: Microbiology of the Built Environment

22 Microbiology of the Built Environment

## Chapter 22 Microbiology of the Built Environment

IV Indoor Microbiology and Microbially Influenced Corrosion

Sending Microbes to Clean Up after Polluters

The chlorinated solvents tetrachloroethene and trichloroethene have long been used as degreasing agents, dry cleaning fluids, and chemical feedstocks. Unfortunately, their improper disposal has contributed to extensive contamination of groundwater at thousands of sites throughout the United States. Since these solvents can be widely dispersed in the subsurface, the only feasible approach to their cleanup is to deploy microbes that can degrade them in place, a process called in situ bioremediation.

Bioremediation of chlorinated contaminants exploits the ability of some bacteria to use these chemicals as electron acceptors through reductive dechlorination, a form of anaerobic respiration. Although dehalogenating bacteria are common, most fail to remove all of the chlorines, leaving the even more toxic and carcinogenic products dichloroethylene and vinyl chloride behind. Thus, the discovery of the bacteria Dehalococcoides and Dehalogenimonas that can completely dechlorinate these solvents and produce the benign gas ethene was critical to effective cleanup.

If these dehalogenating bacteria are naturally present, then biostimulation by injecting organic carbon is used to trigger bioremediation. However, if these microbes are absent, bioaugmentation (the addition of microorganisms expressing a desired activity) is needed to jump-start cleanup. At a remediation site, large numbers of Dehalococcoides are injected along with an inexpensive carbon source such as molasses (shown here in the photo of a contaminated manufacturing site). Fermentation of the molasses by various bacteria produces the acetate and H2 needed by Dehalococcoides to dehalogenate the solvents. It was initially unclear how long Dehalococcoides would continue to degrade solvents remaining in the groundwater after injection stopped. However, environmental microbiology research showed that these physiologically specialized microbial cleanup agents remain active for at least four years after injection is stopped, all the time continuing to protect humans from our past poor environmental stewardship.

Source: Schaefer, C.E., Lavorgna, G.M., Haluska, A.A., and Annable, M.D. 2018. Long-term impacts on groundwater and reductive dechlorination following bioremediation in a highly characterized trichloroethene DNAPL source area. Groundwater Monit. Rem. 38: 65.

In this chapter we address the microbiology of “built” systems. These include the infrastructure for drinking water and wastewater distribution and treatment, gas and oil transmission, building materials, private and public spaces, and environments modified for mineral extraction or for the cleanup of pollutants. By their very nature, built systems create new microbial habitats, and these promote both desired and undesired microbial activities. From the standpoint of microbial ecology, these activities are simply the natural result of microbes exploiting resources provided to them.

Microbial activity in the built environment is the proverbial double-edged sword. Examples of built systems that promote desirable microbial activities include biological reactors for the treatment of wastewater and the stimulation of microbial activity in aquifers to clean up environmental pollutants. By contrast, an undesirable activity is microbially influenced corrosion of the pipelines used for the transmission of wastewater, drinking water, and oil, activities that destroy billions of dollars’ worth of essential infrastructure yearly in the United States alone.

I Mineral Recovery and Acid Mine Drainage

It is often remarked that microorganisms are “Earth’s greatest chemists,” and the activities of these tiny biochemical factories have been exploited in many ways. Here we consider how microbial activities help extract valuable metals from low-grade ores, and the environmental consequences thereof.

22.1 Mining with Microorganisms

One of the most common forms of iron in nature is pyrite (FeS2), which is often present in coal and in metal ores. Sulfide (HS−) also forms insoluble minerals with many metals, and many ores mined as sources of these metals are sulfide ores. If the concentration of metal in the ore is low, it may be economically feasible to mine the ore only if the desired metals are first concentrated by microbial leaching (Figure 22.1). The promotion of acid production and dissolution of FeS2 by acidophilic bacteria such as Acidithiobacillus ferrooxidans is used to leach the metal ores in large-scale mining operations. Leaching is especially useful for copper ores because copper sulfate (CuSO4), formed during the oxidation of copper sulfide ores, is very water-soluble. Indeed, approximately a quarter of all copper mined worldwide is obtained by microbial leaching.

Figure 22.1 The leaching of low-grade copper ores using iron-oxidizing bacteria.


(a) A typical leaching dump. The low-grade ore has been crushed and dumped in such a way that the surface area exposed is as high as possible. Pipes distribute the acidic leach water over the surface of the pile. The acidic water slowly percolates through the pile and exits at the bottom. (b) Effluent from a copper leaching dump. The acidic water is very rich in Cu2+. (c) Recovery of copper as metallic copper (Cu0) by passage of the Cu2+-rich water over metallic iron in a long flume. (d) A small pile of metallic copper removed from the flume, ready for further purification.

The Leaching Process

The susceptibility to oxidation varies among minerals, and those minerals that are most readily oxidized are most amenable to microbial leaching. Thus, iron and copper sulfide ores such as pyrrhotite (FeS) and covellite (CuS) are readily leached, whereas lead and molybdenum ores are much less so. In microbial leaching, low-grade ore is dumped in a large pile called the leach dump and a dilute sulfuric acid solution at pH 2 is percolated down through the pile (Figure 22.1a). The liquid emerging from the bottom of the pile (Figure 22.1b) is rich in dissolved metals and is transported to a precipitation plant (Figure 22.1c) where the desired metal is precipitated and purified (Figure 22.1d). The liquid is then pumped back to the top of the pile and the cycle repeated. As needed, acid is added to maintain an acidic pH.

We illustrate microbial leaching of copper with the common copper ore CuS, in which copper exists as Cu2+. A. ferrooxidans oxidizes the sulfide in CuS to SO4 2−, releasing Cu2+ as shown in Figure 22.2. However, this reaction can also occur spontaneously. Indeed, the key reaction in copper leaching is actually not the bacterial oxidation of sulfide in CuS but the spontaneous oxidation of sulfide by ferric iron (Fe3+) generated from the bacterial oxidation of ferrous iron (Fe2+) (Figure 22.2). In any copper ore, FeS2 is also present, and its oxidation by bacteria leads to the formation of Fe3+. The spontaneous reaction of CuS with Fe3+ proceeds in the absence of O2 and forms Cu2+ plus Fe2+ (Figure 22.2).

