ofamino acids commonly found in proteins). Theside group may be a hydrogen atom, or a morecomplex organic molecule, such as a sulfhydryl

Nucleic acids

Nucleic acids consist of basic structural monomerscalled nucleotides, where each nucleotide con-tains a nitrogen-containing base, a pentose sugar,and an organic phosphate group. Two principaltypes of polynucleotides are deoxyribonucleicacid (DNA) and ribonucleic acid (RNA). DNAcontains the genetic information of the cell. RNA plays three crucial roles: (i) messenger RNAcarries select genetic information from DNA in asingle-stranded molecule; (ii) transfer RNA areadaptor molecules that translate mRNA duringprotein synthesis; and (iii) ribosomal RNA, ofwhich several types exist, are catalytic componentsof the ribosome that carry out the synthesis of

(−SH) group or an aromatic ringed structure. The side chains also impact the surface charac-teristics of the amino acid because insertion of acarboxyl group makes it anionic, while insertionof methyl groups (CH3) makes them more hydro-phobic. Dipeptides result from the covalent bond-ing of the carboxyl group of one amino acid to the amino group of another, releasing watervia dehydration. Further addition of amino acidswould result in the formation of a long chain-likemolecule called a polypeptide. Polypeptides cantake on any number of more complex structuresresulting from the way they fold in accordancewith the positioning of the side groups and the way different polypeptides interact with oneanother in the same protein. This gives rise to theincredible diversity in protein structures.

proteins. The nitrogen-containing bases are allring structures, with either a single ring (e.g., thepyrimidines: thyamine, cytosine, and uracil) or a double ring (e.g., the purines: adenine andguanine). The pentose in DNA is deoxyribose,while the pentose in RNA is ribose. In anucleotide, a nitrogenous base is attached to a pentose sugar by a glycosidic bond, while the sugars are held together by phosphodiesterbonds. Polynucleotides, such as DNA and RNA,then form by covalent bonding between the phosphate of one nucleotide with the pentose of another. An extremely important nucleotide,aside from being a constituent in nucleic acids, isadenosine triphosphate (ATP). It functions as acarrier of chemical energy.

continued

Dehydration

Hydrolysis

Hydrogenbonding

CN

R1H

H H

R2

HO

OH

O

OH R2 OH

C

Amino acid 1

Aminogroup

Sidechain

Carboxylgroup

CN

H

H

C

R2

H O

CN

H

H H OH

C

Amino acid 2

CN C

Dipeptide

Peptidebond

H2O

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14 CHAPTER 1

Lipids

Lipids are a diverse group of organic compoundsthat are insoluble in water, but dissolve readily in nonpolar solvents such as alcohol. They areessential to the structure and function of cell membranes, as well as serving as fuel reserves.Simple lipids contain two monomers; an alcoholcalled glycerol and a group of hydrocarbons

known as fatty acids. A fat molecule is formedwhen a molecule of glycerol combines with one, two, or three fatty acid molecules to form a monoglyceride, diglyceride, or triglyceride,respectively. The chemical bond that formsbetween a fatty acid and a glycerol molecule is called an ester linkage. Complex lipids formwhen phosphate, carboxylate (COO−), or aminogroups replace a fatty acid molecule – recall the

N

NN

H

N

OH OH

H H

H

P

H

H

N

H

O

O–

O

O

OPhospho-diesterlinkage

OHOrganic

phosphategroup

AdenineH H

H

CH2

Ribose

P

CH2

CH2

Ribose

–O

O

O

H

H

Ribose

N-base

N-base

Dehydration

Hydrolysis

C

C

C

H

H

H

H

H

OH

OH

OH

Glycerol

(Replacing with a phosphate groupmakes instead a phospholipid)

HC

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

H

H

C

O

HO

(CH2)10CH3

OCHO

OH

OO– P

OH

OHO C (CH2)10CH3

Lauric acid (a fatty acid)

Hydrogenbonding

C

C

C

H

H

H

H

H

O

O

O C

O

O

C

O

C (CH2)10CH3 + H2O

(CH2)10CH3 + H2O

(CH2)10CH3 + H2O

Ester linkagecontinued

Box 1.1 continued

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MICROBIAL PROPERTIES AND DIVERSITY 15

1 Passive – As discussed above, prokaryotes haveevolved cell shapes that maximize their diffusionalproperties. The process of passive diffusion involvesthe movement of solutes across the plasma mem-brane due to a concentration gradient, i.e., from an area of high concentration to one that is lower, in order to achieve a state of equilibrium.During the process there is no consumption ofenergy. The rate of diffusion, in turn, is proportionalto the difference in concentration between the external and internal environments, the permeabil-ity, and the total surface area. Cells commonly rely on this process to transport nonpolar, fatty-acid-soluble molecules, such as alcohols, or thosemolecules sufficiently uncharged, such as gases and water.

