Processes In Microbial Ecology PDF

2 PROCESSES IN MICROBIAL ECOLOGY CHAPTER 1 (A)...

2 PROCESSES IN MICROBIAL ECOLOGY CHAPTER 1 (A) (C) Introduction (B) (D) Microbes make up an unseen world, unseen at least by Bacteria and archaea often look quite similar under the naked eye. In the pages that follow, we will explore the microscope, and in fact archaea were once this world and the creatures that inhabit it. We will dis- thought to be a type of bacteria. An old name for cover that processes carried out by microbes in the archaea is “archaebacteria” while bacteria were once unseen world affect our visible world. These processes referred to as “eubacteria”, the “true” bacteria. Now we include virtually every chemical reaction occurring in know that bacteria and archaea occupy separate king- nature, making up the great elemental cycles of carbon, doms, accounting for two of the three kingdoms of life nitrogen, and the rest of the elements in the biosphere. found on earth. The third kingdom, Eukarya (some- The processes also involve interactions between organ- times spelled Eucarya), includes microbes, such as isms, both among microbes and between microbes and fungi, protozoa, and algae (but not blue-green algae, large organisms. more appropriately called cyanobacteria), as well as This chapter will introduce the types of microbes higher plants and animals. Prominent members of found in nature and some basic terms used throughout the third kingdom are protists, which are single-cell Figure 1.1 Examples of some microbes. Panel A: Soil bacteria belonging to the Gemmatimonadetes phylum, each about 1 μm the book. It will also discuss why we should care about eukaryotes. wide. Image courtesy of Mark Radosevich. Panel B: Fungal hyphae. Image courtesy of David Ellis. Panel C: Various eukaryotic microbes in nature. The answers will give some flavor of The diversity in the types of microbes found in nature algae from the summer, Narragansett Bay, 50–100 μm. Image courtesy of Susanne Menden-Deuer. Panel D: A marine ciliate, what microbial ecology is all about. Chapters 2 and 3 is matched by the diversity of processes they carry out. Cyttarocylis encercryphalus, about 100 μm. Image courtesy of John Dolan. continue the introduction to microbes and their envi- Microbes do some things that are similar to the functions ronment. But first, we need to look at a few definitions. of plants and animals in the visible world. Some microbes are primary producers and carry out photosynthesis starts with those that a layperson might give, if asked why being counted as being ill. We know less about the similar to plants, some are herbivores that graze on we should learn about microbes. impact of diseases on smaller organisms, such as the What is a microbe? microbial primary producers, and still others are carni- zooplankton in aquatic systems (Fig. 1.2) or inverte- The microbial world is populated by a diverse collec- vores that prey on herbivores. But microbes do many brates in soils. These small organisms are crucial for tion of organisms, many of which having nothing in more things that have no counterparts among large Microbes cause diseases of macroscopic maintaining the health of natural ecosystems, which is common except their small size. Microbes include by organisms. These things, these processes, are essential organisms, including humans now being threatened on many fronts by climate definition all organisms that can be observed only with for life on this planet. Most people probably think “germs” when asked how change. There is some evidence that diseases in the a microscope and are smaller than about 100 μm. microbes affect their life. Of course, some microbes do ocean are becoming more common (Lafferty et al., Microbes and its synonym “microorganisms” include cause diseases of humans and other macroscopic 2004), and amphibians on land are now declining Why study microbial ecology? bacteria, archaea, fungi, and other types of eukaryotes organisms. The role of infectious diseases in control- worldwide due to infections by chytrid fungi, perhaps (Fig. 1.1). The microbial world also houses viruses, The main reason has already been implied: microbes ling population levels of macroscopic plants and ani- linked to global warming (Rohr and Raffel, 2010). though arguably they are not alive and are not microbes. mediate many processes essential to the operation of mals in nature is recognized to be important (Ostfeld But pathogens are exceptions rather than the rule. The Ignoring viruses for now, bacteria and archaea are the the biosphere. But there are other reasons for studying et al., 2008), but its impact probably is still underesti- microbiologist John Ingraham pointed out that there are simplest and usually the smallest microbes in nature. microbial ecology. The following list of seven reasons mated. Sick animals in nature are most likely to be more murders among humans than pathogens among killed off by predators, or simply disappear before microbes (Ingraham, 2010). The vast majority of microbes 1  INTRODUCTION 3 4 PROCESSES IN MICROBIAL ECOLOGY (A) (B) Box 1.1 Two founders of microbiology Fungus One of the founders of microbiology, Pasteur, made (Chapter 5). Pasteur also explored the role of bacteria many contributions to chemistry and biology during in causing diseases, but it was a contemporary of the early days of these fields. His more important Pasteur, Robert Koch (1843–1910), who defined the contributions include work showing that life does criteria, now known as Koch’s postulates, for show- not arise from spontaneous generation, a theory held ing that a particular microbe causes a disease. Koch, in the mid-nineteenth century to explain organic another founder of microbiology, developed the agar matter degradation. Decomposition of organic mate- plate method for isolating bacteria, a method still rial is still examined today in microbial ecology used today in microbial ecology. 