Figure 22.2 Arrangement of a leaching pile and reactions in the microbial leaching of copper sulfide minerals to yield metallic copper.


Reaction 1 occurs both biologically and chemically. Reaction 2 is strictly chemical and is the most important reaction in copper-leaching processes. For reaction 2 to proceed, it is essential that the Fe2+ produced from the oxidation of sulfide in CuS to sulfate be oxidized back to Fe3+ by iron chemolithotrophs (see chemistry in the oxidation pond).

Mastering Microbiology

Art Activity: Figure 22.2 Arrangement of a leaching pile and reactions in the microbial leaching of copper sulfide minerals to yield metallic copper

Metal Recovery

The precipitation plant is where the Cu2+ from the leaching solution is recovered (Figure 22.1c, d). Shredded scrap iron (a source of elemental iron, Fe0) is added to the precipitation pond to reduce Cu2+ (abiotically) and recover metallic copper (Cu0) from the leach liquid by the chemistry shown in the lower part of Figure 22.2. This produces a Fe2+-rich liquid that is pumped to a shallow oxidation pond where iron-oxidizing chemolithotrophs oxidize the Fe2+ to Fe3+. This now ferric-iron-rich acidic liquid is pumped to the top of the pile and the Fe3+ is used to oxidize more CuS (Figures 22.1 and 22.2). The entire CuS leaching operation is thus driven by the oxidation of Fe2+ to Fe3+ by iron-oxidizing bacteria.

Temperatures rise in a leaching dump and this leads to shifts in the iron-oxidizing microbial community. A. ferrooxidans is a mesophile, and when heat generated by microbial activities raises temperatures above about 30 °C inside a leach dump, this bacterium is outcompeted by mildly thermophilic, iron-oxidizing chemolithotrophic Bacteria such as Leptospirillum ferrooxidans and Sulfobacillus. At even higher temperatures (60–80 °C), hyperthermophilic Archaea such as Sulfolobus (Section 17.10) predominate in the leach dump.

Other Microbial Leaching Processes: Uranium and Gold

Bacteria are also used in the leaching of uranium (U) and gold (Au) ores. In uranium leaching, A. ferrooxidans oxidizes U4+ to U6+ with O2 as an electron acceptor. However, U leaching depends more on the abiotic oxidation of U4+ by Fe3+ with A. ferrooxidans contributing to the process mainly through the reoxidation of Fe2+ to Fe3+, as in copper leaching (Figure 22.2). The reaction observed is as follows: UO2+Fe2(SO4)3→UO2SO4+2 FeSO4(U4+)(Fe3+)(U6+)(Fe2+)

Unlike UO2, the uranyl sulfate (UO2SO4) formed is highly soluble and is concentrated by other processes.

Gold is typically present in nature in deposits associated with minerals containing arsenic (As) and FeS2. A. ferrooxidans and related bacteria can leach the arsenopyrite minerals, releasing the trapped Au: 2 FeAsS[Au]+7 O2+2 H2O+H2SO4→Fe2(SO4)3+2 H3AsO4+[Au]

The Au is then complexed with cyanide (CN−) by traditional gold-mining methods. Unlike copper leaching, which is done in a huge dump (Figure 22.1a), gold leaching is done in small bioreactor tanks (Figure 22.3), where more than 95% of the trapped Au can be released. Moreover, the potentially toxic As and CN− residues from the mining process are removed in the gold-leaching bioreactor. Arsenic is removed as a ferric precipitate, and CN− is removed by its bacterial oxidation to CO2 plus urea in later stages of the Au recovery process. Small-scale microbial-bioreactor leaching has thus become popular as an alternative to the environmentally devastating gold-mining techniques that leave a toxic trail of As and CN− at the extraction site. Pilot processes are also being developed for bioreactor leaching of zinc, lead, and nickel ores.

Figure 22.3 Gold bioleaching.

Gold leaching tanks in Ghana (Africa). Within the tanks, a mixture of Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, and Leptospirillum ferrooxidans solubilizes the pyrite/arsenic mineral containing trapped gold, which releases the gold.

We now examine the other side of the coin—environmental damage from microbial mining.

Check Your Understanding

What is required to oxidize CuS under anaerobic conditions?

What key role does Acidithiobacillus ferrooxidans play in the copper leaching process?

22.2 Acid Mine Drainage

Although microbial leaching has tremendous value in mining operations, the same process has contributed to extensive environmental destruction where mining operations improperly handle or dispose of pyrite-containing coal and mineral deposits. Bacterial and spontaneous oxidation of sulfide minerals is the major cause of acid mine drainage, an environmental problem worldwide caused by surface mining operations. As described for the oxidation of copper sulfides promoted in microbial mining (Section 22.1), the oxidation of FeS2 is a combination of chemically and bacterially catalyzed reactions, and two electron acceptors participate in the process: O2 and Fe3+. When FeS2 is first exposed in a mining operation (**Figure 22.4a,*b***), a slow chemical reaction with O2 begins (Figure 22.4c). This reaction, called the initiator reaction, leads to the oxidation of HS− to SO4 2− and the development of acidic conditions as Fe2+ is released. Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans then oxidize Fe2+ to Fe3+, and the Fe3+ formed under these acidic conditions, being soluble, reacts spontaneously with more FeS2 and oxidizes the HS− to sulfuric acid (H2SO4), which immediately dissociates into SO4 2− and H+: FeS2+14 Fe3++8 H2O→15 Fe2++2 SO4 2−+16 H+

Figure 22.4 Coal and pyrite.


(a) Coal from the Black Mesa formation in northern Arizona (USA); the gold-colored spherical discs (about 1 mm in diameter) are particles of pyrite (FeS2). (b) A coal seam in a surface coal-mining operation. Exposing the coal to oxygen and moisture stimulates the activities of iron-oxidizing bacteria growing on the pyrite in the coal. (c) Reactions in pyrite degradation. The primarily abiotic initiator reaction sets the stage for the primarily bacterial oxidation of Fe2+ to Fe3+. The Fe3+ attacks and oxidizes FeS2 abiotically in the propagation cycle.