2 Facilitated – The hydrophobic nature of the inside membrane prevents the passive movement of soluble ions and simple organic molecules. Even asubstance as small as a proton (H+) is restrictedbecause it is always hydrated as the charged hydronium ion (H3O

+). If the ions or organic mole-cules have external concentrations in excess of thecytosol, then they can diffuse into the cell along aconcentration gradient with the aid of specializedmembrane transport proteins called permeases.Three kinds of permeases exist (Booth, 1988).Uniporters transfer single ions from one side of the plasma membrane to the other. Symportersmove ions or organic molecules, with a coupling ion required for transport of the first, in the samedirection. Antiporters move the ions one way, and

phospholipids in cell membranes. This can makethe macromolecule amphipathic in nature, with ahydrophilic “head” that interacts with water anda hydrophobic “tail” that avoids water.

Carbohydrates

The basic building blocks of carbohydrates con-sist of simple sugars called monosaccharides,with each molecule containing three to seven carbon atoms. Pentoses (five-carbon sugars) andhexoses (six-carbon sugars) are extremely import-ant to life because they include ribose (found inRNA and ATP), deoxyribose (found in DNA),and glucose (the main constituent of cell wallsand an important energy reserve). Derivatives ofmonosaccharides can be formed by replacingone or more of the hydroxyl groups by another

chemical species, such as through N acetylation(N replacing O in sugar and adding −CH3COgroup) to form one of the sugar derivatives inpeptidoglycan. Polysaccharides consist of eightor more monosaccharides covalently bonded to one another via glycosidic bonds. A high proportion of polysaccharides have simple repetitive structures of the same monosaccharide(e.g., starch, cellulose, chitin) or disaccharides(e.g., peptidoglycan). More complicated struc-tures (e.g., heteropolysaccharides) may involvethree or more sugars with perhaps a side-branch,or the repeating sequences can be interrupted (i.e., as in alginates). Polysaccharides can evencombine with other macromolecules, such asprotein or lipids, to form glycoproteins and glycolipids, respectively.

Dehydration

Hydrolysis

Starch

O

OH

H

H

HO

HH

HOH

H

HO

OH

Hydrogenbonding

OH

CH2OH CH2OH CH2OH CH2OH

H2OOH OHOH OH

OH OHH OH

H

OH

H

HO

H H

α-glucose α-glucose

OH

HOH

H

H

HO

H H

Box 1.1 continued

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16 CHAPTER 1

a secondary ion the other. Each permease is highlyspecific to the uptake of certain ions or simpleorganic molecules, and the cell has the means toregulate which permeases are active depending on its nutritional needs (Fig. 1.8).

3 Active – The concentration of most chemicals withinthe cytosol is generally higher than those outside thecell. Therefore, neither passive or facilitated diffu-sion allows the cells to acquire their necessary con-centrations. As an alternative, active transport is anenergy-dependent process that moves solutes orsimple organic molecules against the concentrationgradient. This process similarly uses permeases,with the energy derived from either ATP or an elec-trochemical gradient (see section 2.1.4(b)).

4 Cytosis – As a consequence of their more rigidcytoskeleton, protozoans use an additional mech-anism called cytosis, in which organic molecules or solutes are moved in and out of a cell withoutpassing through the plasma membrane. It essen-tially involves the plasma membrane wrappingaround a substance, engulfing it to form a membrane-bound sphere called a vesicle, whichcan then open inside the cell and release the substance. Alternatively, it can separate from theplasma membrane and remain intact as a vesicle.