1.0 mm mary producers, meaning they use light energy to con- base upon which the fisheries of the world depend. Figure 1.2 A common freshwater zooplankton Daphnia pulicaria (the common name is “water flea”), uninfected (panel A), vert carbon dioxide to organic material (Chapter 4). Consequently, there is a general relationship between and infected (panel B) by a fungus. The fungi are the small numerous dots visible in the transparent body cavity of the Daphnia. These microbes, the “phytoplankton”, include cyano- microbial production and fishery yields (Conti and Taken from Johnson et al. (2006). Used with permission from the Ecological Society of America. bacteria and eukaryotic algae. Phytoplankton are not Scardi, 2010). directly eaten by fish, not even by the small, young Another important connection with our food is the stages of fish, the fish larvae. Rather, mostly microscopic role of microbes in producing the inorganic nutrients animals (zooplankton) and protists are the main herbiv- that are essential for growth and biomass production in nature are not pathogenic, including those living on the first microbiologists, who could be called microbial ores in lakes and oceans. Zooplankton and protists in by higher plants in terrestrial environments and by and in us. The human body is host to abundant and ecologists, worked on topics in what we now call food turn are eaten by still larger zooplankton and fish larvae, phytoplankton in aquatic environments. Essential inor- diverse microbial communities. In fact, most of the cells microbiology. Louis Pasteur (1822–1895) was hired by as part of a food chain leading eventually to adult fish ganic nutrients, such as ammonium and phosphate, in the human body are not human, but rather are the wine industry to figure out why some wines turned (Fig. 1.3). There can be more direct connections between come from microbes as they degrade organic material bacteria. An average adult has about 1 × 1014 microbes, sour and became undrinkable. The problem, as Pasteur microbes and fish. In some aquaculture farms shrimp (Chapter 5). Other microbes change (“fix”) nitrogen gas, tenfold more than the number of human cells. Microbes found out, was a classic one of competition between feed on “bioflocs” which form from bacteria growing on which cannot be used by plants as a nitrogen source, inhabiting our skin and mucous membranes help to pre- two types of microbes, one that produced alcohol added wheat flour and ammonium from the shrimp. into ammonium, which can (Chapter 12). The fertility of vent invasion by pathogens, and the bacteria in the gas- (good wine) and the other organic acids (sour, undrink- The simple, linear food chain shown in Figure 1.3 is soils depends on microbes in other ways. Organic mate- trointestinal tract do the same as well as aiding digestion. able wine). To this day, food microbiologists try to accurate in only some aquatic systems. But even in rial from higher plants, partially degraded by microbes, Disruption of the microbial community in the colon, for understand the complex microbial interactions and waters with more complex food webs, microbes are the and other organic compounds directly from microbes example, allows the pathogen Clostridium difficile to processes that affect our favorite things to eat and make up soil organic material. This organic component flourish, which often leads to severe diarrhea. One cure drink, an important job in applied microbial ecology. of soil contains essential plant nutrients and affects is “bacteriotheorapy”, also called fecal transplantation, in Microbes are also involved in meat and dairy products. Fish water flow, fluxes of oxygen and other gases, pH, and which a normal microbial community is “transplanted” Ruminants, such as cows, goats, and sheep, depend on many other physical-chemical properties of soils that into the colon of a diarrhea-suffering patient (Khoruts complex microbial consortia to digest the polysaccha- Zooplankton directly contribute to the growth of crop plants. So, our Nutrients CO2 et al., 2010). A huge project is now examining the rides in the grasses they eat (Chapter 14). A branch of food indirectly and directly depends on microbes and (N, P, etc) genomes of human-associated microbes, the “human microbial ecology can be traced to microbiologists Small grazers what they do. microbiome” (http://www.hmpdacc.org/), using metage- such as Robert Hungate (1906–2004), who studied Phytoplankton nomic approaches (Chapter 10) first designed for soils microbe-ruminant interactions and the mainly anaer- and oceans. obic processes carried out by these microbes (Hungate, Figure 1.3 A simple food chain (solid arrows), from Microbes degrade and detoxify pollutants 1966). phytoplankton to fish, common in many aquatic habitats. The modern environmental movement is often said to Note that microbes (phytoplankton) are at the base of this Microbes are also important in supporting life in lakes have started with the publication of Silent Spring in food chain, and other microbes (small grazers) make up the Much of our food depends on microbes and the oceans, and eventually make possible the fish first few transfers. Still other microbes (mainly bacteria), not 1962 by Rachael Carson (1907–1964). The book chroni- Microbes produce several things that we eat and drink we may eat. Microbes take over the role of macroscopic shown, contribute to the release of nutrients used by cled the damage to wildlife and ecosystems caused by every day, including yogurt, wine, and cheese. Some of plants in aquatic environments and are the main pri- phytoplankton. the pesticide dichloro-diphenyl-trichloroethane, better  INTRODUCTION 5 6 PROCESSES IN MICROBIAL ECOLOGY known by its initials, DDT. Fortunately, the concentra- Microbes can be useful model systems for exploring Just as we can learn about large organisms from mals and plants did not appear until about a billion tions of DDT have been decreasing over time, in part general principles in ecology and evolution microbes, the flow of ideas can go the other way. General years ago, two to three billion years after microbes had due to regulations banning it in most developed coun- Microbes have served as models for exploring many theories developed for exploring the ecology of plants invented via evolution most of the various strategies tries, following publication of Silent Spring. In addition, questions in biochemistry, physiology, and molecular and animals often are useful for exploring questions in now known for existing on earth. We can gain insights microbes, mostly bacteria, degrade DDT and other biology. They are good models because they grow