Biogeochemical Cycling

Chapter 14 Biogeochemical Cycling

Raina M. Maier

14.1 Introduction 14.3.3 Ammonia Assimilation 14.4.4 Sulfur Reduction 14.1.1 Biogeochemical (Immobilization) 14.5 Iron Cycle Cycles and Ammoniﬁ cation 14.5.1 Iron Reservoirs 14.1.2 Gaia Hypothesis (Mineralization) 14.5.2 Iron in Soils and 14.2 Carbon Cycle 14.3.4 Nitriﬁ cation Sediments 14.2.1 Carbon Reservoirs 14.3.5 Nitrate Reduction 14.5.3 Iron in Marine 14.2.2 Carbon Fixation and 14.4 Sulfur Cycle Environments Energy Flow 14.4.1 Sulfur Reservoirs 14.5.4 Iron Oxidation 14.2.3 Carbon Respiration 14.4.2 Assimilatory Sulfate 14.5.5 Iron Reduction 14.3 Nitrogen Cycle Reduction and Sulfur Questions and Problems 14.3.1 Nitrogen Reservoirs Mineralization References and Recommended 14.3.2 Nitrogen Fixation 14.4.3 Sulfur Oxidation Readings

14.1 INTRODUCTION copper or uranium from low-grade ores. There are also det- rimental aspects of the cycles that can cause global envi- 14.1.1 Biogeochemical Cycles ronmental problems such as the formation of acid rain and acid mine drainage, metal corrosion processes, and for- What happens to the vast array of organic matter that is mation of nitrous oxide, which can deplete Earth’s ozone produced on Earth during photosynthetic processes? This layer (see Chapter 15). As these examples illustrate, the material does not keep accumulating. Rather, it is con- microbial activities that drive biogeochemical cycles are sumed and degraded, and a delicate global balance of car- highly relevant to the field of environmental microbiology. bon is maintained; carbon dioxide is removed from the Thus, the knowledge of these cycles is increasingly critical atmosphere during photosynthesis and released during res- as the human population continues to grow and the impact piration. This balance is a result of the biologically driven, of human activity on Earth’s environment becomes more characteristic cycling of carbon between biotic forms such significant. In this chapter, the biogeochemical cycles per- as sugar or other cellular building blocks and abiotic forms taining to carbon, nitrogen, sulfur, and iron are delineated. such as carbon dioxide. Cycling between biotic and abiotic Although our discussion will be limited to these cycles, it forms is not limited to carbon. All of the major elements should be noted that there are a number of other cycles. found in biological organisms (see Table 14.1 ), as well as These include the phosphorus cycle, the calcium cycle, and some of the minor and trace elements, are similarly cycled more ( Dobrovolsky, 1994 ). in predictable and definable ways. Taken together, the various element cycles are called the biogeochemical cycles. Understanding these cycles 14.1.2 Gaia Hypothesis allows scientists to understand and predict the development of microbial communities and activities in the envi- In the early 1970s, James Lovelock theorized that Earth ronment. There are many activities that can be harnessed behaves like a superorganism, and this concept developed in a beneficial way, such as for remediation of organic and into what is now known as the Gaia hypothesis. To quote metal pollutants or for recovery of precious metals such as Lov elock (1995): “ Living organisms and their material

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environment are tightly coupled. The coupled system is a to the Gaia hypothesis, this is the development and con- superorganism, and as it evolves there emerges a new prop- tinued presence of life. Microbial activity, and later the erty, the ability to self-regulate climate and chemistry.” The appearance of plants, have changed the original heat-trapping basic tenet of this hypothesis is that Earth’s physicochemi- carbon dioxide–rich atmosphere to the present oxidizing, cal properties are self-regulated so that they are maintained carbon dioxide–poor atmosphere. This has allowed Earth to in a favorable range for life. As evidence for this, consider maintain an average surface temperature of 13°C, which is that the sun has heated up by 30% during the past 4–5 favorable to the life that exists on Earth. billion years. Given Earth’s original carbon dioxide–rich How do biogeochemical activities relate to the Gaia atmosphere, the average surface temperature of a lifeless hypothesis? These biological activities have driven the Earth today would be approximately 290°C (Table 14.2). In response to the slow warming of the sun, resulting in the fact, when one compares Earth’s present-day atmosphere major atmospheric changes that have occurred over the last with the atmospheres found on our nearest neighbors 4–5 billion years. When Earth was formed 4–5 billion years Venus and Mars, one can see that something has drastically ago, a reducing (anaerobic) atmosphere existed. The initial affected the development of Earth’s atmosphere. According reactions that mediated the formation of organic carbon were abiotic, driven by large influxes of ultraviolet (UV) light. The resulting reservoir of organic matter was utilized by early TABLE 14.1 Chemical Composition of an E. coli Cell anaerobic heterotrophic organisms. This was followed by the development of the ability of microbes to fix carbon diox- Elemental breakdown % Dry mass of an E. coli cell ide photosynthetically. Evidence from stromatolite fossils Major elements suggests that the ability to photosynthesize was developed at Carbon 50 least 3.5 billion years ago. Stromatolites are fossilized lami- Oxygen 20 nated structures that have been found in Africa and Australia Hydrogen 8 Nitrogen 14 (Fig. 14.1). Although the topic is hotly debated, there is evi- Sulfur 1 dence that these structures were formed by photosynthetic Phosphorus 3 microorganisms (first anaerobic, then cyanobacterial) that Minor elements grew in mats and entrapped or precipitated inorganic mate- Potassium 2 rial as they grew (Bosak et al., 2007). Calcium 0.05 The evolution of photosynthetic organisms tapped Magnesium 0.05 into an unlimited source of energy, the sun, and provided Chlorine 0.05 a mechanism for carbon recycling, that is, the first carbon Iron 0.2 cycle (Fig. 14.2). This first carbon cycle was maintained Trace elements for approximately 1.5 billion years. Geologic evidence Manganese All trace elements combined then suggests that approximately 2 billion years ago, comprise 0.3% of dry weight of cell photosynthetic microorganisms developed the ability to Molybdenum produce oxygen. This allowed oxygen to accumulate in the Cobalt atmosphere, resulting, in time, in a change from reducing Copper to oxidizing conditions. Further, oxygen accumulation in Zinc the atmosphere created an ozone layer, which reduced the Adapted from Ages of Gaia: A Biography of our living earth by James Lovelock. influx of harmful UV radiation, allowing the development of higher forms of life to begin.

TABLE 14.2 Atmosphere and Temperatures Found on Venus, Mars, and Earth Plane Gas Venus Mars Earth without life Earth with life Carbon dioxide 96.5% 95% 98% 0.03% Nitrogen 3.5% 2.7% 1.9% 9% Oxygen Trace 0.13% 0.0 21% Argon 70 ppm 1.6% 0.1% 1% Methane 0.0 0.0 0.0 1.7 ppm Surface temperature (°C) 459 53 290 50 13

Adapted from Lovelock, 1995.

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FIGURE 14.1 An example of a living stromatolite (left) and a stromatolite fossil (right). From (left) Reynolds, 1999 and (right) Fa rabee, 2008.

At the same time that the carbon cycle evolved, the Carbon cycle nitrogen cycle emerged because nitrogen was a limiting element for microbial growth. Although molecular Autotrophs nitrogen was abundant in the atmosphere, microbial cells could not directly utilize nitrogen as N2 gas. Cells require

organic nitrogen compounds or reduced inorganic forms of CO2 Organic carbon nitrogen for growth. Therefore, under the reducing conditions found on early Earth, some organisms developed a mechanism for fixing nitrogen using the enzyme nitroge- Heterotrophs nase. Nitrogen fixation remains an important microbio- logical process, and to this day, the majority of nitrogenase What is relative contribution of microorganisms and enzymes are totally inhibited in the presence of oxygen. plants to photosynthesis? When considered over this geologic time scale of several billion years, it is apparent that biogeochemical FIGURE 14.2 The carbon cycle is dependent on autotrophic organisms activities have been unidirectional. This means that the that fix carbon dioxide into organic carbon and heterotrophic organisms predominant microbial activities on earth have evolved that respire organic carbon to carbon dioxide. over this long period of time to produce changes and to respond to changes that have occurred in the atmosphere, namely, the appearance of oxygen and the decrease in car- 14.2 CARBON CYCLE bon dioxide content. Presumably these changes will con- 14.2.1 Carbon Reservoirs tinue to occur, but they occur so slowly that we do not have the capacity to observe them. A reservoir is a sink or source of an element such as car- One can also consider biogeochemical activities on bon. There are various global reservoirs of carbon, some of a more contemporary time scale, that of tens to hundreds which are immense in size and some of which are relatively of years. On this much shorter time scale, biogeochemi- small (Table 14.3). The largest carbon reservoir is carbonate cal activities are regular and cyclic in nature, and it is rock found in Earth’s sediments. This reservoir is four orders these activities that are addressed in this chapter. On the of magnitude larger than the carbonate reservoir found in one hand, the presumption that Earth is a superorganism the ocean and six orders of magnitude larger than the car- and can respond to drastic environmental changes is heart- bon reservoir found as carbon dioxide in the atmosphere. ening when one considers that human activity is effecting If one considers these three reservoirs, it is obvious that the unexpected changes in the atmosphere, such as ozone carbon most available for photosynthesis, which requires depletion and buildup of carbon dioxide. However, it is carbon dioxide, is in the smallest of the reservoirs, the atmo- important to point out that the response of a superorganism sphere. Therefore, it is the smallest reservoir that is most is necessarily slow (thousands to millions of years), and as actively cycled. It is small, actively cycled reservoirs such residents of Earth we must be sure not to overtax Earth’s as atmospheric carbon dioxide that are subject to perturba- ability to respond to change by artificially changing the tion from human activity. In fact, since global industrializa- environment in a much shorter time frame. tion began in the late 1800s, humans have affected several

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TABLE 14.3 Global Carbon Reservoirs TABLE 14.4 Net Carbon Flux between Selected Carbon Reservoirs Carbon reservoir Metric tons Actively carbon cycled Carbon source Flux (metric tons carbon/year) Atmosphere 11 9 CO 2 6.7 1 0 Yes Release by fossil fuel 7 1 0 combustion Ocean Biomass 4.0 1 09 No Land clearing 3 1 09 Carbonates 3.8 1 013 No 9 Dissolved and 2.1 1 012 Yes Forest harvest and decay 6 1 0 particulate organics Forest regrowth 4 1 09 Land Net uptake by oceans (diffusion) 3 1 09 Biota 5.0 1 011 Yes Humus 1.2 1 012 Yes Annual ﬂ ux 9 1 09 Fossil fuel 1.0 1 013 Yes Earth’s crust a 1.2 1 0 17 No Adapted from Atlas and Bartha, 1993.

Data from Dobrovolsky, 1994. a This reservoir includes the entire lithosphere found in either terrestrial or ocean environments. into organic matter ( Fig. 14.2 ). Photosynthetic organisms, also called primary producers, include plants and microor- of the smaller carbon reservoirs. Utilization of fossil fuels ganisms such as algae, cyanobacteria, some bacteria, and (an example of a small, inactive carbon reservoir) and defor- some protozoa. As shown in Figure 14.3 , the efficiency of estation (an example of a small, active carbon reservoir) are sunlight trapping is very low; less than 0.1% of the sunlight two activities that have reduced the amount of fixed organic energy that hits Earth is actually utilized. As the fixed sun- carbon in these reservoirs and added to the atmospheric car- light energy moves up each level of the food chain, up to bon dioxide reservoir ( Table 14.4 ). 90% or more of the trapped energy is lost through respira- The increase in atmospheric carbon dioxide has not tion. Despite this seemingly inefficient trapping, photoauto- been as great as expected. This is because the reservoir of trophic primary producers support most of the considerable carbonate found in the ocean acts as a buffer between the ecosystems found on Earth. Productivity varies widely atmospheric and sediment carbon reservoirs through the among different ecosystems depending on the climate, equilibrium equation the type of primary producer, and whether the system is a managed one (Table 14.5). For example, one of the most H CO&& HCO CO 23 3 2 productive natural areas are the coral reefs. Managed agricultural systems such as corn and sugarcane systems are Thus, some of the excess carbon dioxide that has been also very productive, but it should be remembered that a released has been absorbed by the oceans. However, there significant amount of energy is put into these systems in has still been a net efflux of carbon dioxide into the atmo- terms of fertilizer addition and care. The open ocean has sphere of approximately 7 1 0 9 metric tons/year. The much lower productivity, but it covers a majority of Earth’s problem with this imbalance is that because atmospheric surface and so is a major contributor to primary produc- carbon dioxide is a small carbon reservoir, the result of a tion. In fact, aquatic and terrestrial environments contribute continued net efflux over the past 100 years or so has been almost equally to global primary production. Plants pre- a 28% increase in atmospheric carbon dioxide from 0.026% dominate in terrestrial environments, but with the exception to 0.033%. A consequence of the increase in atmospheric of immediate coastal zones, microorganisms are responsi- carbon dioxide is that it contributes to global warming ble for most primary production in aquatic environments. through the greenhouse effect. The greenhouse effect is It follows that microorganisms are responsible for approxi- caused by gases in the atmosphere that trap heat from the sun mately one-half of all primary production on Earth. and cause Earth to warm up. This effect is not solely due to carbon dioxide; other gases such as methane, chlorofluoro- carbons (CFCs), and nitrous oxide contribute to the problem. 14.2.3 Carbon Respiration Carbon dioxide that is fixed into organic compounds as a 14.2.2 Carbon Fixation and Energy Flow result of photoautotrophic activity is available for consump- tion or respiration by animals and heterotrophic microor- The ability to photosynthesize allows sunlight energy to be ganisms. This is the second half of the carbon cycle shown trapped and stored. In this process carbon dioxide is fixed in Figure 14.2 . The end products of respiration are carbon