Again, the bacteria oxidize Fe2+ to Fe3+, and this Fe3+ reacts with more FeS2. Thus, there is a progressive, rapidly increasing rate at which FeS2 is oxidized, called the propagation cycle (Figure 22.4c). Under natural conditions some of the Fe2+ generated by the bacteria leaches away and is subsequently carried by anoxic groundwater into surrounding streams. However, bacterial or spontaneous oxidation of Fe2+ then takes place in the aerated streams, and because O2 is present, the insoluble Fe(OH)3 is formed.

As we have seen (Figure 22.4c), the breakdown of FeS2 ultimately leads to the formation of H2SO4 and Fe2+; in waters in which these products have formed, pH values can be lower than 1. Mixing of acidic mine waters into rivers (Figure 22.5) and lakes seriously degrades water quality because both the acid and the dissolved metals (iron, aluminum, and heavy metals such as cadmium and lead) are toxic to aquatic organisms.

Figure 22.5 Acid mine drainage from a surface coal-mining operation.

The yellowish-red color is due to the precipitated iron oxides in the drainage (see Figure 22.4c for the reactions in acid mine drainage).

The O2 requirement for the oxidation of Fe2+ to Fe3+ explains how acid mine drainage develops. As long as the pyritic material is not mined, FeS2 cannot be oxidized because O2, water, and the bacteria cannot reach it. However, when a mineral or coal seam is exposed (Figure 22.4b), O2 and water are introduced, making both spontaneous and bacterial oxidation of FeS2 possible. The acid formed can then leach into surrounding aquatic systems (Figure 22.5).

Where acid mine drainage is extensive and Fe2+ levels high, a strongly acidophilic species of Archaea, Ferroplasma, is often present. This aerobic iron-oxidizing organism is capable of growth at pH 0 and at temperatures up to 50 °C. Cells of Ferroplasma lack a cell wall and are phylogenetically related to Thermoplasma, also a cell-wall-lacking and strongly acidophilic (but chemoorganotrophic) member of the Archaea (Section 17.3).

In Part II, we explore the use of microbes in cleaning up several undesirable agents that, one way or the other, end up in the environment.

Check Your Understanding

In what oxidation state is iron in the mineral Fe(OH)3? In FeS? How is Fe(OH)3 formed?

Natural pyritic deposits, such as underground coal seams, do not contribute to acid mine drainage; why not?

II Bioremediation

Bioremediation is the microbial cleanup of oil, toxic chemicals, or other environmental pollutants by stimulating the degradative activities of indigenous microbes or by the addition of microbes having the needed degradative capacity. These pollutants include both natural materials, such as petroleum products, and synthetic chemicals new to nature.

Bioremediation is the process by which toxic or otherwise dangerous materials are cleaned up using microbes as the cleaning agents. Major successes thus far have been registered with spills of crude oil or the leakage of hydrocarbons from bulk storage tanks because the microbiology of hydrocarbon cleanup is well founded. But many other materials can be bioremediated, including chlorinated pollutants such as solvents and pesticides, and even radioactive waste in uranium-contaminated environments, a legacy of past mining of uranium for nuclear fuel and weapons. We begin with a consideration of this very toxic pollutant.

22.3 Bioremediation of Uranium-Contaminated Environments

22.3 Bioremediation of Uranium-Contaminated Environments

22.3 Bioremediation of Uranium-Contaminated Environments

Major classes of inorganic pollutants are metals and radionuclides that cannot be destroyed but only altered in chemical form. Often the extent of environmental pollution is so great that physical removal of the contaminated material is impossible. Thus, containment is the only real option, and a common goal in the bioremediation of inorganic pollutants is to change their mobility, making them less likely to move in groundwater and contaminate surrounding environments. The containment of uranium is a prime example.

Bioremediation of Uranium

Uranium contamination of groundwater has occurred at sites in the United States and elsewhere where uranium ores have been processed or stored (Figure 22.6), and the movement of radioactive materials offsite via groundwater is a threat to environmental and human health. Because the contamination is often widespread, making mechanical methods of recovery very expensive, microbiologists have joined forces with engineers to develop biological treatments that exploit the ability of some bacteria to reduce U6+ to U4+. Uranium as U6+ is soluble, whereas U4+ forms an immobile uranium mineral called uraninite (UO2), thus limiting the movement of U into groundwater and potential contact with humans and other animals.

Figure 22.6 Uranium bioremediation.


An experimental plot at a United States Department of Energy uranium-contaminated site. Organic carbon (acetate) is being infused into the site (see inset photo) and travels in groundwater in the direction of the arrow shown in the main photo. Acetate is an electron donor for reduction of U6+ to U4+, which immobilizes the uranium.

Bacterial Transformations of Uranium

In order to change the oxidation state of U in major uranium contaminants to a form that will stabilize the element, Bacteria, including metal-reducing Shewanella and Geobacter species (Section 15.13) and sulfate-reducing Desulfovibrio species (Section 15.11), couple the oxidation of organic matter and H2 to the reduction of U6+ to U4+.

Field studies in which organic electron donors such as acetate have been injected into uranium-contaminated aquifers to stimulate U6+ reduction have shown that this can lower U levels to below the U.S. Environmental Protection Agency’s drinking water standard of 0.126 μM. Natural levels of organic matter are typically very low in the subsurface (Section 20.8), but can be sufficient to support microbial activities including anaerobic respiration using U6+ as electron acceptor. However, even though uraninite is stable under reducing conditions, if conditions become oxic, it reoxidizes (primarily abiotically by manganese oxide minerals) and once again becomes mobile.

Much ongoing uranium bioremediation research is focused on questions of whether microbially reduced uranium is stable if the composition of the microbial community changes or if oxidants, such as O2, NO3 −, and Fe3+, are introduced via groundwater. This is obviously an important question because uraninite stability must be targeted for the long term in order to account for the long half-life of nuclear decay of uranium; U238, the most common isotope of uranium, has a half-life approximately equal to the age of Earth (4.5 billion years)!