rap- microbial ecology. For example, microbial ecologists into the early evolution of life by looking at microbes in organic pollutants to innocuous compounds and even- idly and can be manipulated easily in laboratory experi- have used island biogeography theory, which was first today’s environments that may mimic those on early tually to CO2 ( Alexander, 1999) in spite of many organic ments. For similar reasons, microbes also are used as conceived for large animals (MacArthur and Wilson, earth (Chapter 13). pollutants being recalcitrant and difficult to degrade models to explore general questions in ecology, popula- 1967), to examine the dispersal of microbes and rela- In addition to looking at life millions of years ago, because of complex chemical structures. With very few tion genetics, and evolution. Virus-bacteria interactions, tionships between microbial diversity and habitat size today’s microbes may provide insights into life on planets exceptions, bacteria and fungi are quite adept at for example, have been used to examine questions and (Chapter 9). Likewise, models about stability and diver- millions of kilometers from earth. Studying microbes in degrading organic compounds, even those quite toxic models of predator-prey interactions (Chapter 8). sity developed for animal communities are now being extraterrestrial-like environments on earth is the main to macroscopic organisms. Experiments with protozoa and bacteria were crucial for applied to microbial communities and processes. focus of the field of “astrobiology”. These environments Inorganic pollutants, such as heavy metals, cannot establishing Gause’s competitive exclusion principle Microbial ecologists look at microbial diversity for pat- are extreme ones where only microbes, and often only be removed by microbial activity, but microbes can (Fig. 1.4), which states that only one species can occupy terns that have been seen for plant and animal diversity, bacteria and archaea, the “extremophiles”, survive change the electrostatic charge of these pollutants a niche at a time. Experiments with both bacteria and such as how diversity varies with latitude. Not all large (Chapter 3). Microbes live in hot springs and deserts, which affects their mobility through the environment. fungi have demonstrated basic principles about natural organism-based theories are applicable to thinking polar ice, permafrost of the tundra, and within rocks— An example of this process is the action of the bacte- selection and adaptation in varying environments about microbes, but many are. unworldly habitats where it is hard to imagine life exist- rium Geobacter on the spread of uranium in groundwa- (Beaumont et al., 2009, Schoustra et al., 2009). Richard ing. Perhaps these earthly extremophiles are similar to ter and subsurface environments near waste dumps for Lenski and colleagues have explored the evolution of life on other planets and perhaps insights gained from radioactive material (Lovley, 2003). In this case, the Escherichia coli over 50 000 generations by following this Some microbes are examples of early life on earth and astrobiological studies on this planet will help in the most oxidized form of uranium, U(VI), moves easily bacterium in cultures that have been transferred into perhaps of life on other planets search for life on other planets. But the work would be through subsurface environments. When U(VI) is fresh media every day, including weekends and holidays, Microbial ecologists examine microbial processes now worthwhile even if there are no extraterrestrial microbes. reduced by Geobacter and probably other bacteria, the since 1988 (Lenski, 2011, Woods et al., 2011). Genome occurring in various environments in order to under- Extreme environments and extremophiles are often resulting U(IV) is less mobile. So, while microbial activ- sequencing (Chapter 10) has revealed exactly how these stand how today’s biosphere operates and to predict bizarre and always fascinating. ity does not remove the contamination in this case, it organisms have changed over time, providing insights how it may be altered in the future due to climate can reduce its spread. into evolution not possible with large organisms. change. But what we learn about microbes living today can also help us understand life in the distant past. The Microbes mediate many biogeochemical processes that first life form on earth undoubtedly was a microbe-like affect global climate creature, and its microbial descendants went on to rule This reason for studying microbial ecology is arguably 160 the planet without large organisms for the first three bil- the most important one. It shapes many topics appearing P. aurelia lion years of earth’s history (Fig. 1.5). Multicellular ani- in this book. The role of microbes in degrading pollut- 140 120 Biomass (relative units) 100 80 lar als s e llu nim ts urs rm te c te a n sa ll ti 60 P. caudatum fo ce st ryo ul ryo le pla o t r th st Fir ka M ka imp nd Din ou Ea Fir eu e u S La di e 40 Only No life Only prokaryote-like cells Today 20 microbes 4500 3500 1700 1000 450 65 0 0 5 10 15 20 25 Millions of years before the present Time (days) Figure 1.5 A few key dates during the history of life. For much of this history only microbes were present, as multicellular Figure 1.4 Experimental evidence for the competitive exclusion principle, which states that no two species can occupy the eukaryotes appeared only one billion years ago, after 3.5 billion years had passed. “Prokaryote” refers to bacteria and archaea. same niche at the same time. Here, two species of the protozoan Paramecium are forced to compete for the same food source, Based on Czaja (2010), Humphreys et al. (2010), Payne et al. (2009), and Rasmussen et al. (2008). the bacterial prey (Bacillus pyocyaneus). Only one Paramecium species wins. Data from Gause (1964).  