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Energy Flow 0.1% Information Box 14.1 The Role of Soil Microbes in Carbon Sequestration

Primary producers 100% Currently there is debate about how soil microbial activity CO2 may influence global warming (Knorr et al., 2005; Rice, 2006). Depending on the relative rates of microbial respiration ver- Grazers 15% sus photosynthetic activity, soils could be either a source or

2 sink for CO2. The estimated amount of carbon sequestered Predators or stored in world soil organic matter ranges from 1.1 to COminerals and 2%

1.6 1012 metric tons (see Table 14.3). This is more than twice Decomposers the carbon in living vegetation (ϳ5.6 1011 metric tons) or in Predators 0.3% the atmosphere (ϳ6.7 1011 metric tons) (Sundquist, 1993). Hence, even relatively small changes in soil carbon storage could have a significant impact on the global carbon balance. FIGURE 14.3 Diagram of the efficiency of sunlight energy flow from primary producers to consumers. In the last 7,800 years, the net carbon reservoir in the soil has decreased by 5.0 1010 metric tons largely due to conversion of land to agriculture (Lai, 2004). It is estimated that some of this lost carbon could be recovered through strategic TABLE 14.5 Net Primary Productivity of Some management practices. For example, agricultural practices

Natural and Managed Ecosystems that enhance crop productivity (CO2 uptake) while decreasing microbial decomposition rates (CO release) could be opti- Description of ecosystem Net primary productivity 2 mized to maximize the sequestration of carbon in the soil res- (g dry organic ervoir. Such practices, which include conservation set-aside, 2 matter/m /year) reduced tillage, and increased crop productivity have been Tundra 400 estimated to account for the sequestration of 1.1 to 2.1 107 metric tons carbon annually (Lokupitiva and Paustian, 2006). Desert 200 However, global warming could also result in enhanced rates Temperate grassland Up to 1500 of microbial decomposition of the carbon stored in the soil. For example, Bellamy et al. (2005) documented carbon losses Temperate or deciduous forest 1200–1600 from all soils across England and Wales from the recent period Tropical rain forest Up to 2800 1978–2003 during which global warming has occurred. Overall, Cattail swamp 2500 the debate is as yet unresolved, and many subtle feedback effects may affect the final outcome, including for example the Freshwater pond 950–1500 C:N ratio within soil organic matter and the mandatory C:N Open ocean 100 requirements of soil microbes. Coastal seawater 200 Upwelling area 600 organic polymers, humus, and C 1 compounds such as meth- Coral reef 4900 ane (CH 4). It is important to understand the fate of these naturally occurring organic compounds. This is because Corn ﬁ eld 1000–6000 degradative capabilities that have evolved for these com- Rice paddy 340–1200 pounds form the basis for degradation pathways that may be Sugarcane ﬁ eld Up to 9400 applicable to organic contaminants that are spilled in the environment (see Chapter 20). But before looking more closely at the individual carbon compounds, it should be pointed out that the carbon cycle is actually not quite as simple as depicted in dioxide and new cell mass. An interesting question to con- Figure 14.2. This simplified figure does not include anaero- sider is the following: if respiration were to stop, how long bic processes, which were predominant on early Earth and would it take for photosynthesis to use up all of the carbon remain important in carbon cycling even today. A more dioxide reservoir in the atmosphere? Based on estimates of complex carbon cycle containing anaerobic activity is shown global photosynthesis, it has been estimated that it would in Figure 14.4 . Under anaerobic conditions, which predomi- take 30 to 300 years. This illustrates the importance of both nated for the first several billion years on Earth, some cellu- legs of the carbon cycle in maintaining a carbon balance (see lar components were less degradable than others ( Fig. 14.5 ). Information Box 14.1). This is especially true for highly reduced molecules such The following sections discuss the most common organic as cellular lipids. These components were therefore left compounds found in the environment and the microbial cat- over and buried with sediments over time and became the abolic activities that have evolved in response. These include present-day fossil fuel reserves. Another carbon compound

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polymers found in the environment, and the phenylpro- Aerobic Anaerobic pene-based polymer lignin. Fossil fuels Carbohydrate-Based Polymers Photosynthesis Fermentation Cellulose is not only the most abundant of the plant polymers, it is also the most abundant polymer found on Earth. It is a linear molecule containing -1,4 linked glucose subunits CO2 H2OO2 CH2O CH2O Alcohols, acids, ( Fig. 14.6A ). Each molecule contains 1000 to 10,000 sub- H CO 2 2 units with a resulting molecular weight of up to 1.8 1 0 6 . These linear molecules are arranged in microcrystalline Respiration fibers that help make up the woody structure of plants. Methanogenesis Cellulose is not only a large molecule, it is also insoluble in CH 4 water. How then do microbial cells get such a large, insolu- FIGURE 14.4 The carbon cycle, showing both aerobic and anaerobic ble molecule across their walls and membranes? The answer contributions. is that they have developed an alternative strategy, which is to make and release enzymes, called extracellular enzymes, that can begin the polymer degradation process outside the (A) Octane cell (Deobald and Crawford, 1997). There are two extracellu- (C8H18) lar enzymes that initiate cellulose degradation: the cellulases -1,4-endoglucanase and -1,4-exoglucanase. The endoglucanase hydrolyzes cellulose molecules randomly within the (B) Cyclopentane polymer, producing smaller and smaller cellulose molecules (C5H10) ( Fig. 14.7 ). The exoglucanase consecutively hydrolyzes two glucose subunits from the reducing end of the cellulose molecule, releasing the disaccharide cellobiose. A third enzyme, known as -glucosidase or cellobiase, then hydrolyzes cel- (C) Naphthalene lobiose to glucose. Cellobiase can be found as both an extra- (C10H8) cellular and an intracellular enzyme. Both cellobiose and glucose can be taken up by many bacterial and fungal cells. FIGURE 14.5 Examples of petroleum constituents: (A) an alkane, (B) an alicyclic compound, and (C) an aromatic compound. A crude oil Hemicellulose is the second most common plant poly- contains some of each of these types of compounds but the types and mer. This molecule is more heterogeneous than cellulose, amounts vary in different petroleum reservoirs. consisting of a mixture of several monosaccharides including various hexoses and pentoses as well as uronic acids. In addition, the polymer is branched instead of linear. An produced under anaerobic conditions is methane. Methane example of a hemicellulose polymer is the pectin molecule is produced in soils as an end product of anaerobic respira- shown in Figure 14.6B , which contains galacturonic acid tion (see Eq. 3.20, Chapter 3). Methane is also produced in and methylated galacturonic acid. Degradation of hemi- significant quantities under the anaerobic conditions found cellulose is similar to the process described for cellulose in ruminants such as cows, as well as termite guts. except that, because the molecule is more heterogeneous, many more extracellular enzymes are involved. 14.2.3.1 Organic Polymers In addition to the two major plant polymers, several other important organic polymers are carbohydrate based. One What are the predominant types of organic carbon found in of these is starch, a polysaccharide synthesized by plants the environment? They include plant polymers, polymers to store energy ( Fig 14.6C ). Starch is formed from glucose used to build fungal and bacterial cell walls, and arthropod subunits and can be linear ( -1,4 linked), a structure known exoskeletons (Fig. 14.6). Because these polymers con- as amylose, or can be branched ( -1,4 and -1,6 linked), a stitute the majority of organic carbon, they are the basic structure known as amylopectin. Amylases ( -1,4-linked food supply available to support heterotrophic activity. The exo- and endoglucanases) are extracellular enzymes pro- three most common polymers are the plant polymers cel- duced by many bacteria and fungi. Amylases produce the lulose, hemicellulose, and lignin ( Table 14.6 ) ( Wagner and disaccharide maltose, which can be taken up by cells and Wolf, 1998 ). The various other polymers produced include metabolized. Another common polymer is chitin, which is starch (plants), chitin (fungi, arthropods), and peptidogly- formed from -1,4-linked subunits of N -acetylglucosamine can (bacteria). These various polymers can be divided into (Fig. 14.6D). This linear, nitrogen-containing polymer is an two groups on the basis of their structures: the carbohy- important component of fungal cell walls and of the exoskel- drate-based polymers, which include the majority of the eton of arthropods. Finally, there is peptidoglycan, a polymer

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Cellulose

Molecular weight up to 1.8 106 Glucose subunits β-1, 4-linked n Glucose subunit (A) Hemicellulose 3 (e.g., pectin)

Molecular weight ϳ40,000

Starch n Galacturonic Methylated Amylopectin acid galacturonic acid (B) Glucose subunits α-1,6 linked branch points

Amylose Amino linkage Acetyl group glucose subunits α-1,4 linked Chitin

N-acetylglucosamine subunits n β-1,4 linked (C) (D)

N-Acetylglucosamine (NAG) N-Acetylmuramic acid (NAM) Peptidoglycan FIGURE 14.6 Common organic polymers found in the environment. (A) Cellulose is the most common plant polymer. It is a linear polymer of NAG-NAM subunits β β-1,4 linked -1,4-link ed glucose subunits. Each polymer contains 1000 to 10,000 subunits. (B) Hemicellulose is the second most common polymer. This molecule is more heterogeneous, consisting of hexoses, pentoses, and Lysozyme - sensitive bond uronic acids. An example of a hemicellulose polymer is pectin. (C) Starch is a polysaccharide synthesized by plants to store energy. Starch is formed from glucose subunits and can be linear ( -1,4-linked), a Peptide bond structure known as amylose, or can be branched ( -1,4 and -1,6 L–Alanine linked), known as amylopectin. (D) Chitin is formed from subunits of β D–Glutamic acid N-acetylglucosamine linked -1,4. This polymer is found in fungal cell walls. (E) Bacterial cell walls are composed of polymers of N-acetylglu- Meso–Diaminopimelic acid cosamine and N-acetylmuramic acid connected by β-1,4 linkages. D–Alanine

(E)

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of N-acetylglucosamine and N -acetylmuramic acid, which is polymerized, resulting in the formation of the amorphous an important component of bacterial cell walls ( Fig. 14.6E ). aromatic polymer known as lignin. In plants lignin sur- rounds cellulose microfibrils and strengthens the cell wall. Lignin Lignin also helps make plants more resistant to pathogens. Biodegradation of lignin is slower and less complete Lignin is the third most common plant polymer and is than degradation of other organic polymers. This is shown strikingly different in structure from all of the carbohy- experimentally in Figure 14.8A, and visually in Figure drate-based polymers. The basic building blocks of lignin 14.8B . Lignin degrades slowly because it is constructed as are the two aromatic amino acids tyrosine and phenylala- a highly heterogeneous polymer and in addition contains nine. These are converted to phenylpropene subunits such aromatic residues rather than carbohydrate residues. The as coumaryl alcohol, coniferyl alcohol, and sinapyl alco- great heterogeneity of the molecule precludes the evolu- hol. Then 500 to 600 phenylpropene subunits are randomly tion of specific degradative enzymes comparable to those for cellulose. Instead, a nonspecific extracellular enzyme, H2 O2 -dependent lignin peroxidase, is used in conjunc- TABLE 14.6 Major Types of Organic Components tion with an extracellular oxidase enzyme that generates of Plants H2 O2 . The peroxidase enzyme and H 2 O2 system gener- Plant component % Dry mass of plant ate oxygen-based free radicals that react with the lignin polymer to release phenylpropene residues (Morgan et al. , Cellulose 15–60 1993). These residues are taken up by microbial cells and Hemicellulose 10–30 degraded as shown in Figure 14.9. Biodegradation of intact Lignin 5–30 lignin polymers occurs only aerobically, which is not surprising because reactive oxygen is needed to release lignin Protein and nucleic acids 2–15 residues. Once residues are released, however, they can be degraded under anaerobic conditions.

Cellulose

β 1-4 exoglucanase β 1-4 endoglucanase

(shorter pieces)

Cellobiose (can be transported into cell) β 1-4 glucosidase (cellobiase)

Glucose

Transport across membrane

Aerobic ercA TCA cycle obina

Fermentation

FIGURE 14.7 The degradation of cellulose begins outside the cell with a series of extracellular enzymes called cellulases. The resulting smaller glucose subunit structures can be taken up by the cell and metabolized.

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100

60 Lignin

40 Wheat straw Percentage remining Percentage 20 Cellulose

0 Hemicellulose 0 100 200 (B) (A) Days FIGURE 14.8 (A) Decomposition of wheat straw and its major constituents in a silt loam. The initial composition of the wheat straw was 50% cellulose, 25% hemicellulose, and 20% lignin. Adapted from Wagner and Wolf, 1998. (B) Leaves from a rain forest in different stages of decomposition. Cellulose and hemicellulose are degraded first, leaving the lignin skeleton. Photo taken in Henry Pittier National Park, Venezuela, courtesy C. M. Miller.