Check Your Understanding

Which reaction, oxidation or reduction, is key to uranium bioremediation?

Why is immobilization a good strategy for dealing with uranium pollution?

22.4 Bioremediation of Organic Pollutants: Hydrocarbons

22.4 Bioremediation of Organic Pollutants: Hydrocarbons

22.4 Bioremediation of Organic Pollutants: Hydrocarbons

Organic pollutants, unlike inorganic pollutants, can generally be completely degraded by microorganisms, eventually to CO2. This is true of petroleum released in oil spills (Figure 22.7), which can be attacked by many different microorganisms. These organisms have been exposed to complex mixtures of hydrocarbons through natural or accidental oil and gas seeps (Figures 20.23 and 20.24) for millennia and thus have evolved the catabolic machinery necessary to degrade this naturally occurring pollutant. In contrast, artificial pollutants (xenobiotics, see Section 22.5) tend to be more persistent and are degraded by more specialized groups of microorganisms. In this section we focus on hydrocarbons and in the next section on xenobiotics.

Figure 22.7 Environmental consequences of large oil spills and the effect of bioremediation.


(a) A contaminated beach along the coast of Alaska containing oil from the Exxon Valdez spill of 1989. (b) The rectangular plot (arrow) was treated with inorganic nutrients to stimulate bioremediation of spilled oil by microorganisms, whereas areas above and to the left were untreated. (c) Oil spilled into the Mediterranean Sea from the Jiyeh (Lebanon) power plant that flowed to the port of Byblos during the 2006 war in Lebanon.

Petroleum and Hydrocarbon Bioremediation

Petroleum is a rich source of organic matter, and because of this, microorganisms readily attack hydrocarbons when petroleum is pumped to Earth’s surface and comes into contact with air and moisture. Under some circumstances, such as in bulk petroleum storage tanks, microbial growth is undesirable. However, in oil spills, biodegradation is desirable and can be promoted by the addition of inorganic nutrients to balance the huge influx of organic carbon from the oil (Figure 22.7b).

The biochemistry of hydrocarbon catabolism was covered in Sections 14.23 and 14.24. Both aerobic and anaerobic biodegradation is possible. We emphasized that under oxic conditions, oxygenase enzymes play an important role in introducing oxygen atoms into the hydrocarbon. Our discussion here will focus on aerobic processes because it is only when O2 is present that oxygenase enzymes can function and hydrocarbon bioremediation can be effective in a relatively short time.

Diverse bacteria, fungi, and a few green algae can oxidize petroleum products aerobically. Small-scale oil pollution of aquatic and terrestrial ecosystems from human as well as natural activities is common. Oil-oxidizing microorganisms develop rapidly on oil films and slicks, and hydrocarbon oxidation is most extensive if the temperature is warm enough and supplies of inorganic nutrients (primarily N and P) are sufficient. Moreover, because oil is insoluble in water and is less dense, it floats to the surface and forms slicks. There, hydrocarbon-degrading bacteria attach to the oil droplets (Figure 22.8) and eventually decompose the oil and disperse the slick. Certain oil-degrading bacteria are specialist species. For example, the bacterium Alcanivorax borkumensis grows only on hydrocarbons, fatty acids, or pyruvate. This organism produces surfactant chemicals that help break up the oil and solubilize it. Once solubilized, the oil can be incorporated more readily and catabolized as an electron donor and carbon source.

Figure 22.8 Hydrocarbon-oxidizing bacteria in association with oil droplets.


The bacteria are concentrated in large numbers at the oil–water interface but are actually not within the droplet itself.

In large surface oil spills such as those shown in Figure 22.7, volatile hydrocarbons, both aliphatic and aromatic, evaporate quickly without bioremediation, leaving nonvolatile components for cleanup crews and microorganisms to tackle. Microorganisms consume oil by oxidizing it to CO2. When bioremediation activities are promoted by inorganic nutrient application, oil-oxidizing bacteria typically develop quickly after an oil spill (Figure 22.7b), and under ideal conditions, 80% or more of the nonvolatile oil components can be oxidized within one year. However, certain oil fractions, such as those containing branched-chain and polycyclic hydrocarbons, are not preferred microbial substrates and remain in the environment much longer. Spilled oil that finds its way into sediments is even more slowly degraded and can have a significant long-term impact on fisheries that depend on unpolluted waters for productive yields.

A notable exception to the more common surface spill of oil was the 2010 sinking of the Deepwater Horizon offshore drilling platform in the Gulf of Mexico, resulting in the rupture of the wellhead at a depth of 1.5 km and the release of over 4 million barrels (635 million liters) of oil and hydrocarbon gases into the deep ocean (Section 20.10 and Figure 20.23). About 35% of the resulting hydrocarbon plume was comprised of low-molecular-weight components and natural gas (methane, ethane, propane). The availability of these more easily degraded oil components likely accelerated the natural degradation process by stimulating the development of a bloom of bacteria having the capacity to oxidize both the easily degraded and more recalcitrant hydrocarbon components. Chemical dispersants were also added directly to the oil plume to accelerate microbial degradation, and much of the oil eventually disappeared through a combination of volatilization and microbial activities.

Degradation of Stored Hydrocarbons

Interfaces where oil and water meet often form on a large scale. Besides water that separates from crude petroleum during storage and transport, moisture can condense inside bulk fuel storage tanks (Figure 22.9) where there are leaks. This water eventually accumulates in a layer beneath the petroleum. Gasoline and crude oil storage tanks are thus potential habitats for hydrocarbon-oxidizing microorganisms. If sufficient sulfate (SO4 2−) is present in the oil, as is often the case with crude oils, sulfate-reducing bacteria can grow in the tanks, consuming hydrocarbons under anoxic conditions (Sections 14.12, 14.24, and 15.11). The sulfide (H2S) produced is highly corrosive and causes pitting and subsequent leakage of the tanks along with souring of the fuel. Aerobic degradation of stored fuel components is less of a problem because the storage tanks are sealed and the fuel itself contains little dissolved O2.

Figure 22.9 Bulk petroleum storage tanks.

Fuel tanks often support microbial growth at oil–water interfaces.