INTRODUCTION 7 8 PROCESSES IN MICROBIAL ECOLOGY ants was already mentioned, but microbes are involved any greenhouse gases and is even colder (–55 °C). High Table 1.1 Some greenhouse gases and how they are affected by microbes. Concentrations are for 2005 and are expressed as in an even more serious “pollution” problem. concentrations of greenhouse gases, mainly carbon parts per million (ppm), per billion (ppb), or per trillion (ppt). Data from Forster et al. (2007). Humans have been polluting the earth’s atmosphere dioxide, in Venus’s atmosphere, along with its proximity Gas Concentrations Greenhouse effect* Microbes or Process with various gases that affect our climate. These gases are to the sun, explain why that planet has an average sur- Carbon dioxide (CO2) 379 ppm 1 Algae and heterotrophic microbes called “greenhouse gases” because they trap long wave face temperature of 460 °C. Greenhouse gases have been Methane (CH4) 1774 ppb 21 Methanogens and methanotrophs radiation, better known as heat, from the sun. Most of increasing in earth’s atmosphere since the start of the Nitrous oxide (N6O) 319 ppb 270 Denitrification and nitrification these gases also have natural sources, and the earth industrial revolution in the early 1800s (Fig. 1.6). Water Halocarbons ** 3–538 ppt 5– >10 000 Degradation by heterotrophs? always has had greenhouse gases, fortunately. Because vapor is the dominant greenhouse gas, but human soci- * Relative to CO2 of greenhouse gases, the average global temperature is ety has a bigger, more direct impact on other gases, most ** Examples include CFC-11 and CF4 16 °C (Schlesinger, 1997), much warmer than the chilly notably carbon dioxide. Other important greenhouse –21 °C earth would be without them. Mars does not have gases include methane (CH4) and nitrous oxide (N2O) ( Table 1.1). Although the concentrations of these gases in with the seasons, as already seen for carbon dioxide, and the atmosphere are much lower, they trap more heat per have varied greatly over geological time, independent of molecule than does CO2. Because of higher greenhouse human intervention. So, the challenge is to separate the 400 gases, average temperatures for the planet are about 1 °C natural changes from those affected by humans and to (A) Atmospheric CO2 warmer now than in the nineteenth century (Fig. 1.6). understand the consequences of these changes. 380 Microbial ecology has an essential role in understand- Microbial ecologists cannot solve the greenhouse Atmospheric CO2 (ppm) 360 ing the impact of greenhouse gases on our climate and problem. But many of the topics discussed in this book the response of ecosystems to climate change, one rea- can help us understand the problem. One job of micro- 340 son being that nearly all of these gases are either used or bial ecologists and other scientists studying the earth produced or both by microbes ( Table 1.1). Carbon diox- system is to figure out the impact of increasing green- 320 Mauna Loa ide, for example, is used by higher plants on land and by house gases and other global changes on the biosphere. Observatory 300 phytoplankton in aquatic ecosystems. This gas is released How will an increase in global temperature affect the by heterotrophic microbes in both terrestrial and aquatic balance between photosynthesis and respiration? How 280 Law Dome Ice Cores ecosystems. The impact of this biological activity can be will aquatic ecosystems respond to increases in dissolved 260 seen in the yearly oscillations of carbon dioxide in Figure CO2 and resulting decreases in pH? How much CO2 and 1.4; it goes down in the summer when plant growth is CH4 will be released if the permafrost of the tundra in (B) high and it increases in winter when carbon dioxide pro- Alaska and Siberia melt? Answering these and other 0.6 Global temperature anomaly duced by respiration exceeds carbon dioxide use by questions depends on the work of microbial ecologists. Temperature Anomaly (°C) 0.4 plant growth. Fluxes of methane, another gas that has been increasing in the atmosphere, are nearly entirely 0.2 controlled by microbes (Chapter 11). Methane and Microbes are everywhere, doing nearly everything nitrous oxide are both produced in anoxic environments The reasons discussed so far for studying microbial ecol- 0.0 which have increased over the years, mainly due to the ogy have focused on practical problems facing human –0.2 growth in agriculture. society. But microbial ecology would be an exciting field What complicates our understanding of these green- even if all of these problems were solved tomorrow. One –0.4 house gases is that nearly all are produced and consumed overall goal of this book is to show the importance of by natural processes mediated by microbes, in addition microbial ecology in explaining basic processes in the –0.6 1860 1880 1900 1920 1940 1960 1980 2000 to the anthropogenetic inputs. For nearly all of these biosphere even if they may appear to be far from any Year gases, the natural processes are much larger than the practical problem facing us today. We should want to human-driven ones, although that is changing. Production know about the most numerous and diverse organisms Figure 1.6 Atmospheric CO2 concentrations (A) and global temperature anomaly (B) since the nineteenth century. of the important plant nutrient ammonium, for example, on the planet, the microbes. Concentrations were estimated from ice cores (Etheridge et al. 1996) or measured directly at the Mauna Loa Observatory, Hawaii, provided by Pieter Tans at the NOAA Earth System Research Laboratory and used with permission (http://www.esrl. directly by humans (fertilizer synthesis) or aided by As a general rule, the smaller the organism, the more noaa.gov/gmd/ccgg/trends/#mlo_data). The global temperature is expressed as the difference between the average tempera- humans (microbial production in agriculture) rivals the numerous it is (Fig. 1.7). Viruses are the smallest and also ture for a year minus the average over 1951–1980. Data from Hansen et al. (2006). natural production of ammonium by microbes (Chapter the most abundant biological entity in both aquatic 12). To complicate things further, greenhouse gases vary habitats and soils, whereas large organisms, such as  INTRODUCTION 9 10 PROCESSES IN MICROBIAL ECOLOGY zooplankton and earthworms, are rare, being 1010 less We already heard about the many microbes living on Table 1.2 Biomass of bacteria and archaea versus plants in the biosphere. Taken from Whitman et al. (1998). “Pg” is petagrams abundant than viruses. A typical milliliter of water from and in macroscopic organisms, including humans. or 1015 grams. the surface of a lake or the oceans contains about 107 Overall, the biomass of bacteria and archaea rivals