(b) Energy

All life forms require a source of energy to drivecellular biosynthesis and function. The ways inwhich organisms metabolize are covered in detailin Chapter 2, but at this point it is sufficient topoint out that the energy can come either fromthe conversion of radiant energy into chemicalenergy via the process of photosynthesis or fromoxidation-reduction reactions using organic orinorganic molecules. The former behave photo-trophically, while the latter behave chemo-trophically. The nutritional patterns among allorganisms can thus be distinguished on the basisof both the carbon and energy sources (Table 1.1).Those that use light as an energy source and carbon dioxide as their chief source of carbon are called photoautotrophs. Those phototrophsthat cannot convert CO2 to organic carbon, but instead use organic compounds, are known as photoheterotrophs. Organisms that use in-organic compounds for energy and CO2 as theircarbon source are referred to as chemolitho-autotrophs (or simply chemoautotrophs), while

K+K+

Na+

H+

H+

H+

H+

Na+

H+

OH–

OH–

OH–

H+OH–

2H+

2H+SO4

2–

SO42–

Cytoplasm Plasma membrane External environment

K uniporter

Na+/H+ antiporter

H+/SO42– symporter

Figure 1.8 The three types of permeases;uniporters, symporters, and antiporters. Energyto drive the transport of ions (or simple organicmolecules) towards the cytoplasm comes fromthe pH gradient between the outside and insideof the cell.

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MICROBIAL PROPERTIES AND DIVERSITY 17

those that use organic compounds for their car-bon source are called chemolithoheterotrophs(or mixotrophs). When organic compounds areused for both energy and carbon the organism is a chemoorganoheterotroph (or simply chemo-heterotroph). As we will see in later chapters,chemoheterotrophy is very important for ele-ment cycling because it couples the oxidation oforganic carbon to the reduction of an oxidizedcompound, referred to as the terminal electronacceptor (TEA). The major TEAs are O2, nitrate(NO3

−), Mn(IV) oxides, Fe(III) oxyhydroxides,sulfate (SO4

2−), or CO2.

1.3.3 Growth rates

As a microbial population colonizes a new envir-onment (whether in a laboratory experiment or in a natural setting) the microorganisms willcollectively undergo a growth cycle that consistsof the lag, exponential (or log), stationary, anddeath phases (Fig. 1.9). The lag phase marks the initial period of time when the cells adjust to their new surroundings, perhaps requiring thesynthesis of new enzymes. Once acclimatized,the cells begin to reproduce very rapidly duringthe exponential phase, with, for instance, thebacteria Escherichia coli dividing on the order ofevery 20–30 minutes under typical laboratorybatch culture conditions. Not only is this phasemarked by significant increases in biomass, but it is also at this stage that the cells exhibit theirmost visible characteristics: the shape, color,density, and the way their colonies aggregate(Madigan et al., 2003). If exponential phase were

to continue unchecked, an astronomical num-ber of cells would result. In the example above,E. coli would produce a population of 2144 cells injust 48 hours, a population weighing about 4000times the weight of the Earth! This of course doesnot happen because the cells would soon run outof required nutrients, their wastes would build upto toxic levels, and there may even be significantchanges in the localized aqueous composition thatwould impact negatively on the cells. In reality,growth rates in nature are considerably slowerthan estimated in the lab, often less than 1% ofthe maximal experimental rates, because physio-chemical conditions in natural environments are rarely ideal and indigenous populations mustcontend with competition for a limited suite ofnutrients.

Table 1.1 Nutritional classification of organisms.

Nutritional pattern Carbon source Energy source

Photoautotroph CO2 SunlightPhotoheterotroph Organic compounds SunlightChemolithoautotroph CO2 Inorganic compoundsChemoheterotroph Organic compounds Organic compounds

Time0

Log

num

ber o

f cel

ls

Lag phaseStationary

phaseExponential

phaseDeathphase

Figure 1.9 Typical growth curve for a bacterialpopulation based on batch culture. Time (0)represents inoculation.

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18 CHAPTER 1

Bacillus spores apparently have been resuscitatedafter preservation in amber for 25–40 millionyears (Cano and Borucki, 1995) and in brineinclusions within salt crystals that are some 250 million years old (Vreeland et al., 2000),although these claims are controversial. Speciesof Azotobacter, Bdellovibrio, Myxococcus and somecyanobacteria instead form protective structurescalled cysts. These are thick-walled structuresthat, like spores, protect the microorganism from harm, but they are somewhat less durablethan spores.