Phenylpropene residues are aromatic in nature (Fig. 14.9), surprising, then, that some of the reactive residues released similar in structure to several types of organic pollutant mol- during polymer degradation might repolymerize and result ecules such as the BTEX (benzene, toluene, ethylbenzene, in the production of humus. In addition, nucleic acid and xylene) and polyaromatic hydrocarbon compounds found protein residues that are released from dying and decaying in crude oil and gasoline, and creosote compounds found in cells contribute to the pool of molecules available for wood preservatives (see Fig. 20.13). These naturally occur- humus formation. This process is illustrated in Figure ring aromatic biodegradation pathways are of considerable 14.10. Considering the wide array of residues that can importance in the field of bioremediation. In fact, a compari- contribute to humus formation, it is not surprising that son of the pathway shown in Figure 14.9 with the pathway humus is even more heterogeneous than lignin. Table 14.7 for degradation of aromatics presented in Chapter 20 shows compares some different properties of these two complex that they are very similar. Lignin is degraded by a variety molecules. of microbes including fungi, actinomycetes, and bacteria. Humus is the most complex organic molecule found The best studied organism with respect to lignin degradation in soil, and as a result it is the most stable organic mole- is the white-rot fungus Phanerochaete chrysosporium . This cule. The turnover rate for humus ranges from 2 to 5% per organism is also capable of degrading several pollutant mol- year, depending on climatic conditions (Wagner and Wolf, ecules with structures similar to those of lignin residues (see 1998). This can be compared with the degradation of lig- Information Box 2.9 and Case Study 20.1). nin shown in Figure 14.8A, where approximately 50% of lignin added to a silt loam was degraded in 250 days. Thus, humus provides a very slowly released source of carbon 14.2.3.2 Humus and energy for indigenous autochthonous microbial popu- Humus was introduced in Section 4.2.2.6 and its structure lations. The release of humic residues most likely occurs in is shown in Figure 4.10. How does humus form? It takes a manner similar to release of lignin residues. Because the a two-stage process that involves the formation of reactive humus content of most soils does not change, the rate of monomers during the degradation of organic matter, fol- formation of humus must be similar to the rate of its turn- lowed by the spontaneous polymerization of some of these over. Thus, humus can be thought of as a molecule that is monomers into the humus molecule. Although the majority in a state of dynamic equilibrium ( Haider, 1992 ). of organic matter that is released into the environment is respired to form new cell mass and carbon dioxide, a small amount of this carbon becomes available to form humus. 14.2.3.3 Methane To understand this spontaneous process, consider the degradation of the common organic polymers found in soil Methanogenesis that were described in the preceding sections. Each of these The formation of methane, methanogenesis, is predomi- polymers requires the production of extracellular enzymes nantly a microbial process, although a small amount of that begin the polymer degradation process. In particular, methane is generated naturally through volcanic activity for lignin these extracellular enzymes are nonspecific and (Table 14.8) (Ehrlich, 1996). Methanogenesis is an anaerobic produce hydrogen peroxide and oxygen radicals. It is not process and occurs extensively in specialized environments

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Lignin polymer

Extracellular enzymes

Lignin monomers (transported into the cell)

Other phenols and various portions of lignin molecules

Coniferyl alcohol Ferulic acid

Vanillic acid Caffeic acid Conyferyl aldehyde

Adjacent hydroxyl groups allow ring Vanillin cleavage

β Protocatechuic -carboxy-cis, cis- acid muconic acid

2 2 222 Acetic β-oxyadipic acid acid Succinic acid

TCA CO22 + H O

FIGURE 14.9 Lignin degradation. Adapted from Wagner and Wolf, 1998 .

including water-saturated areas such as wetlands and paddy At present a substantial amount of methane is released fields; anaerobic niches in the soil; landfills; the rumen; and to the atmosphere as a result of energy harvesting and uti- termite guts. Methane is an end product of anaerobic deg- lization. A second way in which methane is released is radation (see Section 3.4.2) and as such is associated with through landfill gas emissions. It is estimated that landfills petroleum, natural gas, and coal deposits. in the United States alone generate 10 1 0 6 metric tons

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Lignin and other plant Organic residues polyphenolic compounds in general

Microbial degradation Microbial degradation

Smaller polyphenolic units, methoxyphenols, and Sugars, organic acids, intermediate phenols and other simple organic compounds

Oxidation of side chains Microbial synthesis & Degradation Microbial and methyl groups, synthesis hydroxylation of rings, transformations and decarboxylation

CO2, H2O, NH3, and Microbial other simple inorganic cells and compounds products

Ring cleavage, Release and alteration oxidation of microbial phenolic polymers

Numerous mono-, di-, and trihydroxyphenols and benzoic acids

Peptides, amino acids, Enzymatic and amino sugars, and autoxidation polysaccharides containing amino sugars from decomposing organic residues and microbial Phenolic radicals and cell autolysis hydroxybenzoquinones

Polymerization/condensation

HUMIC MATERIAL

FIGURE 14.10 Possible pathways for the formation of soil humus. Adapted with permission from Wagner and Wolf, 1998 .

of methane per year produced as a result of anaerobic deg- avoid accidents, the methane generated in a landfill must radation processes. Although methane makes a relatively be managed in some way. If methane is present in con- minor carbon contribution to the global carbon cycle (com- centrations higher than 35%, it can be collected and used pare Table 14.8 with Table 14.3), methane emission is of for energy. Alternatively, the methane can be burned off at concern from several environmental aspects. First, like concentrations of 15% percent or higher. Most commonly, carbon dioxide, methane is a greenhouse gas and contrib- however, it is simply vented to the atmosphere to prevent it utes to global warming. In fact, it is the second most com- from building up in high enough concentrations to ignite. mon greenhouse gas emitted to the atmosphere. Further, it Although venting landfill gas to the atmosphere does help is 22 times more effective than carbon dioxide at trapping prevent explosions, it clearly adds to the global warming heat. Second, localized production of methane in land- problem. fills can create safety and health concerns. Methane is an The organisms responsible for methanogenesis are a explosive gas at concentrations as small as 5%. Thus, to group of obligately anaerobic archaebacteria called the

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energy for the fixation of carbon dioxide. Methanogens TABLE 14.7 Chemical Properties of Humus and that utilize CO 2 /H2 are therefore autotrophic. In addition to Lignin the autotrophic reaction shown in Eq. 14.1, methanogens Characteristic Humic material Lignin can produce methane during heterotrophic growth on a limited number of other C 1 and C 2 substrates including acetate, Color Black Light brown methanol, and formate. Since there are very few carbon

Methoxyl (–OCH3 ) Low High compounds that can be used by methanogens, these organ- content isms are dependent on the production of these compounds Nitrogen content 3–6% 0% by other microbes in the surrounding community. As such, an interdependent community of microbes typically devel- Carboxyl and phenolic High Low hydroxyl content ops in anaerobic environments. In this community, the more complex organic molecules are catabolized by populations Total exchangeable 150 0.5 acidity (cmol/kg) that ferment or respire anaerobically, generating C1 and C2 carbon substrates as well as CO2 and H2 that are then used -Amino nitrogen Present 0 by methanogens. Vanillin content 1% 15–25%

D ata from Wagner and Wolf 1998. Methane Oxidation Clearly, methane as the end product of anaerobiosis is found extensively in nature. As such, it is an available food source, TABLE 14.8 Estimates of Methane Released into and a group of bacteria called the methanotrophs have the Atmosphere developed the ability to utilize methane as a source of carbon and energy. The methanotrophs are chemoheterotrophic 6 Source Methane emission (10 and obligately aerobic. They metabolize methane as shown metric tons/year) in Eq. 14.2. Biogenic Ruminants 80–100 methane monoxygenase Termites 25–150 →→→ CH42 O CH3 OH HCHO Paddy fi elds 70–120 formaldeehye Natural wetlands 120–200 methane methanol → Landfi lls 5–70 HCOOH CO22 H O Oceans and lakes 1–20 formic acid carbon dioxide Tundra 1–5 (Eq. 14.2) Abiogenic Coal mining 10–35 The first enzyme in the biodegradation pathway is called Natural gas fl aring and venting 10–35 Industrial and pipeline losses 15–45 methane monooxygenase. Oxygenases in general incorpo- Biomass burning 10–40 rate oxygen into a substrate and are important enzymes in Methane hydrates 2–4 the initial degradation steps for hydrocarbons (see Section Volcanoes 0.5 20.6.2.1). However, methane monooxygenase is of particu- Automobiles 0.5 lar interest because it was the first of a series of enzymes Total 350–820 isolated that can cometabolize highly chlorinated solvents Total biogenic 302–665 (81–86% of total) such as trichloroethene (TCE) (see Section 20.6.2.1). Until this discovery it was believed that biodegradation of highly Total abiogenic 48–155 (13–19% of total) chlorinated solvents could occur only under anaerobic con- Adapted from Madigan et al. , 1997. ditions as an incomplete reaction. The application of methanogens for cometabolic degradation of TCE is a strategy under development for bioremediation of groundwater contaminated with TCE. This example is a good illustration of methanogens. The basic metabolic pathway used by the the way in which naturally occurring microbial activities can methanogens is be harnessed to solve pollution problems.

4HCOCHHO→ 2 G 130. 7 kJ 2 242 (Eq. 14.1) 14.2.3.4 Carbon Monoxide and Other C1 Compounds

This is an exothermic reaction where CO2 acts as the terminal Bacteria that can utilize C1 carbon compounds other than electron acceptor and H 2 acts as the electron donor providing methane are called methylotrophs. There are a number of

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TABLE 14.9 C1 Compounds of Major Environmental Importance Compound Formula Comments

Carbon dioxide CO 2 Combustion, respiration, and fermentation end product, a major reservoir of carbon on Earth Carbon monoxide CO Combustion product, common pollutant, product of plant, animal, and microbial respiration, highly toxic

Methane CH 4 End product of anaerobic fermentation or respiration

Methanol CH 3 OH Generated during breakdown of hemicellulose, fermentation by-product Formaldehyde HCHO Combustion product, intermediate metabolite Formate HCOOH Found in plant and animal tissues, fermentation product

Formamide HCONH2 Formed from plant cyanides

Dimethyl ether CH 3 OCH3 Generated from methane by methanotrophs, industrial pollutant Cyanide ion CN Generated by plants, fungi, and bacteria, industrial pollutant, highly toxic

Dimethyl sulﬁ de (CH3 )2 S Most common organic sulfur compound found in the environment, generated by algae

Dimethyl sulfoxide (CH3 )2 SO Generated anaerobically from dimethyl sulﬁ de

22HO HO (Eq. 14.4) important C1 compounds produced from both natural and 22 2 anthropogenic activities (Table 14.9). One of these is car-

bon monoxide. The annual global production of carbon This organism is a chemoautotroph and fixes the CO2 gen- monoxide is 3 – 4 1 0 9 metric tons/year ( Atlas and Bartha, erated in Eq. 14.3 into organic carbon. The oxidation of the 1993). The two major carbon monoxide inputs are abiotic. hydrogen produced provides energy for CO2 fixation (see Approximately 1.5 1 0 9 metric tons/year result from atmo- Eq. 14.4). Under anaerobic conditions, methanogenic bac- spheric photochemical oxidation of carbon compounds such teria can reduce carbon monoxide to methane: as methane, and 1.6 1 0 9 metric tons/year results from burning of wood, forests, and fossil fuels. A small proportion, CO3 H CH H O (Eq. 14.5) 242 0.2 1 0 9 metric tons/year, results from biological activity in

ocean and soil environments. Carbon monoxide is a highly A number of other C1 compounds support the growth toxic molecule because it has a strong affinity for cyto- of methylotrophic bacteria ( Table 14.9 ). Many, but not all, chromes, and in binding to cytochromes, it can completely methylotrophs are also methanotrophic. Both types of bac- inhibit the activity of the respiratory electron transport chain. teria are widespread in the environment because these C1 Destruction of carbon monoxide can occur abiotically compounds are ubiquitous metabolites and, in response to by photochemical reactions in the atmosphere. Microbial their presence, microbes have evolved the capacity to metab- processes also contribute significantly to its destruction, olize them under either aerobic or anaerobic conditions. even though it is a highly toxic molecule. The destruction of carbon monoxide seems to be quite efficient because the level of carbon monoxide in the atmosphere has not risen 14.3 NITROGEN CYCLE significantly since industrialization began, even though CO emissions have increased. The ocean is a net producer of In contrast to carbon, elements such as nitrogen, sulfur, and carbon monoxide and releases CO to the atmosphere. In iron are taken up in the form of mineral salts and cycle oxi- contrast, the terrestrial environment is a net sink for carbon doreductively. For example, nitrogen can exist in numerous 9 monoxide and absorbs approximately 0.4 1 0 metric oxidation states, from 3 in ammonium (NH4 ) to 5 in tons/year. Key microbes found in terrestrial environments nitrate (NO3 ). These element cycles are referred to as the that can metabolize carbon monoxide include both aero- mineral cycles. The best studied and most complex of the bic and anaerobic organisms. Under aerobic conditions mineral cycles is the nitrogen cycle (Fig. 14.11). There is Pseudomonas carboxydoflava is an example of an organ- great interest in the nitrogen cycle because nitrogen is the ism that oxidizes carbon monoxide to carbon dioxide: mineral nutrient most in demand by microorganisms and plants. It is the fourth most common element found in CO H O CO H (Eq. 14.3) cells, making up approximately 12% of cell dry weight. 222

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Aerobic N2 Nitrogen fixation Nitrification NH4 NO2 N2O Ammonium assimilation

noitacifirtineD Mineralization noitacifirtiN

Assimilatory NO R-NH

DNRA 2 reduction

NO 3

NO2 NO3 Dissimilatory NO3 reduction Anaerobic

FIGURE 14.11 The nitrogen cycle.