that of Organism Habitat No. of cells (× 1028) Pg of carbon viruses, 106 bacteria, 104 protists, and 103 or fewer phyto- all plants in the biosphere ( Table 1.2), and certainly is Bacteria and archaea Aquatic 12 2.2 plankton cells, depending on the environment. A typical greater than animal biomass. Oceanic subsurface 355 303 gram of soil or sediment likewise contains about 1010 Microbes are found where macroscopic organisms Soil 26 26 viruses, 109 bacteria, and so on for larger organisms. Even are not, in environments with extremes in temperatures, Terrestrial subsurface 25–250 22–215 deep environments, kilometers below the earth’s sur- pH, or pressure: “extreme” for humans, but quite normal Total 415–640 355–546 face, have thousands of microbes. The deep ocean and for many microbes (Chapter 3). Some hyperthermophilic Plants Terrestrial — 560 Marine — 51 probably deep subsurface environments also have rela- bacteria and archaea thrive in near boiling water (>80 °C), tively large numbers of archaea. Even seemingly impen- which kills all other organisms, including eukaryotic etrable rocks can harbor dense microbial communities. microbes. The hot springs of Yellowstone are famous for harboring dense and exotic microbial communities that methods. It is a great intellectual puzzle to figure out the 1011 not only thrive at high temperatures but also low pH; actions and creatures in the unseen world and how they 1010 (A) Viruses these microbes live in boiling acid baths. At the other affect our visible world. This book will introduce some of 109 Bacteria extreme, both eukaryotic and prokaryotic microbes live the methods used in microbial ecology so that readers in the brine channels of sea ice where water is still liquid can gain deeper insights and appreciation of the bound- 108 but very salty (20% versus 3.5% for seawater) and cold aries between the known and unknown. By learning a bit Organisms per liter 107 Cyanobacteria (–20 °C). The deep ocean may be extreme to us with its about the methodology of microbial ecology, readers 106 Protists high hydrostatic pressure, one hundredfold higher at will also understand better why some seemingly simple 105 1000 m than at sea level, and perennially cold tempera- questions are difficult to answer. 104 tures (about 3 °C), but this is one of the largest ecosys- Here we start with one of the most basic questions: 103 tems on the planet; 71% of the globe is covered by the how many bacteria are in an environment? One of the 102 Other Phytoplankton oceans of which 75% (by volume) is deeper than 1000 m. first answers came from the plate count method, which 101 Many microbes thrive and grow albeit slowly in these consists of growing or cultivating organisms on solid 100 Zooplankton deep waters. agar media (Fig. 1.8). (The terms “to culture” and “to cul- 10–1 In addition to being numerous, there are many differ- 0.01 0.1 1 10 100 1000 ent types of microbes, some with strange and weird (from 1011 Viruses our biased perspective) metabolisms on which the bio- 1010 (B) sphere depends. In addition to plant- or animal-like Bacteria 109 metabolism, some microbes can live without oxygen and “breathe” with nitrate (NO3–) or sulfate (SO42–) (Chapter 11). 108 Organisms per gram Compounds like hydrogen sulfide (H2S) are deadly to 107 macroscopic organisms, but they are essential comestibles 106 Fungi for some microbes. Several metabolic reactions, such as 105 methane production and the synthesis of ammonium Protists from nitrogen gas, are carried out only by microbes. Other 104 microbes are capable of producing chemicals, like acetone Figure 1.8 The plate count method. A sample from the 103 and butane, that seem incompatible with life. Microbes environment is usually diluted first by adding 1.0 ml or 0.1 g Nematodes 102 are truly capable of doing nearly everything. of soil to 9.0 ml of an appropriate buffer, then diluted again by adding 1.0 ml of the first dilution to a new tube containing 101 0.01 0.1 1 10 100 1000 9.0 ml of the buffer. Then 0.1 ml from the second dilution Size (mm) How do we study microbes in nature? tube is spread on an agar plate. After a few days, if 10 colonies grew up after two dilutions (more may be needed Figure 1.7 Size distribution of some microbes and other organisms in a typical aquatic habitat (A) and in soils (B). The size The facts about microbes in nature discussed above for some environments), we could deduce that there were given for fungi is the diameter of the hyphae. Some fungi can be several meters long. came from many studies using many approaches and 104 culturable bacteria per ml or gram in the original habitat.  I N T R O D U C T I ON 11 12 PROCESSES IN MICROBIAL ECOLOGY 2. Stain with magnitude lower than the abundance determined by growing them in the lab have many consequences for DAPI direct microscopic observations. In seawater, for exam- microbial ecology. For starters, it means that most ple, the plate count method indicates that there is about microbes cannot be identified by traditional methods. 3. Filter through 103 bacteria per milliliter, a thousand-fold less than the Even if they can be identified by other methods polycarbonate filter number determined by direct microscopic counts (Chapter 9), the physiology of these microbes cannot ( Jannasch and Jones, 1959). This difference has been be studied by traditional laboratory approaches. This 4. Count with called the “Great Plate Anomaly”. One explanation for lack of information about physiology hinders under- epifluorescence the anomaly is that the uncultured microbes are dead, standing the ecological and biogeochemical roles of microscope since the microbe must be viable and capable of growing specific microbes in nature. Fortunately, much can be bacteria in 1. Preserve sample with enough to form a macroscopic colony if it is to be learned about microbes as a whole in nature by using counting grid formaldehyde. The microbes polycarbonate filter with counted by the plate count method. For this reason, the approaches that examine processes and bulk proper- are the dots here counting grid plate count method sometimes is called misleadingly the ties of microbes. For example, methods are available “viable count method”. In contrast, a particle must only to examine the contribution of bacteria and fungi ver- have