1.4 Microbial diversity

Microorganisms are the most ubiquitous lifeforms. Unlike multicellular life that is restrictedto the Earth’s surface, the adaptable nature ofmicroorganisms has allowed them to inhabit the most diverse environments imaginable, oftenrepresenting the only life forms. In most aquaticsystems, microbial cell densities are remarkablysimilar, ranging from 105 to 106 cells ml−1. Suchconsistency reflects less the overall microbialproductivity than it does control of numbers by grazing (Fenchel et al., 2000). Much highercell densities can, however, exist, whereverabundant nutrients and energy are available, and often where predation is minimized. One suchsetting is in the waters immediately surroundingwarm, deep-sea hydrothermal vents, where celldensities up to 109 cells ml−1 have been reported(e.g., Corliss et al., 1979). The high microbialdensities, in general, have important implica-tions for global dispersal because microorganismsare unlikely to be restricted by any geograph-ical barriers. Yet ubiquitous dispersal also meansrelatively low global species richness when com-pared with more complex organisms that have geographically restricted ranges (Finlay, 2002).In terms of global biomass, the total amount ofmicrobial carbon is 60–100% of the estimatedtotal carbon found in plants, with their totalbiomass estimated to be roughly 4–6 × 1030 cellsor 350–550 Pg of C, where 1 Pg = 1015 g

In the stationary phase, there is no net increaseor decrease in overall cell number – some cells inthe group actively grow while others die. But, ifconditions continue to deteriorate for the entirepopulation, a large number of cells enter the final death phase. Microorganisms are, however,extremely adaptable to adverse conditions, and ifthe supply of nutrients becomes diminished, theywill employ a variety of compensatory strategies(Sunda, 2000). One typical response is for thecells to become smaller, thereby increasing theirsurface area to volume ratios to maximize theirdiffusional capabilities. This is observed in thesurface waters of the oceans where there is a shift in species dominance from large cells tothose that are extremely small (<2 µm), such asthe so-called picoplankton. Cells also respond by growing at a slower rate, which increases cellular concentrations at a given uptake rateand decreases the metabolic demand for metal-loenzymes. Some species also decrease theirrequirements for limiting metals by alteringmetabolic pathways or by changing the type ofmetal-containing enzyme in key pathways. Forinstance, under iron-limiting conditions, manymarine species are able to replace ferrodoxin, an iron-containing protein, with flavodoxin, anonmetalloprotein (e.g., La Roche et al., 1996).

If conditions worsen still, and the micro-organism is potentially faced with starvation, itmay respond by forming protective structures.Certain bacterial genera, such as Bacillus andClostridium, form endospores (spores formedintracellularly) that are released into the envir-onment once the parent cell decays. Then, asconditions improve, the endospore convertsback to a vegetative cell. Other bacteria, such as Methylosinus species, form exospores (sporesformed outside the cell – extracellularly) bygrowing or budding out from one end of the cell. Both types of spores are extremely durable to heat and chemicals, so much so that they caneven survive conditions in space, leading to thehypothesis that life may have been transportedto Earth from elsewhere in the solar system (seesection 1.5.7). What is truly amazing is that some

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(Whitman et al., 1998). In addition, micro-organisms contain 85–130 Pg of N and 9–14 Pgof P, which amounts to roughly ten times morenutrients than those stored in plants.

The vast majority of microorganisms in aquaticsystems grow in microcolonies attached to sub-merged surfaces in the form of biofilms. Thebiofilms consist mainly of highly hydrated extra-cellular polymers (EPS) that are secreted by themicroorganisms embedded within it. Benthicstrategies can include growing on suspended sediment particles, plants and mineral surfaces(epilithic mode), the latter often leads to mineralweathering and metal corrosion (see Chapter 5).Biofilms are remarkably resilient communitieswhere the cells live and are retained within protected adherent microcolonies, while takingadvantage of the inorganic and organic com-pounds that preferentially accumulate at inter-faces. As microbial populations grow, theyeventually enshroud available surfaces, while atthe same time dispatching mobile “swarmer” cellsto reconnoitre neighboring niches and to estab-lish new microcolonies in the most favorable ofthem (Costerton et al., 1994). Under ideal condi-tions, the biofilms thicken into what is commonlyreferred to as a microbial mat (see Chapter 6).These natural ecosystems contain many types of microbial species, and in any given part of themat there exists a highly organized communitywhere nutrients and metabolites are continuouslyrecycled between cells in close proximity.

One of the primary goals of microbial eco-logists has been the isolation and culture ofmicroorganisms of interest. During enrichmentculture techniques, specific media and growthconditions are chosen to duplicate as closely as possible the natural conditions of the desiredmicroorganism. Unfortunately, the enrichmentcultures favor some species over others, and a frequent outcome is that the microorganismsthat thrive and dominate the culture conditionsmay only have comprised a small component of the natural population. Indeed, estimates suggest that some 99% of the microorganismsvisualized microscopically in environmental

samples are not cultivated by routine techniques(Amann et al., 1995). This has led to one of thegreatest obstacles to understanding the diversityof natural microbial communities – our inabilityto make sound ecological inferences based on the metabolic properties of a few cultivatablespecies.