The nitrogen cycle includes the microbially catalyzed processes of nitrogen fixation, ammonium oxidation, assimi- TABLE 14.10 Global Nitrogen Reservoirs latory and dissimilatory nitrate reduction, ammonification, Nitrogen reservoir Metric tons Actively cycled and ammonium assimilation. nitrogen Atmosphere 15 14.3.1 Nitrogen Reservoirs N2 3.9 1 0 No Ocean 8 Nitrogen in the form of the inert gas, dinitrogen (N2 ), has Biomass 5.2 1 0 Yes accumulated in Earth’s atmosphere since the planet was Dissolved and 3.0 1 011 Yes particulate organics formed. Nitrogen gas is continually released into the atmo- 11 Soluble salts (NO 3 , 6.9 1 0 Yes sphere from volcanic and hydrothermal eruptions, and NO2 , NH4 ) 13 is one of the major global reservoirs of nitrogen (Table Dissolved N 2 2.0 1 0 No 14.10). A second major reservoir is the nitrogen that is Land found in Earth’s crust as bound, nonexchangeable ammo- Biota 2.5 1 0 10 Yes nium. Neither of these reservoirs is actively cycled; the Organic matter 1.1 1 0 11 Slow a 14 nitrogen in Earth’s crust is unavailable and the N 2 in the Earth’s crust 7.7 1 0 No atmosphere must be fixed before it is available for biologi- Adapted from Dobrovolsky, 1994. cal use. Because nitrogen fixation is an energy-intensive a This reservoir includes the entire lithosphere found in either terrestrial or ocean process and is carried out by a limited number of microor- environments. ganisms, it is a relatively slow process. Smaller reservoirs of nitrogen include the organic nitrogen found in living biomass and in dead organic matter and soluble inorganic nitrogen salts. These small reservoirs tend to be actively aquatic environments are compared with human fertilizer cycled, particularly because nitrogen is often a limiting inputs in T able 14.11. Approximately 65% of the N fixed nutrient in the environment. For example, soluble inorganic 2 annually is from terrestrial environments, including both nitrogen salts in terrestrial environments can have turnover natural systems and managed agricultural systems. Marine rates greater than once per day. Nitrogen in plant biomass ecosystems account for a smaller proportion, 20%, of N turns over approximately once a year, and nitrogen in 2 fixation. Nitrogen fixation is an energy-intensive process organic matter turns over once in several decades. and until recently was performed only by selected bacteria and cyanobacteria. However, a substantial amount of 14.3.2 Nitrogen Fixation nitrogen fixation, 15% of the total N 2 fixed, now occurs through the manufacture of fertilizers. Because this is an Ultimately, all fixed forms of nitrogen, NH 4 , NO3 , and energy-driven process, fertilizer prices are tied to the price organic N, come from atmospheric N2 . The relative contri- of fossil fuels. As fertilizers are expensive, management butions to nitrogen fixation of microbes in terrestrial and alternatives to fertilizer addition have become attractive.

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TABLE 14.11 Relative Inputs of Nitrogen Fixation TABLE 14.13 Rates of Nitrogen Fixation from Biological Sources N2 -fi xing system Nitrogen fi xation Source Nitrogen fi xation (kg N/hectare/year) (metric tons/year) Rhizobium –legume 200–300 8 Terrestrial 1.35 1 0 Anabaena–Azolla 100–120 7 Aquatic 4.0 1 0 Cyanobacteria–moss 30–40 7 Fertilizer manufacture 3.0 1 0 Rhizosphere associations 2–25 Free-living 1–2

TABLE 14.12 Representative Genera of Free-Living plant root fix small amounts of nitrogen (1 to 2 kg N/hect- Nitrogen-Fixers are/year). Bacterial cells associated with the nutrient-rich Status with Mode of energy Genus rhizosphere environment can fix larger amounts of N2 (2 to respect to oxygen generation 25 kg N/hectare/year). Cyanobacteria are the predominant Aerobic Heterotrophic Azotobacter N2 -fixing organisms in aquatic environments, and because Beijerinckia they are photosynthetic, N2 fixation rates are one to two

Acetobacter orders of magnitude higher than for free-living nonpho- Pseudomonas tosynthetic bacteria. An evolutionary strategy developed Facultatively Heterotrophic Klebsiella collaboratively by plants and microbes to increase N2 fixa- anaerobic Bacillus tion efficiency was to enter into a symbiotic or mutualistic relationship to maximize N fixation (see Information Microaerophilic Heterotrophic Xanthobacter 2 Azospirillum Box 14.2). The best studied of these symbioses is the Rhizobium –legume relationship, which can increase N fix- Strictly anaerobic Autotrophic Thiobacillus 2 Heterotrophic Clostridium ation to 200 to 300 kg N/hectare/year. This symbiosis irre-

Desulfovibrio vocably changes both the plant and the microbe involved but is beneficial to both organisms. Aerobic Phototrophic Anabaena (cyanobacteria) Nostoc As the various transformations of nitrogen are discussed in this section, the objective is to understand how they are Facultatively Phototrophic (bacteria) Rhodospirillum interconnected and controlled. As already mentioned, N anaerobic 2 fixation is limited to bacteria and is an energy-intensive Strictly anaerobic Phototrophic (bacteria) Chlorobium process (see Information Box 14.3). Therefore, it does not Chromatium make sense for a microbe to fix N 2 if sufficient amounts are present for growth. Thus, one control on this part of the nitrogen cycle is that ammonia, the end product of N 2 fixation, is an inhibitor for the N 2 fixation reaction. A sec- These include rotation between nitrogen-fixing crops such ond control in some situations is the presence of oxygen. as soybeans and nonfixing crops such as corn. Wastewater Nitrogenase is extremely oxygen sensitive, and some free- reuse is another alternative that has become especially pop- living aerobic bacteria fix N2 only at reduced oxygen ten- ular in the desert southwestern United States for nonfood sion. Other bacteria such as Azotobacter and Beijerinckia crops and uses, such as cotton and golf courses, where both can fix N 2 at normal oxygen tension because they have water and nitrogen are limiting (see Chapter 24). developed mechanisms to protect the nitrogenase enzyme. Nitrogen is fixed into ammonia (NH 3) by over 100 different free-living bacteria, both aerobic and anaerobic, as well as some actinomycetes and cyanobacteria ( Table 14.12 ). Summary for Nitrogen Fixation For example, Azotobacter (aerobic), Beijerinckia (aerobic), Azospirillum (facultative), and Clostridium (anaerobic) ● N2 fixation is energy intensive can all fix N . Because fixed nitrogen is required by all 2 ● End product of N2 fixation is ammonia biological organisms, nitrogen-fixing organisms occur in ● N2 fixation is inhibited by ammonia most environmental niches. The amount of N fixed in 2 ● Nitrogenase is O2 sensitive, some free-living N2 ; fixers each niche depends on the environment ( Table 14.13). require reduced O 2 tension Free-living bacterial cells that are not in the vicinity of a

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Information Box 14.2 The Legume–Rhizobia Symbiosis

Gram-negative heterotrophic bacteria classified within the gen- 2 weeks after infection). Within the root nodule, the rhizobia era Rhizobium , Bradyrhizobium , Sinorhizobium , and Azorhizobium can enlarge and elongate to perhaps five times the normal size of form an intriguing symbiosis with leguminous plants. This sym- rhizobia and change physiologically to forms known as bacter- biosis has been well studied because it can meet or nearly meet oids. The nodule also contains leghemoglobin, which protects the nitrogen requirements of economically important legume the nitrogenase enzyme within the bacteroids from the pres- crops, reducing or eliminating the need for fertilizer applica- ence of oxygen. The leghemoglobin imparts a pink color to tion. Grain legumes such as peas, beans, and soybeans can fix the interior of the nodule and is indicative of active nitrogen about 50% of their total nitrogen requirements, whereas forage fixation. Thus, examination of the interior of a nodule allows legumes such as alfalfa or clover are even more efficient, achiev- instant determination of whether the nodule is active. Prior to ing nitrogen fixation rates as high as 200 to 300 kg per hectare the end of the growing season, nodules begin to break down or per year. The formation of root nodules on the plant host root senesce, at which point they appear white, green, or brown. system is the result of subtle interactions between the host Commercial legume crops are often aided in terms of nitro- and the rhizobial endosymbiont ( Jones et al. , 2007 ). The plant gen fixation through the application of rhizobial inoculants. releases bacteria-specific phenolic compounds called flavo- This is particularly important when a new legume species is noids that in turn signal rhizobia to produce plant-specific lipo- introduced into soils that are free of indigenous rhizobia. In this chitooligosaccharide compounds called Nod factors. The Nod case, rhizobia introduced into the soil through the use of inocu- factors activate a series of host plant responses that prepare lants tend to establish themselves in the available ecological the plant to form infection threads, which the invading bacteria niche and are difficult to displace by any subsequently intro- use to enter the plant. The infection thread is a thin tubule that duced rhizobia. Therefore, in such situations it is important that penetrates into the plant cortex. Plant cells in the cortex receive the originally introduced rhizobia are appropriate. The desired the invading bacteria, after which they mature into structures characteristics include being infective (capable of causing nod- known as symbiosomes. The bacteria in the symbiosome ule initiation and development), effective (capable of efficient then differentiate into bacteroid cells that are basically nitrogen nitrogen fixation), competitive (capable of causing nodule initi- fixation factories. Also contributing to the success of this amaz- ation in the presence of other rhizobia), and persistent (capable ing invasion process is the fact that the rhizobia can evade all of surviving in soil between crops in successive years). Usually host plant immune responses. rhizobia are impregnated into some kind of peat-based carrier As rhizobia are released from the infection thread, cell divi- with approximately 109 rhizobia per gram of peat. Production of sion occurs, and a visible root nodule begins to be seen (1 to commercial rhizobium inoculants is a big business globally.

Nod Factor nod genes Rhizobium

Flavoniod Colonized curled Nod root hair factor receptor

Cortical cell division

The process of legume root hair infection by rhizobia.

14.3.3 Ammonia Assimilation near-neutral pH. Thus, it is generally the ammonium form (Immobilization) and Ammoniﬁ cation that is assimilated by cells into amino acids to form pro- (Mineralization) teins, cell wall components such as N-acetylmuramic acid, and purines and pyrimidines to form nucleic acids. This The end product of N2 fixation is ammonia (Fig. 14.11). process is known as ammonium assimilation or immobiliza- In the environment, there exists an equilibrium between tion. Nitrogen can also be immobilized by the uptake and ammonia (NH3 ) and ammonium (NH4 ) that is driven by incorporation of nitrate into organic matter, a process known pH. This equilibrium favors ammonium formation at acid or as assimilatory nitrate reduction (Section 14.3.5.1). Because

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Information Box 14.3 The Process of Nitrogen Fixation

Nitrogen gas is a very stable molecule that requires a large oxygen. One oxygen-insensitive system has been reported from amount of energy (946 kJ per mole) to fix into ammonia. The the actinomycete Streptomyces thermoautotrophicus . energy for this process arises from the oxidation of carbon The dinitrogenase reductase and dinitrogenase form a sources in the case of heterotrophs or from light in the case complex during which an electron is transferred and the two of photosynthetic diazotrophs. A diazotroph is a microorgan- MgATPs are hydrolyzed to MgADP inorganic phosphate (P i ). ism that can fix nitrogen and so does not require fixed nitrogen The two proteins then dissociate and the process repeats. After for growth. Central to biological nitrogen fixation is the enzyme the dinitrogenase protein has collected sufficient electrons, complex nitrogenase ( Zhao et al. , 2006 ). The overall nitrogenase it binds a molecule of nitrogen gas and reduces it, producing

complex consists of two protein components, which in turn ammonia and hydrogen gas. Thus, during reduction of one N 2 consist of multiple subunits. The iron protein termed dinitro- molecule, the two proteins must complex and then dissociate a genase reductase is thought to function in the reduction of total of eight times. This is the rate-limiting step of the process the molybdenum–iron protein dinitrogenase, which reduces and takes considerable time. In fact, it takes 1.25 seconds for a

nitrogen gas to ammonia. In the 1980s, it was shown that some molecule of enzyme to reduce one molecule of N 2 (Sylvia et al., nitrogenase enzymes do not contain molybdenum and that 2004). This is why nitrogen-fixing bacteria require a great deal vanadium and other metals can substitute for molybdenum. To of the nitrogenase enzyme, which can constitute 10–40% of the date, three nitrogenase systems have been identified including bacterial cell’s proteins. Nif, Vnf, and Anf, all of which are sensitive to and inhibited by

Fd (oxidized) FeMo-cofactor

N2 8H Fd (reduced) 8e

nMg-ATP

2NH3 H2

nMg-ADP nPi C2H2 2H C2H4 (n 16) Dehydrogenase Dinitrogenase reductase (iron-molybdenum (iron protein) protein)

Overall reaction: N2 8H 8e 16Mg-ATP 2NH3 H2 16Mg-ADP 16Pi

nitrate must be reduced to ammonium before it is incorpo- availability. At high ammonium concentrations ( 0.1 m M rated into organic molecules, most organisms prefer to take or 0.5 mg N/kg soil), in the presence of reducing up nitrogen as ammonium if it is available. The process that equivalents (reduced nicotinamide adenine dinucleotide, reverses immobilization, the release of ammonia from dead NADH), ammonium is incorporated into -ketoglutarate and decaying cells, is called ammonification or ammonium to form glutamate. However, in most soil and many aquatic mineralization. Both immobilization and mineralization of environments, ammonium is present at low concentrations. nitrogen occur under aerobic and anaerobic conditions. Therefore microbes have a second ammonium uptake pathway that is energy dependent. This reaction is driven by ATP and two enzymes, glutamine synthase and glutamate 14.3.3.1 Ammonia Assimilation (Immobilization) synthetase (GOGAT, Fig. 14.12B). The first step in this There are two pathways that microbes use to assimilate reaction adds ammonium to glutamate to form glutamine, ammonium. The first is a reversible reaction that incorpo- and the second step transfers the ammonium molecule rates or removes ammonium from the amino acid gluta- from glutamine to -ketoglutarate, resulting in the forma- mate ( Fig. 14.12A ). This reaction is driven by ammonium tion of two glutamate molecules.