Check Your Understanding

Why do petroleum-degrading bacteria need to attach to the surface of oil droplets?

What is unusual about the physiology of the bacterium Alcanivorax?

22.5 Bioremediation and Microbial Degradation of Major Chemical Pollutants: Chlorinated Organics and Plastics

22.5 Bioremediation and Microbial Degradation of Major Chemical Pollutants: Chlorinated Organics and Plastics

22.5 Bioremediation and Microbial Degradation of Major Chemical Pollutants: Chlorinated Organics and Plastics

Unlike hydrocarbons, many chemicals that humans put into the environment have never been there before; these are xenobiotics, substances not previously present on Earth and new to microbial communities.

Catabolism of Organohalogens: The Chlorinated Organics

Major chemical pollutants are primarily xenobiotics. These include pesticides, polychlorinated biphenyls (PCBs), munitions, dyes, and chlorinated solvents, among many other chemicals. Some xenobiotics differ chemically in such major ways from anything organisms have experienced in nature that they are slow to biodegrade. In these cases, human intervention is often required to promote and accelerate degradation. We focus here on microbial degradation of major chlorinated compounds that are among the most common of environmental pollutants, either through widespread targeted application, as for pesticides, or through improper disposal or manufacture of chemicals used primarily in industrial applications.

Microorganisms that degrade chlorinated compounds are widespread in nature and evolved a natural halogen cycle long before the introduction of manufactured chemicals. Some natural chlorinated compounds originate from forest fires and volcanic eruptions. Others are the products of microbial synthesis of specific bioactive compounds, including haloalkanes and secondary metabolites produced by algae, fungi, bacteria, plants, and some animals. These likely provide the producing organisms a competitive advantage over predators or competitors, but few have been fully characterized. Today, well over 5000 different natural organohalogens have been identified and some are being studied for use in clinical medicine. A notable example is a bioactive secondary metabolite produced by the marine actinobacterium Salinispora tropica; the metabolite, salinosporamide A (Figure 22.10), is in clinical development for treatment of myeloma, a cancer of white blood cells.

Figure 22.10 Examples of xenobiotic compounds.

Although none of these compounds is a natural compound (except for salinosporamide A), microorganisms exist that can break them down.

The microbial synthesis and degradation of naturally occurring organohalogens provides a rich natural source of enzymes for the degradation of the manufactured organohalogens that now pollute the environment. Although some organohalogens are degraded only very slowly, most function as either electron donors or terminal electron acceptors for microbial growth, and virtually all are metabolized by microorganisms.

Over 1000 pesticides have been marketed worldwide and include herbicides, insecticides, and fungicides. Pesticides display a wide variety of chemistries and include chlorinated, aromatic, and nitrogen- and phosphorus-containing compounds (Figure 22.10 and see Explore the Microbial World, “Solving the Marine Methane Paradox,” in Chapter 21). Highly chlorinated compounds are typically the pesticides most resistant to microbial attack. For example, chlorinated compounds such as DDT persist relatively unaltered for years in soils, whereas compounds such as 2,4-D (Figure 22.10) are significantly degraded in just a few weeks. Environmental factors, such as temperature, pH, aeration, and organic content of the soil, influence the rate of pesticide decomposition, and some pesticides can disappear from soils nonbiologically by volatilization, leaching, or spontaneous chemical breakdown. In addition, some pesticides are degraded only when other organic material is present that can be used as the primary energy source, a phenomenon called cometabolism. In many cases, pesticides that are cometabolized are only partially degraded, generating new xenobiotic compounds that may be even more toxic or difficult to degrade than the original compound. Thus, from an environmental standpoint, cometabolism of a pesticide is not always a good thing.

Aerobic and Anaerobic Dechlorination

The microbial degradation of chlorinated xenobiotics (Figure 22.10) is generally initiated by dechlorination. In aerobes, dechlorination is most often catalyzed by halogenase enzymes having specific substrate requirements. For example, a specific monooxygenase catalyzes removal of the first chlorine from the aromatic ring of the insecticide pentachlorophenol (Figure 22.11). This is followed by reductive elimination of two additional chlorines, dioxygenase ring cleavage, and elimination of the final chlorine to generate products that enter the citric acid cycle (Section 3.6). In contrast to this very specific pathway for catabolism of pentachlorophenol, oxidative degradation of trichloroethylene (TCE) and vinyl chloride is initiated by cometabolism in which a relatively nonspecific monooxygenase generates an unstable epoxide that decomposes abiotically to form harmless products (see Figure 22.12).

Figure 22.11 Pentachlorophenol metabolism by *Sphingobium chlorophenolicum*.


A monooxygenase catalyzes the first chlorine removal from pentachlorophenol, followed by two reductive dehalogenase steps before the aromatic ring is cleaved by a dioxygenase. A subsequent dehalogenation reaction yields a product that can enter central metabolism.

Although the aerobic breakdown of chlorinated xenobiotics is undoubtedly ecologically important, reductive dechlorination may be even more so because of the rapidity with which anoxic conditions develop in polluted microbial habitats. We previously described reductive dechlorination as a form of anaerobic respiration in which chlorinated organic compounds such as chlorobenzoate (C7H4O2Cl−) are terminal electron acceptors that when reduced release chloride (Cl−), a nontoxic substance (Section 14.13).

Many compounds can be reductively dechlorinated, including dichloro-, trichloro-, and tetrachloro- (perchloro-) ethylene, chloroform, dichloromethane, and polychlorinated biphenyls (some of these are shown in Figure 22.10). In addition, several brominated and fluorinated organic compounds can be dehalogenated in analogous fashion. Many of these halogenated compounds are highly toxic and some have been linked to cancer (particularly trichloroethylene and vinyl chloride). Some of these compounds, such as PCBs, have been widely used as insulators in electrical transformers and enter anoxic environments from slow leakage of the transformer or from leaking storage containers. Moreover, perchloroethylene (PCE) is commonly used as a dry-cleaning solvent. Eventually these compounds end up in groundwater or sediment, where they are among the most common contaminants detected in the United States. There is therefore great interest in reductive dechlorination as a bioremediation strategy for their removal from anoxic environments, as we now consider.