DNA to be included in the direct count method sus larger organisms in degrading organic material Number of microbes per ml= (Fig. 1.9). Dead bacteria could still have DNA and be (Chapter 5). The methods include those that yield esti- counted. So, the discrepancy between direct and plate mates of bacterial and fungal biomass and activity, Number of microbes per grid x counts was first thought to be due to large numbers of although the identity of the bacteria and fungi remains (Area of filter)/(area of grid) ÷ dead or at least inactive bacteria that were included in unknown. This approach is sometimes called “black box direct count methods but not by the plate count method. microbial ecology” because bacteria and fungi, in this Volume of sample filtered There are similar problems with other microbes, although case, are being treated as black boxes whose contents the methods differ (Chapter 9). (the types of microbes) we cannot see. Opening up the In fact, to explain the discrepancy, nearly all bacteria black box and connecting specific microbes with spe- Figure 1.9 How microbes are examined by epifluorescence microscopy, the direct count method. “DAPI” is 4’,6-diamidino- 2-phenylindole, a stain specific for double-stranded nucleic acids. While under the microscope, the sample is exposed (the would have to be dead. If most bacteria were dead or cific processes or functions is an important topic in DAPI is “excited”) to UV light (in the case of DAPI staining) and cells stained by DAPI fluoresce—they give off light, resulting dormant, it would have huge consequences for under- microbial ecology today. in bright spots of light on a black background. The “epi” part of epifluorescence comes from the fact that the excitation light standing the role of microbes in nature. is above rather than below the sample. We now know that the discrepancy between plate The three kingdoms of life: Bacteria, Archaea, and direct counts is not due mainly to dead or dormant and Eukarya bacteria (Chapter 5). Microbial ecologists still argue about the numbers of viable, dormant, and dead bacte- One solution to the problem of identifying microbes tivate” mean the same). The assumption behind the ria, but problems with the plate count method explain without cultivation is to use sequences of genes. Box 1.2 Able assistance with agar “plate count method” is that each microbe in the origi- most of the discrepancy. The basic problem is that an These genes are often called “phylogenetic markers”, plates nal sample will grow on the solid media and form a agar plate is a very foreign habitat for most bacteria and because the gene sequences are also used to deduce macroscopic clump of cells or colony, which can be other microbes. Even plates with “minimal media”, to cite evolutionary relationships among microbes in addition Agar plates are made by pouring molten agar counted by eye or with a low power microscope. The one problem, have organic compounds in concentra- to determining their taxonomy. For reasons discussed amended with various compounds into Petri dishes. bacterium, now isolated on the agar plate, can be iden- tions much higher than encountered by microbes in in Chapter 9, the favorite phylogenetic marker of Once cool, the agar solidifies and becomes a porous tified by examining its response to a battery of bio- nature. Also, many microbes are not adapted to grow in microbial ecologists and microbiologists is the gene support on which microbes grow to form macro- chemical tests. These tests provide some of the first aggregates and to form macroscopic colonies, necessary coding for a type of ribosomal RNA (rRNA) found in scopic colonies. The added compounds may provide clues about the bacterium’s physiology and thus its eco- for a microbe to be counted by the traditional plate the small subunit of ribosomes (SSU rRNA). More spe- necessary organic material for microbial growth or logical and biogeochemical roles in nature. The physiol- count method. There are some problems also with the cifically, the 16S rRNA gene is used for bacteria and they may inhibit growth of some microbes, allow- ogy and genetics of isolated bacteria in “pure culture” direct count method, such as confusing inert particles archaea while the 18S rRNA gene and others are used ing only the targeted microbes to grow up. Although (cultures with only a single microbe) can then be exam- with real microbes due to non-specific staining. But for eukaryotes. Before the development of rRNA gene- the approach is usually attributed to Robert Koch, ined in great detail. overall, many particles observed by epifluorescence based methods to identify uncultivated microbes two assistants of Koch came up with the key parts. The problem is that most microbes are very difficult to microscopy are active bacteria, other microbes, or (Chapter 9), rRNA genes from cultivated microbes were Petri dishes were thought up by Julius Richard isolate and to grow on agar plates. This problem became viruses. sequenced. Petri while Koch’s wife, Fannie Hesse, suggested apparent when the abundance of bacteria determined Regardless of what explains the Great Plate Anomaly, In the 1970s, Carl Woese first championed the use of agar, which was used at the time to make jam. by the plate count method was found to be orders of the difficulties in isolating microbes from nature and rRNA molecules for categorizing microbes ( Woese and  I N T R O D U C T I ON 13 14 PROCESSES IN MICROBIAL ECOLOGY Fox, 1977). Although he started by examining 5S rRNA which is an early stage in the Precambrian, some 2000– Table 1.3 Characteristics defining Bacteria, Archaea, and Eukarya (eukaryotes). molecules, he soon switched to 16S rRNA because its 4000 million years ago. We now know that Archaea are Characteristic Bacteria Archaea Eukarya larger size made it more informative than the smaller 5S not any more ancient than Bacteria, but the name stuck molecule (1500 versus 120 nucleotides). Using these anyway. 