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(A) COO COO

HC NH3 C O glutamate CH H O CH 2 2 dehydrogenase 2 NH4

CH2 CH2 COO NAD NADH COO glutamate α-ketoglutarate

(B) COO

HC NH3 COO ADP Pi NH4 CH2 C O ATP CH2 CH COO glutamine C 2 synthetase O NH2 CH2 HC NH3 glutamine COO CH2 α - ketoglutarate CH 2 Ferredoxin C 2e O O glutamate- glutamate synthase (GOGAT) 2H

COO HC NH3

CH2

CH2 COO Transamination glutamate

FIGURE 14.12 Pathways of ammonium assimilation and ammonification (Paul and Clark, 1996). Assimilation: (A) The enzyme glutamate dehydrogenase catalyzes a reversible reaction that immobilizes ammonium at high ammonium concentrations. (B) The enzyme system glutamine synthase–glutamate synthetase (GOGAT) that is induced at low ammonium concentrations. This ammonium uptake system requires ATP energy. Ammonification: Ammonium is released from the amino acid glutamate as shown in (A). (C) Ammonia is also released from urea, a molecule that is found in animal waste and in some fertilizers. Note that the first of these reactions (A and B) occurs within the cell. In contrast, urease enzymes are extracellular enzymes, resulting in release of ammonia to the environment.

14.3.3.2 Ammoniﬁ cation (Mineralization) Section 14.2.3.1, microorganisms release a variety of extra- Ammonium mineralization can occur intracellularly by the cellular enzymes that initiate degradation of plant poly- reversible reaction shown in Figure 14.12A . Mineralization mers. Microorganisms also release a variety of enzymes reactions can also occur extracellularly. As discussed in including proteases, lysozymes, nucleases, and ureases that

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can initiate degradation of nitrogen-containing molecules found outside the cell such as proteins, cell walls, nucleic TABLE 14.14 Chemoautotrophic Nitrifying Bacteria acids, and urea. Some of these monomers are taken up by Genus Species the cell and degraded further, but some of the monomers are acted on by extracellular enzymes to release ammonium Ammonium oxidizers Nitrosomonas europaea directly to the environment, as shown in Figure 14.12C eutrophus for urea and the extracellular enzyme urease. marina Which of these two processes, immobilization or miner- Nitrosococcus nitrosus alization, predominates in the environment? It depends on mobilis whether nitrogen is the limiting nutrient. If nitrogen is lim- oceanus iting, then immobilization will become the more important Nitrosospira briensis process. For environments where nitrogen is not limiting, Nitrosolobus multiformis mineralization will predominate. Nitrogen limitation is dic- Nitrosovibrio tenuis tated by the carbon/nitrogen (C/N) ratio in the environment. Nitrite oxidizers Generally, the C/N ratio required for bacteria is 5 and for Nitrobacter winogradskyi fungi is 10. So a typical average C/N ratio for soil micro- hamburgensis vulgaris bial biomass is 8 ( Sylvia et al., 2004 ). It would then seem Nitrospina gracilis logical that at the C/N ratio of 8, there would be a net bal- Nitrococcus mobilis ance between mineralization and immobilization. However, Nitrospira marina one must take into account that only approximately 40% of the carbon in organic matter is actually incorporated into cell mass (the rest is lost as carbon dioxide during respiration). Thus, the C/N ratio must be increased by a fac- 14.3.4 Nitriﬁ cation tor of 2.5 to account for the carbon lost as carbon dioxide Nitrification is the microbially catalyzed conversion of ammo- during respiration. Note that nitrogen is cycled more effi- nium to nitrate ( Fig. 14.11 ). Although this is predominantly an ciently than carbon, and there are essentially no losses in aerobic chemoautotrophic process, some methylotrophs can its uptake. In fact, a C/N ratio of 20 is not only the theo- use the methane monooxygenase enzyme to oxidize ammo- retical balance point but also the practically observed one. nium and a few heterotrophic fungi and bacteria can also per- When organic amendments with C/N ratios less than 20 are form this oxidation. The autotrophic nitrifiers are a closely added to soil, net mineralization of ammonium occurs. In related group of bacteria (Table 14.14). The best studied contrast, when organic amendments with C/N ratios greater nitrifiers are from the genus Nitrosomonas, which oxidizes than 20 are added, net immobilization occurs. ammonium to nitrite, and Nitrobacter, which oxidizes nitrite There are numerous possible fates for ammonium that is to nitrate. The oxidation of ammonium is shown in Eq. 14.6: released into the environment as a result of ammonium min- → eralization. It can be taken up by plants or microorganisms NH42215. O NO2 H H 2 O and incorporated into living biomass, or it can become G 267. 5 kJ/mol bound to nonliving organic matter such as soil colloids (Eq. 14.6) or humus. In this capacity, ammonium adds to the cat- This is a two-step energy-producing reaction and the ion-exchange capacity (CEC) of the soil. Ammonium can energy produced is used to fix carbon dioxide. There are become fixed inside clay minerals, which essentially traps two things to note about this reaction. First, it is an inef- the molecule and removes the ammonium from active ficient reaction, requiring 34 moles of ammonium to fix cycling. Also, because ammonium is volatile, some miner- 1 mole of carbon dioxide. Second, the first step of this alized ammonium can escape into the atmosphere. Finally, reaction is catalyzed by the enzyme ammonium mono- ammonium can be utilized by chemoautotrophic microbes oxygenase. This first step is analogous to Eq. 14.2, where in a process known as nitrification. the enzyme methane monooxygenase initiates oxidation of methane. Like methane monooxygenase, ammonium monooxygenase has broad substrate specificity and can be Summary for Ammonia Assimilation and used to oxidize pollutant molecules such as TCE cometa- Mineralization bolically (see Section 20.6.2.1). The second step in nitrification, shown in Eq. 14.7, is ● Assimilation and mineralization cycles ammonia even less efficient than the first step, requiring approxi- between its organic and inorganic forms mately 100 moles of nitrite to fix 1 mole of carbon dioxide. ● Assimilation predominates at C:N ratios > 20, NO0.. 5 O→ NO G 86 96 kJ/mol mineralization predominates at C:N ratios < 20 223 (Eq. 14.7)

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These two types of nitrifiers (i.e., those that carry out the and surface waters as one possible fate. In addition, nitrate reactions shown in Eqs. 14.6 and 14.7 ) are generally found can be taken up and incorporated into living biomass by together in the environment. As a result, nitrite does not plants and microorganisms. The uptake of nitrate is fol- normally accumulate in the environment. Nitrifiers are sen- lowed by its reduction to ammonium, which is then incor- sitive populations. The optimum pH for nitrification is 6.6 porated into biomass. This process is called assimilatory to 8.0. In environments with pH 6.0, nitrification rates nitrate reduction or nitrate immobilization. Finally, micro- are slowed, and below pH 4.5, nitrification seems to be organisms can utilize nitrate as a terminal electron acceptor completely inhibited. in anaerobic respiration to drive the oxidation of organic Heterotrophic microbes that oxidize ammonium include compounds. There are two separate pathways for this dis- some fungi and some bacteria. These organisms gain no similatory process, one called dissimilatory nitrate reduc- energy from nitrification, so it is unclear why they carry tion to ammonium, where ammonium is the end product, out the reaction. The relative importance of autotrophic and one called denitrification, where a mixture of gaseous and heterotrophic nitrification in the environment has not products including N 2 and N2 O is formed. yet been clearly determined. Although the measured rates of autotrophic nitrification in the laboratory are an order 14.3.5.1 Assimilatory Nitrate Reduction of magnitude higher than those of heterotrophic nitrifica- Assimilatory nitrate reduction refers to the uptake of tion, some data for acidic forest soils have indicated that nitrate, its reduction to ammonium, and its incorporation heterotrophic nitrification may be more important in such into biomass (see Fig. 14.12A and B). Most microbes uti- environments ( Syliva et al., 2004 ). lize ammonium preferentially, when it is present, to avoid Nitrate does not normally accumulate in natural, undis- having to reduce nitrate to ammonium, a process requir- turbed ecosystems. There are several reasons for this. One is ing energy. So if ammonium is present in the environment, that nitrifiers are sensitive to many environmental stresses. assimilatory nitrate reduction is suppressed. Oxygen does But perhaps the most important reason is that natural eco- not inhibit this activity. In contrast to microbes, for plants systems do not have much excess ammonium. However, in that are actively photosynthesizing and producing energy, agricultural systems that have large inputs of fertilizer, nitri- the uptake of nitrate for assimilation is less problematic fication can become an important process resulting in the in terms of energy. In fact, because nitrate is much more production of large amounts of nitrate. Other examples of mobile than ammonium, it is possible that, in the vicin- managed systems that result in increased nitrogen inputs into ity of the plant roots, nitrification of ammonium to nitrate the environment are feedlots, septic tanks, and landfills. The makes nitrogen more available for plant uptake. Because nitrogen released from these systems also becomes subject to this process incorporates nitrate into biomass, it is also nitrification processes. Because nitrate is an anion (negatively known as nitrate immobilization (see Section 14.3.3). charged), it is very mobile in soil systems, which also have an overall net negative charge. Therefore, nitrate moves easily with water and this results in nitrate leaching into ground- Summary for Assimilatory Nitrate Reduction water and surface waters. There are several health concerns related to high levels of nitrate in groundwater, including ● Nitrate taken up must be reduced to ammonia before it methemoglobinemia and the formation of nitrosamines. High is assimilated levels of nitrate in surface waters can also lead to eutrophica- ● Ammonia inhibits this process tion and the degradation of surface aquatic systems. These ● O2 does not inhibit this process consequences as well as the control of nitrification in managed systems are discussed in detail in Section 15.6. 14.3.5.2 Dissimilatory Nitrate Reduction Dissimilatory Nitrate Reduction to Ammonium Summary for Nitriﬁ cation There are two separate dissimilatory nitrate reduction processes, both of which are used by facultative chemohet- ● Nitrification is an aerobic process erotrophic organisms under microaerophilic or anaerobic ● Nitrification is sensitive to a variety of chemical inhibitors and is inhibited at low pH conditions (Fig. 14.11). The first process, called dissimilatory nitrate reduction to ammonium (DNRA), uses nitrate ● Nitrification processes in managed systems can result in nitrate leaching and groundwater contamination as a terminal electron acceptor to produce energy to drive the oxidation of organic compounds. The end product of DRNA is ammonium:

14.3.5 Nitrate Reduction → NO3242 H H NH 42 3 H O What are the possible fates of nitrate in the environment? G 603kJ/ 8e transfer We have just discussed nitrate leaching into groundwater (Eq. 14.8)

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The first step in this reaction, the reduction of nitrate to nitrite, is the energy-producing step. The further reduc- TABLE 14.15 Bacteria That Utilize Dissimilatory tion of nitrite to ammonium is catalyzed by an NADH- Nitrate or Nitrite to Ammonium (DRNA) dependent reductase. This second step provides no addi- Genus Typical habitat tional energy, but it does conserve fixed nitrogen and also regenerates reducing equivalents through the reoxidation of Obligate anaerobes Clostridium Soil, sediment NADH to NAD . These reducing equivalents are then used Desulfovibrio Sediment to help in the oxidation of carbon substrates. In fact, it has Selenomonas Rumen been demonstrated that under carbon-limiting conditions Veillonella Intestinal tract nitrite accumulates (denitrification predominates), whereas Wolinella Rumen under carbon-rich conditions ammonium is the major prod- Facultative anaerobes uct (DNRA predominates). A second environmental fac- Citrobacter Soil, wastewater tor that selects for DNRA is low levels of available electron Enterobacter Soil, wastewater acceptors. It is not surprising therefore that this process is Erwinia Soil Escherichia Soil, wastewater found predominantly in saturated, carbon-rich environments Klebsiella Soil, wastewater such as stagnant water, sewage sludge, some sediments high Photobacterium Seawater in organic matter, and the rumen. Table 14.15 lists a variety Salmonella Sewage of bacteria that perform DNRA. It is interesting to note that Serratia Intestinal tract most of the bacteria on this list have fermentative rather than Vibrio Sediment oxidative metabolisms. Microaerophiles Campylobacter Oral cavity Aerobes Summary for Dissimilatory Reduction of Bacillus Soil, food Nitrate to Ammonia Neisseria Mucous membranes Pseudomonas Soil, water

● Anaerobic respiration using nitrate as TEA Adapted from Tiedje, 1988.