Bioremediation of PCE and TCE

The organohalogens TCE and PCE are poorly soluble, but both can be degraded aerobically by monooxygenase enzymes, such as methane monooxygenase of methanotrophic bacteria (Section 14.16), to form nontoxic organic compounds (Figure 22.12). Early remediation efforts employed this activity to eliminate TCE from polluted sites. However, this is a cometabolic process that yields no energy for growth of methanotrophs, and maintaining activity required the continuous injection of both oxygen and methane—an explosive gas mixture—into the subsurface.

Figure 22.12 Anaerobic and aerobic pathways for PCE and TCE degradation.


Top panel: Anaerobic pathway for reductive dechlorination by Dehalococcoides species. Lower panel: cometabolic epoxide formation and abiotic product decay catalyzed aerobically by a monooxygenase enzyme. Monooxygenases generate unstable epoxides of both TCE and PCE.

More successful remediation is now based on stimulating the growth of organisms capable of using organohalogens as electron acceptors (anaerobic respiration), thus avoiding the need to inject gases into the subsurface. Many facultative or obligate anaerobes capable of organohalogen respiration are known (Figure 22.13). Obligate organohalide-respiring bacteria grow only by organohalide respiration, using H2 as an electron donor, and with the exception of Dehalobacter are members of the Chloroflexi (Figure 22.13). In contrast, facultative organohalide-respiring bacteria are more physiologically and phylogenetically diverse (Figure 22.13), being able to use organic electron donors and common electron acceptors such as Fe3+, SO4 2−, and NO3 −.

Figure 22.13 Phylogeny of organohalide-respiring bacteria.

Facultative organohalide respirers use other electron acceptors, such as nitrate, in addition to organohalides, and a greater variety of electron donors, including pyruvate, lactate, ethanol, and butyrate. Obligate organohalorespirers are restricted to H2 as electron donor and organohalides as electron acceptors.

Since the biologically available energy from organohalide respiration decreases with decreasing number of chlorines on the molecule, the much less favorable electron acceptor vinyl chloride (VC, Figure 22.12) often accumulates at polluted sites. This chemical is more toxic than its parent compounds, and remediation is successful only if conversion to the fully dechlorinated product, ethene, is achieved. Most organohalide-respiring bacteria are unable to fully dechlorinate PCE or TCE, but the bacterium Dehalococcoides can do this and supports a full bioremediation technology.

Successful bioremediation of PCE or TCE requires hydrogen (H2) to stimulate the growth of Dehalococcoides. This is achieved by the injection of an inexpensive fermentable carbon source to the contaminated site, most commonly molasses or emulsified vegetable oil (EVO). Fermentation of these generates H2 and acetate used as the electron donor for dehalogenation and as a carbon source, respectively, by Dehalococcoides. Nutrient (nitrogen and phosphate) injection coupled with groundwater extraction and recirculation accelerates solvent degradation. Bioaugmentation—the injection of an active Dehalococcoides culture into the subsurface—also accelerates the process or provides an inoculum if a resident Dehalococcoides population is absent (see MicrobiologyNow).

Plastics

Plastics are classic examples of xenobiotics, and the plastics industry worldwide produces over 300 million tons of plastic per year, almost half of which are discarded rather than recycled. Plastics are polymers of various chemistries (Figure 22.14). Many plastics remain essentially unaltered for long periods in landfills, refuse dumps, and as litter in the environment. A significant amount of synthesized plastic enters the marine environment, and this is of particular environmental concern. Weathering of plastic debris in the ocean causes fragmentation into small particles called microplastics (Section 20.4 and Figure 20.7) that marine invertebrates can ingest, possibly disrupting important marine food webs. Microplastics also clog the baleen filter used by baleen whales to harvest small marine invertebrates and fish.

Figure 22.14 Oil-based and biodegradable plastics.


(a) The monomeric structure of several synthetic plastics. (b) Structure of the copolymer of poly-β-hydroxybutyrate (PHB) and poly-β-hydroxyvalerate (PHV). (c) A brand of shampoo previously marketed in Germany and packaged in a bottle made of the PHB/PHV copolymer. (d) Drinking bottle made from PET, the only oil-based plastic now known to be significantly biodegraded by microorganisms. (e) Pathway of PET degradation by the bacterium Ideonella sakaiensis to generate monomers as a carbon and energy source.

Among commonly used plastics, only polyethylene terephthalate (PET) has hydrolyzable ester bonds that are susceptible to biodegradation (Figure 22.14d, e). Over 50 million tons of PET is produced yearly for plastic products such as soft drink and water bottles and polyester fabrics. Although the fungus Fusarium can slowly cometabolize PET to release terephthalic acid, the bacterium Ideonella sakaiensis (Betaproteobacteria, Section 16.2) can attach to the surface of PET, express enzymes that depolymerize the plastic (Figure 22.14e), and use PET as a carbon and energy source. Two hydrolase enzymes of I. sakaiensis depolymerize PET: the exoenzyme PETase releases mono(2-hydroxyethyl) terephthalic acid (MHET), which is converted into terephthalic acid and glycol by the cell surface enzyme MHETase (Figure 22.14e). These substances then support growth of I. sakaiensis. Since PET is the most common plastic used for bottled drinking water, discovery of the metabolic capacities of I. sakaiensis offers some hope that the tons of PET water bottles that have been casually disposed of into the environment in recent decades may eventually disappear through microbial action.

The persistence of plastic products in the environment has also fueled research into improved biodegradable alternatives to synthetic plastics including natural microbial polymers. Polyhydroxyalkanoates (PHAs) are a common bacterial storage polymer (Section 2.7), are readily biodegraded, and have many of the desirable properties of synthetic plastics. PHAs can be biosynthesized in various chemical forms, each with its own unique physical properties (stiffness, shear and impact strength, and the like). A PHA copolymer containing equal amounts of poly- β-hydroxybutyrate and poly-β- hydroxyvalerate was marketed in the past as a container for shampoo (Figure 22.14b, c). However, because synthetic plastics are currently less expensive than microbially synthesized plastics, plastics such as PET make up virtually the entire plastics market today.