In order to circumvent the inherent culture-based biases, it was suggested that, by extractionof nucleic acids directly from environmentalsamples, genes that were present in all micro-organisms (e.g., rRNA) could be isolated,sequenced, and compared with pre-existing cul-tures (e.g., Pace et al., 1986). Such a phylogeneticapproach is now routinely used to study micro-bial diversity, and is often published in the formof a tree that strictly highlights the evolution-ary relationship between the microorganisms,without actually inferring an evolutionary pathbeyond the species of interest (e.g., Fig. 1.10).Consequently, rRNA studies have provided amuch more comprehensive view of the micro-bial world, particularly in the sense that novelspecies are continuously being reported to existin environments where we previously thoughtwe had a firm understanding of the microbialecology (e.g., Dojka et al., 2000).

Ribosomal RNA genes are obtained fromDNA isolated directly from natural samples.However, these genes need to be separated from all the other genes in the genomic DNA,and the quickest way to this is to specificallyamplify rRNA using the polymerase chain reaction or PCR, a method that multiplies DNAby up to a billion-fold in a test tube – only verysmall amounts of DNA are initially required toobtain workable amounts of rRNA. Once thedifferent 16S rRNA genes have been amplifiedfrom environmental DNA, they have to besorted or separated so their sequences can beidentified. There are numerous approaches toseparate this mixture of 16S rRNA genes, includ-ing cloning, which separates individual genesinto cloning vectors that are then assimilated bya large number of laboratory cultures (also knownas a clone library), or more rapid approaches

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20 CHAPTER 1

such as denaturing gradient gel electrophoresis(DGGE), which separates different genes bysequence in the form of individual bands on an acrylamide gradient gel. In both cases, theenvironmental sequences can then be comparedwith sequences from known cultured isolatesfound in reference databases such as GenBank(http://www.ncbi.nlm.nih.gov) and the RibosomalDatabase Project (RDP, http://rdp.cme.msu.edu).Molecular phylogenetic trees are then con-structed by placing the unknown environmentalsequences within a phylogenetic framework withtheir closest cultured relatives (see Theron andCloete, 2000 for details).

At this stage, we still have very little idea ofwhat the relative abundance of some of theseenvironmental sequences are. To determine this,one can use the sequence obtained from theenvironmental analysis to design a probe thatwill identify the cell from which the sequencewas originally isolated (e.g., Devereux et al.,1992). These probes are short fluorescently

tagged oligonucleotides that bind specifically tothe ribosomal RNAs of the probe-target popula-tion. This method, known as fluorescence in situhybridization (FISH), provides a way to visualizethe community spatial distribution, and the ability to design these probes to detect groups ofrelated microorganisms means that they can beused to identify and enumerate similar types ofmicroorganisms even if they cannot be grown in culture (e.g., Fig. 1.11). However, the amountof rRNA present in a microorganism is propor-tional to its metabolic activity. Inactive cellshave too few rRNA molecules within their cellsto be detected using these techniques and thusonly active members of a microbial communitycontain enough ribosomes to yield a detect-able signal (e.g., DeLong et al., 1989). Natural samples can also be subjected to multiple FISHprobing, where a suite of probes, each deigned to react with a specific microorganism or group,can lead to phylogenetically characterizing thediversity of an entire population.

Sulfurihydrogenibium azorense Az-Fu1T

Sulfurihydrogenibium subterraneum HGMK1T

Hydrogenothermus marinus VM1T

Persephonella marina EX-H1T

Persephonella guaymasensis EX-H2T

Hydrogenobacter thermophilus T3

Aquifex pyrophilus Kol5AT

Environmental clone SRI-40

Strain YNP-SS1

95

95

100

95

55

100

100

100

0–10

Figure 1.10 Maximum-likelihood phylogenetic tree showing the position of various members of the Aquificales,the purported deepest branching lineage within the domain Bacteria. The length of the scale bar representsevolutionary distance by the number of fixed mutations per nucleotide position. The numbers associated with the branches are a statistical measure of the confidence of divergence between two lineages (expressed inpercentages) based on an algorithmic re-sampling known as bootstrapping. (From Aguiar et al., 2004.Reproduced with permission from the Society for General Microbiology.)

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