● Inhibited by O 2 ● Not inhibited by ammonia ● Found in a limited number of carbon rich, TEA poor % N (Denitrification) environments 2 200 40 60 80 100 ● Fermentative bacteria predominate Rumen Digested sludge

Denitriﬁ cation Estuarine sediments The second type of dissimilatory nitrate reduction is known

as denitrification. Denitrification refers to the microbial acceptor Lake sediments reduction of nitrate, through various gaseous inorganic

C/e (relative scale) forms, to N2 . This is the primary type of dissimilatory Soil C nitrate reduction found in soil, and as such is of concern Soil because it cycles fixed nitrogen back into N2 . This process 100 80 60 40 20 0 removes a limiting nutrient from the environment. Further, some of the gaseous intermediates formed during denitrifi- % NH4 (Dissimilatory reduction) cation, for example, nitrous oxide (N O), can cause deple- FIGURE 14.13 Partitioning of nitrate between denitrification and DNRA 2 tion of the ozone layer and can also act as a greenhouse as a function of available carbon/electron (C/e ) acceptor ratio. Adapted from J. M. Tiedje in Biology of Anaerobic Microorganisms, A. J. B. Zehnder, gas contributing to global warming (see Section 15.5). The ed. © 1988. Reprinted by permission of John Wiley & Sons, Inc. overall reaction for denitrification is → 252NO32 HHNHO 22 6 a carbon-limited, electron acceptor-rich environment, denitrification will be the preferred process because it provides G 888kJ/ 8 e transfer more energy than DNRA. The relationship between denitrification and DNRA is summarized in Figure 14.13 . (Eq. 14.9) The four steps involved in denitrification are shown in Denitrif ication, when calculated in terms of energy pro- more detail in Figure 14.14 . The first step, reduction of duced for every eight-electron transfer, provides more nitrate to nitrite, is catalyzed by the enzyme nitrate reduc- energy per mole of nitrate reduced than DNRA. Thus, in tase. This is a membrane-bound molybdenum–iron–sulfur

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Outside cell NO3 NO2 NO N2O N2

Outer membrane

nitrite reductase nitrous oxide N O reductase N NO2 NO 2 2

Periplasm

Inner membrane nitrate reductase

nitric oxide reductase Cytoplasm NO 3 NO2 FIGURE 14.14 The denitrification pathway. Adapted from Myrold, 1998 .

Region of O2 Region of O control of synthesis control of 2 activity

N2O red Hyphomicrobium NO2 red NO3 red Thiobacillus denitrificans

Nar Nir

0 0.32 0.64 0.96 1.28 1.6 3.2 Oxygen concentration (mg/L) FIGURE 14.15 Approximate regions of oxygen concentration that inhibit the enzyme activity and synthesis for three steps in the denitrification pathway. Adapted from J. M. Tiedje in Biology of Anaerobic Microorganisms , A. J. B. Zehnder, ed. © 1988. Reprinted by permission of John Wiley & Sons, Inc.

protein that is found not only in denitrifiers but also in a heme form, both of which are distributed widely in the DNRA organisms. Both the synthesis and the activity environment. Synthesis of nitrite reductase is inhibited by of nitrate reductase are inhibited by oxygen. Thus, both oxygen and induced by nitrate. Nitric oxide reductase, a denitrification and DNRA are inhibited by oxygen. The membrane-bound protein, is the third enzyme in the path- second enzyme in the pathway is nitrite reductase, which way, catalyzing the conversion of nitric oxide to nitrous catalyzes the conversion of nitrite to nitric oxide. Nitrite oxide. The synthesis of this enzyme is inhibited by oxy- reductase is unique to denitrifying organisms and is not gen and induced by various nitrogen oxide forms. Nitrous present in the DNRA process. It is found in the periplasm oxide reductase is the last enzyme in the pathway and con- and exists in two forms, a copper-containing form and verts nitrous oxide to dinitrogen gas. This is a periplasmic

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copper-containing protein. The activity of the nitrous oxide reductase enzyme is inhibited by low pH and is even more TABLE 14.16 Genera of Denitrifying Bacteria sensitive to oxygen than the other three enzymes in the Genus Interesting characteristics denitrification pathway. Thus, nitrous oxide is the final product of denitrification under conditions of high oxy- Organotrophs Alcaligenes Common soil bacterium gen (in a relative sense, given a microaerophilic niche) and Agrobacterium Some species are plant pathogens low pH. In summary, both the synthesis and activity of Aquaspirillum Some are magnetotactic, oligotrophic denitrification enzymes are controlled by oxygen. Enzyme Azospirillum Associative N2 -fi xer, fermentative activity is more sensitive to oxygen than enzyme synthesis, Bacillus Spore former, fermentative, some as shown in Figure 14.15. The amount of dissolved oxy- species thermophilic Blastobacter Budding bacterium, phylogenetically gen in equilibrium with water at 20°C and 1 atm pressure r elated to Rhizobium is 9.3 mg/l. However, as little as 0.5 mg/l or less inhibits Bradyrhizobium Symbiotic N2 -fi xer with legumes the activity of denitrification enzymes. As already stated, Branhamella Animal pathogen nitrous oxide reductase is the most sensitive denitrification Chromobacterium Purple pigmentation enzyme and it is inhibited by dissolved oxygen concentra- Cytophaga Gliding bacterium; cellulose-degrader Flavobacterium Common soil bacterium tions of less than 0.2 mg/l. Flexibacter Gliding bacterium Whereas the denitrification pathway is very sensitive Halobacterium Halophilic to oxygen, neither it nor the DNRA pathway is inhibited Hyphomicrobium Grows on C1 substrates, oligotrophic by ammonium as is the assimilatory nitrate reduction path- Kingella Animal pathogen way. However, the initial nitrate level in an environmental Neisseria Animal pathogen Paracoccus Halophilic, also lithotrophic system can help determine the extent of the denitrifica- Propionibacterium Fermentative tion pathway. Low nitrate levels tend to favor production Pseudomonas Commonly isolated from soil, very of nitrous oxide as the end product. High nitrate levels diverse genus Rhizobium Symbiotic N -fi xer with legumes favor production of N 2 gas, a much more desirable end 2 product. Wolinella Animal pathogen Organisms that denitrify are found widely in the environ- Phototrophs ment and display a variety of different characteristics in terms Rhodopseudomonas Anaerobic, sulfate-reducer of metabolism and activities. In contrast to DNRA organisms, Lithotrophs which are predominantly heterotrophic using fermentative Alcaligenes Uses H2 , also heterotrophic, common soil metabolism, the majority of denitrifiers also are heterotro- isolate phic but use respiratory pathways of metabolism. However, Bradyrhizobium Uses H2 , also heterotrophic, symbiotic Nitrosomonas N -fi xer with legumes as shown in Table 14.16 , some denitrifiers are autotrophic, 2 Paracoccus NH3 -oxidizer some are fermentative, and some are associated with other Pseudomonas Uses H2 , also heterotrophic, halophilic aspects of the nitrogen cycle (e.g., they can fix N 2 ). Thiobacillus Uses H2 , also heterotrophic, common soil Thiomicrospira isolate Thiosphaera S-oxidizer S-oxidizer Summary for Denitrifi cation S-oxidizer, heterotrophic nitrifi er, aerobic denitrifer

● Anaerobic respiration using nitrate as TEA From Myrold, 1998. ● Inhibited by O2 ● Not inhibited by ammonia

● Produces a mix of N2 and N2O ● Many heterotrophic bacteria are denitrifiers (S2 ) (Fig 14.16). In cells, sulfur is required for synthesis of the amino acids cysteine and methionine and is also required for some vitamins, hormones, and coenzymes. In 14.4 SULFUR CYCLE proteins, the sulfur-containing amino acid cysteine is especially important because the formation of disulfide bridges Sulfur is the tenth most abundant element in Earth’s crust. between cysteine residues helps govern protein folding and It is an essential element for biological organisms, making hence activity. All of these compounds contain sulfur in up approximately 1% of the dry weight of a bacterial cell the reduced or sulfide form. Cells also contain organic sul- ( Table 14.1 ). Sulfur is not generally considered a limiting fur compounds in which the sulfur is in the oxidized state. nutrient in the environment except in some intensive agricul- Examples of such compounds are glucose sulfate, choline tural systems with high crop yields. Sulfur is cycled between sulfate, phenolic sulfate, and two ATP-sulfate compounds 2 oxidation states of 6 for sulfate (SO4 ) and 2 for sulfide that are required in sulfate assimilation and can also serve to

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FIGURE 14.16 The sulfur cycle. Aerobic Sulfide oxidation o HS2 S

Sulfur mineralization

noitaripserrufluS noitadixorufluS

noitadixocihportotohP

R-SH Assimilatory sufate reduction reduction

etaflu s yr otali m is si D

o 2 S SO4 Phototrophic oxidation Anaerobic

store sulfur for the cell. Although the sulfur cycle is not as complex as the nitrogen cycle, the global impacts of the sul- TABLE 14.17 Global Sulfur Reservoirs fur cycle are extremely important, including the formation Sulfur reservoir Metric tons Actively of acid rain, acid mine drainage, and corrosion of concrete sulfur cycled and metal (see Section 15.2 and 15.3). Atmosphere 6 SO 2 /H 2 S 1.4 1 0 Yes 14.4.1 Sulfur Reservoirs Ocean Biomass 1.5 1 0 8 Yes Sulfur is outgassed from Earth’s core through volcanic Soluble inorganic ions 1.2 1 0 15 Slow 2 activity. The sulfur gases released, primarily sulfur dioxide (primarily SO4 ) (SO ) and hydrogen sulfide (H S), become dissolved in the 2 2 Land ocean and aquifers. Here the hydrogen sulfide forms spar- Living biomass 8.5 1 0 9 Yes ingly soluble metal sulfides, mainly iron disulfide (pyrite), Organic matter 1.6 1 0 10 Yes and sulfur dioxide forms metal sulfates with calcium, bar- Earth’s crusta 1.8 1 0 16 No ium, and strontium as shown in Eqs. 14.10 and 14.11. Adapted from Dobrovolsky, 1994. a This reservoir includes the entire lithosphere found in either terrestrial or ocean 2S22 Fe FeS (pyrite) (Eq. 14.10) environments. 2 SO 2 Ca2 CaSO (gypsum) (Eq. 14.11) 2 4

This results in a substantial portion of the outgassed sulfur The largest reservoir of sulfur is found in Earth’s being converted to rock. Some of the gaseous sulfur com- crust and is composed of inert elemental sulfur deposits, pounds find their way into the upper reaches of the ocean sulfur–metal precipitates such as pyrite (FeS2 ) and gypsum and the soil. In these environments, microbes take up and (CaSO 4), and sulfur associated with buried fossil fuels. cycle the sulfur. Finally, the small portions of these gases A second large reservoir that is slowly cycled is the sulfate that r310emain after precipitating and cycling find their found in the ocean, where it is the second most common way into the atmosphere. Here, they are oxidized to the anion ( Dobrovolsky, 1994 ). Smaller and more actively water-soluble sulfate form, which is washed out of the cycled reservoirs of sulfur include sulfur found in biomass atmosphere by rain. Thus, the atmosphere is a relatively and organic matter in the terrestrial and ocean environ- small reservoir of sulfur (Table 14.17). Of the sulfur found ments. Two recent practices have caused a disturbance in in the atmosphere, the majority is found as sulfur diox- the global sulfur reservoirs. The first is strip mining, which ide. Currently, one-third to one-half of the sulfur dioxide has exposed large areas of metal sulfide ores to the atmo- emitted to the atmosphere is from industrial and automo- sphere, resulting in the formation of acid mine drainage. bile emissions as a result of the burning of fossil fuels. The second is the burning of fossil fuels, a sulfur reser- A smaller portion of the sulfur in the atmosphere is present voir that was quite inert until recently. This has resulted in as hydrogen sulfide and is biological in origin. sulfur dioxide emissions into the atmosphere with the

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resultant formation of acid rain. These processes are discussed further in Section 15.3. Information Box 14.4 Processing of Sulfate for Uptake into Bacteria

active transport 14.4.2 Assimilatory Sulfate Reduction and 22⎯→⎯⎯ SO4 (outside cell) SO4 (inside cell) Sulfur Mineralization sulfate (Eq. 14.12) The primary soluble form of inorganic sulfur found in soil ATP sulfurylase is sulfate. Whereas plants and most microorganisms incor- 2 ⎯→⎯⎯ ATP SO4 APS PPi porate reduced sulfur (sulfide) into amino acids or other adenosine 5-phosphosulfate sulfur-requiring molecules, they take up sulfur in the oxi- (Eq. 14.13)

dized sulfate form and then reduce it internally ( Widdel, APS phosphokinase 1988). This is called assimilatory sulfate reduction. Cells ATP APS ⎯→⎯⎯ PAPS 35-phosphoadenosine- -phossphosulfate assimilate sulfur in the form of sulfate because it is the (Eq. 14.14) most available sulfur form, and because sulfide is toxic.