1. Membrane-bound nucleus Absent Absent Present 2. Cell wall Muramic acid Muramic acid absent Muramic acid absent rRNA gene sequences, Woese divided all life into three The difference between prokaryotes and eukaryotes is 3. Membrane lipids Ester linked Ether linked Ester linked kingdoms: Bacteria, Archaea, and Eukarya (Fig. 1.10). easily seen microscopically. A prokaryotic cell appears to 4. Ribosomes 70S 70S 80S (in cytoplasm) Bacteria and Archaea make up the prokaryotes (see Box be empty when viewed by light microscopy, and in some 5. Initiator tRNA Formylmethionine Methionine Methionine 1.3), which are those organisms without a nucleus. All sense it is because it lacks a nucleus and all other 6. Introns in tRNA genes Rare Yes Yes other organisms are in the Eukarya kingdom. The term organelles. The genome of prokaryotes is usually in a sin- 7. RNA polymerases One (4 subunits) Several (8–12 subunits each) Three (12–14 subunits each) Sensitivity to: “archaea” came from early ideas about when these gle circular piece of DNA in the cytoplasm (Chapter 10). 8. Diphtheria toxin No Yes Yes microbes first appeared on the planet. Even before The genome of a eukaryote, in contrast, is contained 9. Chlorampheniocol, streptomycin, Yes No No (cytoplasm) Woese’s work on rRNA sequences, microbiologists knew within a nucleus (“karyote” in Greek) and is organized and kanamycin that what we now call archaea had strange metabolisms into chromosomes. In addition to nuclei, eukaryotes that seemed to be advantageous for life on early earth. have compartmentalized some metabolic functions into For this reason, Woese called these microbes “archae- organelles, such as mitochondria and chloroplasts, which under standard light microscopy and fill up the eukaryo- vibrios have a comma-like appearance. In contrast, bac- bacteria”, derived from the geological term Archaean, are absent in prokaryotes. These organelles are visible tic cell. Of special interest, the nucleus of a eukaryote is teria and archaea in most natural environments are much easily seen with epifluorescence microscopy after stain- smaller, about 0.5 μm, and usually appear as simple cocci. ing for DNA. In contrast, in the same epifluorescence There are even reports of even smaller bacteria, called Animals Entamoebae Slime photomicrograph, prokaryotes appear as solid dots with nanobacteria, with cells on the order of 0.1 μm. The Chloroflexi molds Euryarchaeota Fungi no internal structure. When aggregated, the DNA of Martian meteorite ALH84001 was initially thought to Gram Cren- Methanosarcina Methano- Halophlies Plants prokaryotic cells forms a dense body, the nucleoid, which have fossilized nanobacteria (McKay et al., 1996), but this Positive archaeota bacterium Clliates is visible by light microscopy. was later disproven ( Jull et al., 1998). It is hard to fit all Proteobacteria Methan- Thermoproteus ococcus Pyrodictium T.color Among the other important characteristics distin- cellular components necessary for a free-living organism Cyanobacteria Flageltates Bacteroidetes guishing prokaryotes and eukaryotes is size ( Table 1.3). into a 0.1 μm cell. Just one important component, a Trichomonads Because of the space needed for the nucleus and other ribosome, is typically about 25 nm in diameter. The size Thermotogae organelles, eukaryotic cells, even microbial eukaryotes, of microbes is illustrated in Figure 1.11. Mircrosporidia are generally bigger than prokaryotes. There are some The final general characteristic distinguishing prokary- Diplomonads exceptionally large prokaryotic cells (Schulz and otes and eukaryotes is metabolic diversity. Eukaryotes Bacteria Archaea Eukarya Jorgensen, 2001), but these microbes have a vacuole that have two basic types of metabolism, one found in plants pushes the cytoplasm to the outer perimeter of the cell, Figure 1.10 A phylogenetic tree showing the three domains of life: Archaea, Bacteria, and Eukarya (eukaryotes). All bacteria Eukaryotic making the effective volume of these giants more like a cell and archaea are microbes, as are many organisms in the Eukarya domain. Tree based on Olsen and Woese (1993) with updated names for bacterial phyla. “Gram positive” now includes two phyla: Actinobacteria and Firmicutes. Many phyla are not shown typical bacterium. Size is not a useful taxonomic trait for Nucleus to keep this tree simple. distinguishing among organisms, as there is much over- lap in size among prokaryotes and eukaryotes. However, Lab bacterium cell size has a huge impact in ecological interactions, such as in predator-prey interactions (Chapter 7) and in Natural Box 1.3 Is prokaryotes a bad word? the success of bacteria in competing with eukaryotes for bacterium dissolved nutrients. Most bacteria and archaea are small, on the order of a Virus A prominent microbial ecologist, Norman Pace, has “bacteria and archaea”, especially for describing argued strongly that the term “prokaryotes” should cells in nature, about which nothing is known except micron, whereas most microbial eukaryotes are >3 μm, 1 μm not be used because any similarities shared by bac- that they are clearly not eukaryotes. The terms although the marine alga Ostreococcus is 10% in highly microbes use more rare and strange metals such as what makes up a microbial cell. Some of the information ments, they are not large components of biochemical weathered soils (oxisols) in the tropics. tungsten (W) and nickel (Ni). The most important presented here is from basic microbiology and experi- structures in microbes. Of the six most abundant elements in bacteria, two micronutrient is iron (Fe). ments with laboratory-grown microbes. Although we Microbes do require these cations for growth in order (oxygen and hydrogen) are readily obtained from water, Iron is abundant in the earth’s crust and is present in can learn much from these experiments, we will see that to maintain osmotic balance and for some enzyme and and a third (sulfur) from a major anion in natural envi- all cells. But microbes need only relatively low amounts the composition of microbes grown in the lab differs membrane functions, but they make up a very small frac- ronments (sulfate; SO42–). Except in some anaerobic envi- (the C:Fe ratio is on the order of 10 000), mostly for elec- from that of microbes in natural environments. These tion of the average microbial cell. In total, inorganic ions ronments, microbes easily obtain sufficient S from tron transfer reactions, such as in the respiratory path- differences give clues about how microbes make a living make up only about 1% of the dry weight of a microbial assimilatory sulfate reduction; “assimilatory” implies