Check Your Understanding

Why might the addition of inorganic nutrients stimulate oil degradation whereas the addition of glucose would not?

What is reductive dechlorination and how does it differ from the reactions shown in Figure 22.11?

How does the bacterium Ideonella sakaiensis degrade an insoluble substance such as PET that it cannot transport into the cell?

III Wastewater and Drinking Water Treatment

Appropriate treatment of wastewater is essential for maintaining environmental quality and for reducing the spread of waterborne diseases. Methods to purify wastewaters rely on the activities of microbial communities that reduce levels of organic carbon, fixed nitrogen, and phosphorus from the waste stream.

Water is the most important potential common source of infectious diseases because a single water source often serves large numbers of people, as, for example, in major cities. Thus, the microbiology of water, water transport systems, and treatments of both wastewater and drinking water are of the utmost importance to public health.

Here we examine systems built for the chemical and biological treatment of water and the transmission systems used for delivering treated water to consumers. Existing water transmission and treatment systems consume about 4% of all electricity in the USA, and we consider more efficient technologies now being brought into application. We also examine the human health consequences of the microbial communities that develop within the pipes of municipal water distribution systems and premise plumbing.

22.6 Primary and Secondary Wastewater Treatment

Wastewater is domestic sewage or liquid industrial waste that cannot be discarded in untreated form into lakes or streams because of public health, economic, environmental, and aesthetic considerations. Wastewater treatment employs physical and chemical methods as well as industrial-scale use of microorganisms. Wastewater enters a treatment plant and, following treatment, the effluent water—treated wastewater discharged from the wastewater treatment facility—is suitable for release into surface waters such as lakes and streams or to drinking water purification facilities (Figure 22.15).

Figure 22.15 Wastewater treatment processes.


Effective water treatment plants use the primary and secondary treatment methods shown here. Water from primary treatment undergoes aerobic oxidation, while the sludge collected from screening and sedimentation, along with flocs and activated sludge from aerobic oxidation of wastewater, undergoes anaerobic digestion (Section 22.8 and Figure 22.22). Tertiary treatment, not shown, may also be used to reduce nutrient levels in waters released to the environment, reducing biochemical oxygen demand (BOD), nitrogen, and phosphorus to very low to undetectable levels.

Mastering Microbiology

Art Activity: Figure 22.15 Wastewater treatment processes

Wastewater and Sewage

Wastewater from sewage or industrial sources cannot be discarded in untreated form into lakes or streams. Sewage is liquid effluent contaminated with human or animal fecal materials and may contain potentially harmful chemicals as well as pathogenic microorganisms. Domestic wastewater is made up of sewage, “gray water” (the water resulting from washing, bathing, cooking, and drinking), and wastewater from small-scale food processing in homes and restaurants. Industrial wastewater includes liquid discharged from the petrochemical, pesticide, food and dairy, plastics, pulp and paper, pharmaceutical, metallurgical, and manufacturing industries. All of these wastewaters must be treated—typically using physical, chemical, and microbiological processes—to remove or neutralize contaminants.

Industrial wastewater may contain highly toxic substances and are “pretreated” biologically or chemically to remove substances such as cyanide, heavy metals such as arsenic, lead, and mercury, and organic materials such as acrylamide and benzene. These substances are converted to less toxic forms by treatment with chemicals or microorganisms capable of neutralizing, oxidizing, precipitating, or volatilizing these wastes. The pretreated wastewater can then be released to the municipal wastewater treatment facility.

The major objective of wastewater treatment is to reduce organic and inorganic materials in wastewater to a level that no longer supports microbial growth and to eliminate other potentially toxic materials. The efficiency of treatment is expressed in terms of a reduction in the biochemical oxygen demand (BOD), the relative amount of dissolved oxygen consumed by microorganisms to completely oxidize all organic and inorganic matter in a water sample (Section 20.9). High levels of organic and inorganic materials in the wastewater result in a high BOD. Typical BOD values for domestic wastewater, including sewage, are approximately 200 mg of O2 per liter. For industrial wastewater from sources such as dairy plants, the values can be as high as 1500 mg/liter. An efficient wastewater treatment facility reduces BOD levels to less than 5 mg/liter in the final treated water.

Primary Treatment: Physical Removal Processes

Wastewater treatment is a multistep operation that relies on several different physical and biological processes (Figure 22.15). Primary, secondary, and sometimes additional (tertiary) treatments are employed to reduce biological and chemical contamination in the wastewater, and each higher level of treatment employs more complex technologies.

Primary wastewater treatment uses a series of grates and screens to physically remove large particulate materials (bottles and other heavy solids) from the wastewater. The effluent is allowed to settle for a few hours and the solids that settle to the bottom of the separation reservoir are typically treated in an anaerobic digester that consumes these solids and generates methane for energy recovery (see Figure 22.22b). The effluent (Figure 22.16) is drawn off for secondary treatment.

Figure 22.16 Primary treatment of wastewater.


Wastewater is pumped into the reservoir (left) where solids settle. As the water level rises, the water spills through the grates to successively lower levels. Water at the lowest level, now virtually free of solids, enters the spillway (arrow) and is pumped to a secondary treatment facility.

Secondary Treatment: Activated Sludge and Trickling Filters

Municipalities that provide only primary treatment discharge extremely polluted water with high BOD into adjacent waterways, and this triggers undesirable microbial growth and a reduction in water quality. Thus, secondary treatment is a necessity. Secondary wastewater treatment uses oxidative degradation reactions catalyzed by microorganisms under oxic conditions (**Figure 22.17a,*b***) to treat wastewater containing relatively low levels of organic material. In general, wastewaters that originate from residential sources can be treated efficiently using only aerobic treatment. Secondary treatment exploits the tendency of bacteria to attach to particulate material and form biofilms (Sections 4.9, 8.10, and 20.4), producing slimy, extracellular polymeric substances to form activated sludge flocs.

Figure 22.17 Secondary aerobic wastewater treatment processes.