Sulfide toxicity occurs because inside the cell sulfide reacts PAPS reductase 2(R– SH) PAPS ⎯→⎯⎯ with metals in cytochromes to form metal sulfide precipi- thioredoxin (reduced) (Eq. 14.15) tates, destroying cytochrome activity. However, under the sulfitePAP RSSR controlled conditions of sulfate reduction inside the cell, AP-3 -phosphate thioredoxin (oxidized) the sulfide can be removed immediately and incorporated 2 2 SO3 33 NADPH S NADP (Eq. 14.16) into an organic form (see Information Box 14.4). Although sulfite sulfide this process does protect the cell from harmful effects of the sulfide, it is an energy-consuming reaction. After sulfate O-acetylserine sulfhydrylase is transported inside the cell (Eq. 14.12), ATP is used to O-acetyl-L -serine S2 ⎯→⎯⎯ convert the sulfate into the energy-rich molecule adenosine L-cystteine acetate H2 O 5 -phosphosulfate (APS) (Eq. 14.13). A second ATP mol- (Eq. 14.17) ecule is used to transform APS to 3 -phosphoadenosine-5 - phosphosulfate (PAPS) (Eq. 14.14). This allows the sulfate to be reduced to sulfite and then sulfide in two steps (Eqs. Normal biological release of reduced volatile sulfur com- 14.15 and 14.16). Most commonly, the amino acid serine is pounds results in the formation of approximately 1 kg 2 used to remove sulfide as it is reduced, forming the sulfur- SO4 /hectare/year. The use of fossil fuels, which all con- containing amino acid cysteine (see Eq. 14.17). tain organic sulfur compounds, increases the amount of 2 The release of sulfur from organic forms is called sul- sulfur released to the atmosphere to up to 100 kg SO4 / fur mineralization. The release of sulfur from organic mol- hectare/year in some urban areas. Exacerbating this ecules occurs under both aerobic and anaerobic conditions. problem is the fact that reserves of fossil fuels low in sulfur The enzyme serine sulfhydrylase can remove sulfide from are shrinking, forcing the use of reserves with higher sul- cysteine in the reverse of the reaction shown in Eq. 14.17, fur content. Burning of fossil fuels produces sulfur dioxide or a second enzyme, cysteine sulfhydrylase, can remove (SO2) as shown in Eq. 14.20: both sulfide and ammonia as shown in Eq. 14.18: HO 2 Fossil fuel combustion SO2 H23 SO cysteine sulfhydrylase sulfurous acid cysteine ⎯→⎯⎯ serine H S (Eq. 14.18) 2 (Eq. 14.20) Thus, increased emission of sulfur compounds to the atmo- In marine environments, one of the major products of algal sphere results in the formation of sulfur acid compounds. metabolism is the compound dimethylsulfoniopropionate These acidic compounds dissolve in rainwater and can (DMSP), which is used in osmoregulation of the cell. The decrease the rainwater pH from neutral to as low as pH 3.5, major degradation product of DMSP is dimethyl sulfide a process also known as the formation of acid rain. Acid (DMS). Both H2 S and DMS are volatile compounds and rain damages plant foliage, causes corrosion of stone and therefore can be released to the atmosphere. Once in the concrete building surfaces, and can affect weakly buffered atmosphere, these compounds are photooxidized to sulfate soils and lakes (see Section 15.2). (Eq. 14.19): 14.4.3 Sulfur Oxidation UV light HO ⎯→⎯⎯2 ⎯2 →⎯⎯ HS/DMS24 SO HSO24(Eq. 14.19) In the presence of oxygen, reduced sulfur compounds can sulfuric acid support the growth of a group of chemoautotrophic bacteria

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TABLE 14.18 Sulfur-Oxidizing Bacteria Group Sulfur conversion Habitat requirements Habitat Genera

0 Obligate or facultative H2 S → S H 2 S–O2 interface Mud, hot springs, Acidithiobacillus, Sulfobacillus, 0 2– chemoautotrophs S → SO4 mining surfaces, Thiomicrospira, Achromatium, 2– 2– S2 O3 → SO 4 acid mine Beggiatoa, Thermothrix drainage, soil

0 Anaerobic phototrophs H2 S → S Anaerobic, H2 S, light Shallow water, Chlorobium, Chromatium, 0 2– S → SO4 anaerobic sediments, Ectothiorhodospira, Thiopedia, meta- or hypolimnion, Rhodopseudomonas anaerobic water

Adapted from Germida, 1998.

2.0 O2 Air

2.4 Beggiatoa Beggiatoa

2.8 Mineral medium (Depth mm) (Depth with 0.2% agar

HS2 3.2 Mineral medium 0 0.32 0.64 0.96 1.28 1.6 with 1.5% agar 1–8 mM Na S Dissolved O2 (mg/l) 2 FIGURE 14.17 Cultivation of the sulfuroxidizing chemolithotroph Beggiatoa. At right is a culture tube with sulfide agar overlaid with initially sulfide-free soft mineral agar. The air space in the closed tube is the source of oxygen. Stab-inoculated Beggiatoa grows in a narrowly defined gradient of H2 S and oxygen as shown. Adapted from Microbial Ecology by R. M. Atlas and R. Bartha. © 1993, by Benjamin Cummings. Adapted with permission.

under strictly aerobic conditions and a group of photoau- The energy provided by this oxidation is used to fix CO2 trophic bacteria under strictly anaerobic conditions ( Table for cell growth. In examining Eq. 14.21, it is apparent that 14.18). In addition, a number of aerobic heterotrophic these organisms require both oxygen and sulfide. However, microbes, including both bacteria and fungi, oxidize sulfur reduced compounds are generally abundant in areas that to thiosulfate or to sulfate. The heterotrophic sulfur oxidation contain little or no oxygen. So these microbes are micro- pathway is still unclear, but apparently no energy is obtained aerophilic; they grow best under conditions of low oxygen in this process. Chemoautotrophs are considered the pre- tension (Fig. 14.17). Characteristics of marsh sediments dominant sulfur oxidizers in most environments. However, that contain these organisms are their black color due because many chemoautotrophic sulfur oxidizers require to sulfur deposits and their “ rotten egg ” smell due to the a low pH for optimal activity, heterotrophs may be more presence of H 2 S. Most of these organisms are filamentous important in some aerobic, neutral to alkaline soils. Further, and can easily be observed by examining a marsh sedi- heterotrophs may initiate sulfur oxidation, resulting in a low- ment under the microscope and looking for small white ered pH that is more amenable for chemoautotrophic activity. filaments. Some chemoautotrophs, most notably Acidithiobacillus 14.4.3.1 Chemoautotrophic Sulfur Oxidation thiooxidans , can oxidize elemental sulfur as shown in Eq. 14.22. Of the chemoautotrophs, most oxidize sulfide to elemental sulfur, which is then deposited inside the cell as characteristic granules (Eq. 14.21). 0 2 SOOSOH15. 242 2 HSOSHO1 0 G 218 kJ/mol G 532 kJ/mol 222 2 (Eq. 14.21) (Eq. 14.22)

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This reaction produces acid, and as a result, A. thiooxidans is extremely acid tolerant with an optimal growth pH of 2. Summary for Photoautotrophic Sulfur Oxidation It should be noted that there are various Acidithiobacillus species, and these vary widely in their acid tolerance (Baker ● It is an anaerobic process that is limited to the purple and Banfield, 2003 ). However, the activity of A. thiooxi- and green sulfur bacteria dans in conjunction with the iron-oxidizing, acid-tolerant, ● It is responsible for a very small portion of total chemoautotroph Acidithiobacillus ferrooxidans is responsi- photosynthetic activity ble for the formation of acid mine drainage, an undesirable consequence of sulfur cycle activity (see Section 15.3). It should be noted that the same organisms can be harnessed 14.4.4 Sulfur Reduction for the acid leaching and recovery of precious metals from low-grade ore, also known as biometallurgy (see Chapter There are three types of sulfur reduction. The first, already 21). Thus, depending on one’s perspective, these organisms discussed in Section 14.4.2, is performed to assimilate can be very harmful or very helpful. sulfur into cell components (Widdel, 1988). Assimilatory Although most of the sulfur-oxidizing chemoautotrophs sulfate reduction occurs under either aerobic or anaerobic are obligate aerobes, there is one exception, Thiobacillus conditions. In contrast, there are two dissimilatory path- denitrificans , a facultative anaerobic organism that can sub- ways, both of which use an inorganic form of sulfur as a stitute nitrate as a terminal electron acceptor for oxygen as terminal electron acceptor. In this case sulfur reduction shown in Eq. 14.23: occurs only under anaerobic conditions. The two types of sulfur that can be used as terminal electron acceptors are 0 S NO3342 CaCO CaSO N (Eq. 14.23) elemental sulfur and sulfate. These two types of metabolism are differentiated as sulfur respiration and dissimi- In this equation, the sulfate formed is shown as precipi- latory sulfate reduction. Desulfur omonas acetooxidans tating with calcium to form gypsum. A. denitrificans is not is an example of a bacterium that grows on small carbon acid tolerant but has an optimal pH for growth of 7.0. compounds such as acetate, ethanol, and propanol using elemental sulfur as the terminal electron acceptor:

Summary for Chemoautotrophic Sulfur Oxidation 0 2 CH32 COOH24248 H O S CO2 S H acetate ● It is an aerobic process, but most sulfur oxidizers are (Eq. 14.25) microaerophilic ● Some heterotrophs oxidize sulfur but obtain no energy Ho wever, the use of sulfate as a terminal electron acceptor ● This process can result in the formation of acid mine seems to be the more important environmental process. drainage The following genera, all of which utilize sulfate as a ter- ● This process can be used in metal recovery minal electron acceptor, are found widely distributed in the environment, especially in anaerobic sediments of aquatic environments, water-saturated soils, and animal 14.4.3.2 Photoautotrophic Sulfur Oxidation intestines: Desulfobacter, Desulfobulbus, Desulfococcus, Desulfonema, Desulfosarcina Desulfotomaculum Photoautotrophic oxidation of sulfur is limited to green and , , and Desulfovibrio purple sulfur bacteria (Table 14.18). This group of bacteria . Together these organisms are known as the evolved on early Earth when the atmosphere contained no sulfate-reducing bacteria (SRB). These organisms can uti- oxygen. These microbes fix carbon using light energy, but lize H 2 as an electron donor to drive the reduction of sul- instead of oxidizing water to oxygen, they use an analo- fate as shown in Eq. 14.26: 2 2 gous oxidation of sulfide to sulfur. 44HSOS24 HO2 (Eq. 14.26)

0 Thus, SRB compete for available H 2 in the environment, CO22 H S S fixed carbon (Eq. 14.24) as H 2 is also the electron donor required by methanogens. It should be noted that this is not usually a chemoautotro- These organisms are found in mud and stagnant water, sul- phic process because most SRB cannot fix carbon diox- fur springs, and saline lakes. In each of these environments, ide. Instead they obtain carbon from low molecular weight both sulfide and light must be present. Although the contri- compounds such as acetate or methanol. The overall reac- bution to primary productivity is small in comparison with tion for utilization of methanol is shown in Eq. 14.27: aerobic photosynthesis, these organisms are important in the sulfur cycle. They serve to remove sulfide from the sur- 2 2 43438CH342 OH SO CO S H2 O rounding environment, effectively preventing its movement methanol into the atmosphere and its precipitation as metal sulfide. (Eq. 14.27)

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------Fe(III) oxide continuum ------

←← Most labile ←← More reactive Less reactive →→ Oxic Zone Fe3؉ (aq)↔ Fe3؉ (aq) ↔ Fe3؉ Oxides ↔ Fe3؉ Oxides O , NO3 complexed with poorly crystallized well crystallized 2 Carbon (or Mn3؉,4؉ organic matter (Ferrihydrite) ( Goethite (oxidized)

Fe3؉ Oxidation Interfacial Zone Reduction redox cycling) Fe2؉)

H O, NH4 2؉ 2؉ 2؉ 2 ؉ Fe (aq) ↔↔ sorbed Fe Fe solids or Mn2 Carbon Anoxic Zone ------Fe(II) continuum ------(reduced)

Leaching Fe2؉, Fe3؉, organic matter) P and metals) FIGURE 14.18 Conceptual diagram of iron redox cycling in soils and sediments. Iron atoms can cycle many times through oxidized and reduced states before being lost from the soil profile. Consequently, electron fluxes associated with iron may greatly exceed the oxidation capacity represented by the mass of iron oxides present at any given time. Figure courtesy Aaron Thompson.