that way. Consequently, iron concentrations are sufficient for in nature. cell. Use of these cations by microbes is also insignificant the end product is assimilated and used for biosynthesis, microbial growth in most environments, with the impor- Composition also helps to explain the imprint of compared to the large concentrations usually found in in contrast to dissimilatory sulfate reduction (Chapter tant exception of the open oceans, where uptake by microbes on the environment. The contents of microbial natural environments. Another abundant cation, calcium 11). The reduced sulfur from assimilatory sulfate reduc- microbes reduces Fe concentrations to very low levels cells are released by various processes and left behind in (Ca2+), is used only by select algae (coccolithophorids—see (10–12M or pM). The low concentration is also due to the tion is used mostly in the synthesis of two sulfur amino soils and aquatic habitats. These remains can contribute Chapter 4), but again not by bacteria, archaea, and fungi, acids, methionine and cysteine. The remaining three ele- insolubility of Fe oxides (FeIII) in oxygenated water at to large geological formations, such as the White Cliffs except as cationic bridges among polymers. The major ments, C, N, and P, are those thought to limit microbial near-neutral pH (Chapter 3). In some oceans, most of Dover (Chapter 4). More common is the impact of biogenic elements are C, N, P, and sulfur (S) ( Table 2.1). growth most frequently in natural environments. notably the high nutrient-low chlorophyll (HNLC) 19  ELEMENTS, BIOCHEMICALS, AND STRUCTURES OF MICROBES 21 22 PROCESSES IN MICROBIAL ECOLOGY similarities. All things considered, the elemental ratios of aerobic ecosystems. The following equation is built on Table 2.2 Elemental ratios of some microbes, expressed in Box 2.1 The sky is not the limit soils and soil microbes are not that different from the the Redfield ratio: moles. Synechococcus is a cyanobacterium common in the Redfield ratio of freshwaters and the oceans. The impli- oceans and some lakes. Data from Bertilsson et al. (2003); A general question in microbial ecology is about the Primary production Caron et al. (1990); Cleveland and Liptzin (2007); Cross et al. cations of this observation have not been fully explored. factors setting or limiting growth rates and bio- 106 CO2 + 16HNO3 + H3PO4 + 122 H2O ® (2005); Geider and La Roche (2002); Goldman et al. (1987); Elemental ratios can be used to examine a variety of Hunt et al. (1987); Van Nieuwerburgh et al. (2004). mass levels for a microbial population in nature. (CH2O)106(NH3)16(H3PO4) + 138 O2 (2.1). biogeochemical and microbial processes in both aquatic The regulation of biomass and rate processes is due ¬ Microbe C:N C:P and terrestrial ecosystems. For example, high C:P ratios to Liebig-type limitation and Blackman-type limi- Respiration could imply microbial growth is limited by P availability, Aquatic heterotrophic bacteria 3.8 – 6.3 26 – 50 tation, respectively. The men who lent their names and similarly for high C:N ratios and N limitation. Equation 2.1 is not entirely accurate, and it hides some Soil microbes (all) 8.6 59.5 to these terms were not microbiologists. Justus Fungi 5 – 17 300 – 1190 Microbes do have some capacity, termed “homeostasis”, critical reactions. For example, Equation 2.1 implies that von Liebig, a nineteenth century German chemist, Synechococcus 5.4 – 7 130 – 165 however, to maintain elemental ratios even as availabil- nitrate (NO3–) is released during respiration and degrada- worked on crop yields, while F.F. Blackman was a Protozoa 6.7 ± 0.9 102 ± 58 ity changes (Fig. 2.2), and this has profound implications tion of organic material, but in fact ammonium (NH4+) is Eukaryotic algae 7.7 ± 2.6 75 ± 31 British plant physiologist who proposed his epony- for understanding controls of microbial metabolism in the main nitrogenous compound produced during min- Zooplankton 5 – 11 80 – 242 mous law of limiting factors in 1905. nature. Another example discussed in Chapter 12 is the eralization of organic material, which is the conversion of Nematodes 8 – 12 ? use of C:N ratios to deduce whether heterotrophic organic material back to inorganic compounds. A couple microbes are net producers or consumers of ammonium. additional steps, which are part of nitrification (Chapter Finally, ratios of nitrate to phosphate concentrations 12), are needed to oxidize NH4+ to NO3–. Another potential 2002), which do not differ significantly from the ratio for Elemental ratios in biogeochemical studies have been used to identify regions of the oceans where inaccuracy in Equation 2.1 is the stoichiometry implied algae. Likewise, coccoid cyanobacteria appear to have Ecologists and biogeochemists often use elemental denitrification (loss of nitrate to N gases) and nitrogen by it, such as the amount of oxygen needed to oxidize C:N ratios similar to the Redfield ratio. In pure cultures, ratios to explore various questions in food web dynam- fixation are common (Chapter 12). one mole of organic carbon (Kortzinger et al., 2001), a the C:N ratios for strains of two cyanobacterial genera, ics and in elemental cycles in the biosphere (Sterner and Perhaps most elegantly, the Redfield ratio can be crucial ratio for interpreting respiration rates. Nonethe- Synechococcus and Prochlorococcus, range from 5.4 to Elser, 2002). The use of elemental ratios to examine bio- used to explore how primary production and respiration less, Equation 2.1 remains a very powerful and succinct nearly 10 ( Table 2.2). geochemical processes started with Alfred Redfield affect the concentrations of the major nutrients in description of the interactions between microbes and Heterotrophic bacteria are also phosphorus-rich (1890–1983) who was one of the first to compare the the geochemistry of natural environments. compared to algae and fungi. For example, the C:P ratio composition of free-floating organisms (the plankton) in of algal biomass is about 75, much higher (much less P) seawater and of major nutrients (Redfield, 1958). than that for heterotrophic bacteria grown in the lab C:N and C:P ratios for various microbes Redfield found that the ratio of C:N:P was 106:16:1 (in “You are what (26–50). Few investigations have examined the P content ato