Both sulfur and sulfate reducers are strict anaerobic che- 14.5 IRON CYCLE moheterotrophic organisms that prefer small carbon substrates such as acetate, lactate, pyruvate, and low molecular 14.5.1 Iron Reservoirs weight alcohols. Where do these small carbon compounds Iron is the fourth most abundant element in Earth’s crust. come from in the environment? They are by-products of the Iron generally exists in three oxidation states: 0, 2, and 3 fermentation of plant and microbial biomass that occurs in corresponding to metallic iron (Fe 0 ), ferrous iron (Fe2 ), anaerobic regions. Thus, the sulfate reducers are part of an and ferric iron (Fe3 ). In the environment, iron is actively anaerobic consortium of bacteria including fermenters, sul- cycled between the 2 and 3 forms ( Fig. 14.18 ). Under fate-reducers, and methanogens that act together to com- aerobic conditions iron is usually found in its most oxidized pletely mineralize organic compounds to carbon dioxide form (Fe 3 ), which has low aqueous solubility. Under and methane (see Section 3.4.2). More recently, it has been reducing or anaerobic conditions Fe3 is reduced to the found that some SRB can also metabolize more complex ferrous form, Fe 2 , which has higher solubility. Iron is an carbon compounds including some aromatic compounds essential but minor element for biological organisms, mak- and some longer chain fatty acids. These organisms are ing up approximately 0.2% of the dry weight of a bacterial being looked at closely to determine whether they can be cell (Table 14.1). Although the amount of iron in a cell is used in remediation of contaminated sites that are highly low, it has a very important function as a part of enzymes anaerobic and that would be difficult to oxygenate. that are used in respiration and photosynthesis, both pro- The end product of sulfate reduction is hydrogen sul- cesses that require electron transfer. fide. What are the fates of this compound? It can be taken up by chemoautotrophs or photoautotrophs and reoxidized, it can be volatilized into the atmosphere, or it can react with metals to form metal sulfides. In fact, the activity of sulfate- 14.5.2 Iron in Soils and Sediments reducers and the production of hydrogen sulfide are respon- Iron is generally not a limiting nutrient in soil due to its sible for the corrosion of underground metal pipes. In this high abundance in Earth’s crust. However, even though process, the hydrogen sulfide produced reacts with ferrous iron abundance is high, the bioavailability of most iron iron metal to make more iron sulfide (see Section 15.2.1). minerals is quite limited. Thus, microorganisms have developed strategies to obtain iron from its mineral form, Summary for Sulfur Reduction: usually from iron oxides or iron oxyhydroxides. The best studied strategy is the use of iron chelators known as sid- 2 erophores ( Fig. 14.19 ). Siderophores are synthesized and ● Anaerobic respiration using SO4 or S0 as a TEA released from the cell, where they bind Fe 3 and help keep ● Completely inhibited by O2 this low solubility form of iron in solution. The soluble ● Produces H2S which can cause metal corrosion iron–siderophore complex is recognized by and binds to

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O H.N NH O O OO N Fe HN O N O N O N O O O O N H H O Fe N N NH O O H H H HN O HN O O O O O O NH O O H H N N H HN Me N N N H H H RI O NHs O Me O (A) Ferrichrome (B) Pseudo bactin HO Me FIGURE 14.19 Two siderophores showing the coordination of the siderophores with iron. Ferrichrome is produced by fungi including Aspergillus , Ustilago, and Penicillium. Pseudobactin is a bacterial siderophore made by Pseudomonas sp.

Information Box 14.5 Can Iron Fertilization of the Oceans Combat Global Warming?

Since the oceans cover 70% of Earth’s surface, they are of of the ocean with iron could increase primary productivity and

interest from a number of perspectives in trying to control hence uptake of atmospheric CO2 . Will this actually work? Boyd atmospheric CO2 concentrations. One idea is that increasing et al. (2007) point out that more information is needed before photosynthesis (i.e., the productivity of phytoplankton) in the this question can be answered. Information is needed concern-

ocean could help offset increasing anthropogenic CO2 emis- ing the following: the influence of macronutrient/iron ratios on sions. To this end, scientists have been debating what governs the rates and duration of primary productivity; the method and primary productivity, in other words, what is the most limit- form in which to apply iron; and understanding how the carbon ing nutrient? Research suggests that in portions of the ocean and iron cycles are coupled and govern the carbon/iron ratio, where nutrient levels are relatively high but chlorophyll levels which in turn will influence the food web structure (see Fig. 6.2) are low, the single nutrient in shortest supply is iron ( Boyd and lifetime of iron in the near surface of the ocean. et al. , 2007 ). This finding has led to the theory that fertilization

siderophore-specific receptors on the cell surface. The iron TABLE 14.19 Global Iron Fluxes to the Ocean is released from the complex, reduced, and taken up into the cell as Fe2 , its more soluble form. Source Flux, teragrams ؋ 10 9 kg) per year 1) Fluvial particulate total iron 625–962 14.5.3 Iron in Marine Environments Fluvial dissolved iron 1.5 Unlike terrestrial and sediment environments, iron is con- Glacial sediments 34–211 sidered a limiting nutrient in the modern marine environ- Atmospheric 16 ment (Raiswell, 2006 ). In fact the extent of this limitation Coastal erosion 8 has been the focus of a vigorous debate (Information Box 14.5). Recent data seem to indicate that for one-third of Hydrothermal 14 Earth’s oceans, those that are more nutrient-rich and sup- Authigenic (release from deep-sea 5 port higher numbers of phytoplankton, iron is the limit- sediments during diagenesis) ing nutrient to growth (Boyd et al., 2007). How does iron Adapted from Jickells et al ., 2005. Reprinted with permission from AAAS. enter the ocean environment? As shown in Table 14.19, the largest flux of iron entering the oceans is fluvial in nature, meaning that it enters as suspended particles or dissolved not reach the open ocean. Therefore, in the open ocean, the iron carried by rivers or is associated with glacial sedi- major pathway of iron entry is eolian or as dust that is car- ments ( Jickells et al. , 2005 ). For the most part, this iron is ried through the atmosphere mainly from desert and other deposited in the sediments of near-coastal areas and does arid environment land surfaces.

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14.5.4 Iron Oxidation 14.5.4.1 Chemoautotrophs Under aerobic conditions, ferrous iron tends to oxidize to the ferric form. Ferrous iron will autoxidize or spontaneously oxidize under aerobic conditions at pH 5. Iron oxidation also occurs biotically. In fact, reduced iron is an important source of energy for several specialized genera of chemoautotrophic bacteria, the iron-oxidizers ( Ehrlich, 1996 ):

Fe2 H1 O Fe3 1 H O G 40 kJ/mol 4 2 2 2 (Eq. 14.28) Note that compared to oxidation of ammonia (Eq. 14.6) or sulfur (Eqs. 14.21, 14.22) the yield of energy in iron oxidation is quite low. Nevertheless, this is an exploit- FIGURE 14.20 Iron hydroxide– and iron oxyhydroxide–coated stalks able niche and an important biological reaction that can of Gallionella and sheaths of Leptothrix . In this transmission elec- have considerable environmental consequences as will be tron micrograph, dark areas are the metal deposits. From University of California Berkeley Geomicrobiology Program, 2008. explained. Iron oxidation is most often associated with acidic 2 2 environments. For example, the acidophilic thermophile Fe and Mn depositing an iron–manganese encrusted Sulfolobus is an archaean that was isolated from acidic hot coating on its sheath. As can be seen in Fig 14.20 , these springs. But iron oxidation is perhaps best studied in asso- microbes can deposit copious amounts of iron (and man- ciation with acid mine drainage and the acidophilic bac- ganese) minerals on their surfaces. This can have serious terium Acidithiobacillus ferrooxidans (see Section 15.3). economic consequences in some instances. For example, Interestingly, although A. ferrooxidans is best studied this process can cause extensive biofouling and corrosion (because it has been most easily cultured), nonculture- of water pipelines. based analysis suggests that other iron-oxidizers may actually play more important roles in the creation of acid conditions in mine tailings and the formation of acid mine Summary drainage (Baker and Banfield, 2003). These include bacteria such as Leptospirillum ferrooxidans and L. ferriphilum , ● Iron oxidation is a chemoautotrophic, aerobic process usually found under acidic conditions Sulfobacillus acidophilus , S. thermosulfooxidans , and ● This process participates in the formation of acid mine Acidimicrobium ferrooxidans as well as some archaea such drainage (and can be used for metal recovery) as Ferroplasma acidiphilum . ● Neutrophilic iron oxidation occurs in a more limited Iron oxidation has also been described in several marine number of environments where, due to iron mineral genera at neutral pH. This process is problematic at neu- deposition, this process can result in biofouling and tral pH because (1) the energy yield is very low, and (2) at corrosion. neutral pH, spontaneous oxidation of Fe 2 to Fe3 (in the form of iron oxyhydroxides), occurs rapidly in the presence of oxygen and competes with the biological reaction 14.5.4.2 Photoautotrophs ( Fig. 14.18 ). Neutrophilic iron-oxidizers overcome these problems with very specific niche requirements. They posi- Some members of the purple and green bacteria can use Fe 2 as an electron donor to carry out anaerobic tion themselves in regions with low O2 tension and high and constant Fe2 concentrations. One marine environ- photosynthesis coupled to photoautotrophic growth (i.e., ment that meets these requirements occurs in hydrothermal growth involving photosynthesis and oxidation): vents on the sea floor (see Chapter 7). Here, anoxic vent light 2 2 fluids charged with Fe rapidly come in contact with the 41Fe CO221 H O C(H 2 O) cold, oxygenated ocean water. Similar conditions can occur 48Fe(OH) H 3 in municipal and industrial water pipelines. (Eq. 14.29) The best-studied neutrophilic iron-oxidizers are Gallionella and Leptothrix. Gallionella is capable of che- It has been proposed that iron-based photosynthesis moautotrophic growth using Fe 2 as sole energy source represents the transition between anaerobic photosynthe- under microaerobic conditions. Leptothrix is a sheath- sis that developed on early Earth and aerobic photosyn- forming chemoheterotrophic organism that oxidizes both thesis that began approximately 2 billion years ago (Jiao

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and Newman, 2007). In fact, it is thought that Fe2 was Organic carbon the most widespread source of reducing power from 3.8 Fe2 H2 to 1.6 billion years ago, where under reducing conditions Fe3 Fe2 was favored over Fe3 . Under these conditions, the CO2 H2O amount of iron in the marine environment was consider- (A) ably higher (0.1 to 1 mmol/liter) than it is today (0.03 to 1 nmol/liter) ( Jickells et al. , 2005 ; Bosak et al. , 2007 ).

Electron shuttle Organic carbon Fe2 (reduced) H2 Summary Fe3 Electron shuttle CO2 (oxidized) H2O ● Phototrophic iron oxidation is a photoautotrophic, (B) anaerobic process that is limited to purple and green FIGURE 14.21 Two primary strategies are used in iron respiration to sulfur bacteria facilitate electron transfer between microbial cells and iron oxide sur- ● This process is thought to be a key transition step faces. (A) Cells can come in direct contact with the oxide surface. (B) An between anaerobic photosynthesis found on early earth electron shuttle can be used to mediate the electron transfer between the and modern aerobic photosynthesis cell and iron oxide surfaces.

example, Geothrix fermentans releases a quinonelike electron shuttle during growth on lactate (electron donor) and 14.5.5 Iron Reduction poorly crystalline iron oxides (electron acceptor) (Nevin Iron is microbially reduced for two purposes, assimilation and Lovley, 2002 ). and energy generation. Assimilatory iron reduction is the reduction of Fe3 for uptake and incorporation into cell Summary for Iron Reduction constituents. As described in Section 14.5.2, this usually involves the release of siderophores that complex Fe3 in ● Assimilatory iron reduction is generally mediated by a the environment exterior to the cell. The iron–siderophore iron-chelating molecules called siderophores. complex then delivers the Fe3 to the cell, which is reduced ● 2 Dissimilatory iron reduction is the use of ferric iron as a to Fe as it is taken up. terminal electron acceptor during anaerobic respiration. Dissimilatory iron reduction or iron respiration is the Due to the abundance of iron in soils and sediments, it 3 use of Fe as a terminal electron acceptor for the purpose is thought that this is an important process in anaerobic of energy generation during anaerobic respiration. Due to environments. its abundance in Earth’s crust, Fe 3 found in iron oxides and oxyhydroxides serves as an important terminal electron acceptor for anaerobic heterotrophic bacteria. Because QUESTIONS AND PROBLEMS iron respiration has been an important activity during the evolution of Earth, there is wide diversity among the bacte- 1. Give an example for carbon, nitrogen, and sulfur, of ria and archaea capable of carrying out this activity ( Weber a. a small actively cycled reservoir et al., 2006). The problem for the iron-reducers is that b. a large actively cycled reservoir most of the iron in the environment is relatively unavail- c. a large inactively cycled reservoir able ( Fig. 14.18 ). So microorganisms have developed some 2. Describe how the ocean has reduced the expected very interesting strategies to solve this problem (Lovley, rate of increase of CO2 in the atmosphere since 2000 ). As shown in Figure 14.21 , the first is to make direct industrialization began. contact with an iron oxide surface. In this case, the iron 3. What is the concept behind fertilization of the ocean reductase is a membrane-bound enzyme, allowing direct with iron? access of the enzyme with the substrate. A second strategy 4. What strategy is used by microbes to initiate is to use an electron shuttle that can act as an intermediate degradation of large plant polymers such as cellulose? in transferring electrons from the cell to the iron oxide sur- 5. Define what is meant by a greenhouse gas and give face. Possible electron shuttles in the environment include two examples. For each example describe how humic acids or other molecules that contain quinone-like microorganisms mediate generation of the gas, and structures that are reduced to a corresponding hydroqui- then describe how human activity influences generation none form. Humic acids are considered an exogenous of the gas. electron shuttle source or one that is obtained from the 6. Both autotrophic and heterotrophic activities are environment outside the cell. Alternatively, some bacteria important in element cycling. For each cycle discussed can make and release endogenous electron shuttles. For in this chapter (carbon, nitrogen, sulfur, and iron),

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