Parts a and b show the activated sludge method. (a) Aeration tank of an activated sludge installation in a metropolitan wastewater treatment plant. The tank is 30 m long, 10 m wide, and 5 m deep. (b) Wastewater flow through an activated sludge installation. Recirculation of activated sludge to the aeration tank introduces microorganisms responsible for oxidative degradation of the organic components of the wastewater. (c) Trickling filter method. The rotating arms distribute wastewater slowly and evenly on the rock bed. The rocks are 10–15 cm in diameter and the bed is 2 m deep.

Mastering Microbiology

Art Activity: Figure 22.17 Secondary aerobic wastewater treatment process

Activated sludge consists of a community of slime-forming aerobic bacteria, including in particular Zoogloea ramigera (Betaproteobacteria, Section 16.2) (Figure 22.18). Protists, small animals, filamentous bacteria, and fungi also attach to the flocs. The activated sludge is mixed, aerated, and circulated in the treatment tanks to promote oxidation of organic carbon by the bacteria (Figure 22.17a, b). The aerated effluent containing the flocs is then pumped into a holding tank or clarifier where the flocs settle. Some of the activated sludge is then returned to the aerator as inoculum for new wastewater, and the rest is commonly pumped to an anaerobic sludge digester (see Figure 22.22c). Because of the volumes of wastewater needing treatment, wastewaters normally stay in an activated sludge tank for 5–10 hours, insufficient for complete oxidation of all organic matter. However, during this time much soluble organic matter is adsorbed to the flocs and incorporated by microbial cells, reducing the BOD by up to 95% (most of the material with high BOD is now in the settled flocs). The flocs are then transferred to the anoxic sludge digester for anaerobic degradation to CO2 and CH4 (Figure 22.17b).

Figure 22.18 A wastewater floc formed by the bacterium *Zoogloea ramigera*.


Floc formed in the activated sludge process consists of a large number of small, rod-shaped cells of Z. ramigera surrounded by a polysaccharide slime layer and arranged in characteristic fingerlike projections in this phase-contrast photomicrograph of a negatively stained preparation.

The trickling filter is an alternative method of aerobic secondary treatment (Figure 22.17c). A trickling filter is a bed of crushed rocks, about 2 m thick. Wastewater is sprayed on top of the rocks and slowly passes through the bed. The organic material in the wastewater adsorbs to the rocks, and microorganisms grow and form biofilms on the large, exposed rock surfaces. The complete mineralization of organic matter to CO2, ammonia, nitrate, sulfate, and phosphate takes place in the extensive microbial biofilm that develops on the rocks.

Following secondary treatment, whether by activated sludge or trickling filter, and assuming tertiary treatment (Section 22.7) is not undertaken, the effluent is typically chlorinated or treated with ozone to further reduce the possibility of biological contamination. The treated effluent can then be discharged into streams or lakes.

Check Your Understanding

What is biochemical oxygen demand (BOD), and why is its reduction important in wastewater treatment?

How do primary and secondary wastewater treatment methods differ?

Other than treated water, what are the final products of wastewater treatment? How might these end products be used?

22.7 Tertiary Wastewater Treatment: Further Removal of Phosphorus and Nitrogen

22.7 Tertiary Wastewater Treatment: Further Removal of Phosphorus and Nitrogen

22.7 Tertiary Wastewater Treatment: Further Removal of Phosphorus and Nitrogen

The combination of primary and secondary wastewater treatments removes a large amount of the BOD and some of the inorganic nutrients from wastewaters. However, additional treatment can generate effluent water of much higher quality. Such advanced wastewater treatment is called tertiary wastewater treatment. Typical goals of tertiary wastewater treatment include removal of additional BOD, removal of key inorganic nutrients required for microbial growth (in particular, ammonia, nitrate, nitrite, and phosphorus), and degradation of any remaining toxic materials. Tertiary treatment is the most complete method of treating sewage and is common in many European countries but has not been widely adopted in other parts of the world because it is expensive. Here we focus on the biological removal of phosphorus and nitrogen, two key nutrients controlling microbial growth.

Biological Phosphorus Removal

Conventional secondary biological treatment removes only about 20% of phosphorus from wastewater, necessitating additional chemical or biological treatment. Chemical precipitation is the most commonly used process, removing up to 90% of the influent phosphorus. Removal is accomplished by the addition of either Fe or Al as chloride or sulfate salts. At near-neutral pH, Fe3+ forms insoluble ferric phosphate (FePO4) or ferric hydroxide-phosphate complexes. These then precipitate and are removed as sludge.

The chemical precipitation process results in a toxic sludge, contributing to additional disposal problems. As an alternative, tertiary treatment that encourages the growth of phosphorus-accumulating bacteria removes up to 90% of phosphorus, a process called enhanced biological phosphorus removal, and does not generate a metal-rich sludge. Here the waste stream is processed by sequential passage through anaerobic and aerobic bioreactors (Figure 22.19). During the anaerobic phase, phosphorus-accumulating organisms (PAOs) utilize polyphosphate (a polymer of phosphate, Section 2.7) and glycogen reserves to generate ATP and reducing power to fuel the uptake and conversion of volatile fatty acids in the wastewater into another storage material, polyhydroxyalkanoate (PHA) (Figures 22.14b and 22.19a; Section 2.7). In the subsequent aerobic phase, PAOs oxidize the PHA for cell growth and replenish polyphosphate and glycogen reserves. The new biomass (sludge) with high polyphosphate content is then collected for phosphorus removal (Figure 22.19b).

Figure 22.19 Enhanced biological phosphorus removal process.


(a) Carbon and phosphate transitions during the treatment process. (b) Wastewater processing. During the passage of wastewater through the reactor system, the microbial community transitions from anaerobic to aerobic growth. In the anaerobic zone, short-chain fatty acids are taken up, intracellular glycogen is degraded, and internal stores of polyphosphate are released as extracellular orthophosphate. In the aerobic zone, the glycogen reserves are regenerated, extracellular phosphate is reassimilated as polyphosphate, and the intracellular stores of polyhydroxyalkanoates (PHAs) are metabolized. High-phosphorus sludge is harvested for disposal.

The ability to store carbon as PHA under anaerobic conditions and later use this intracellular reserve for aerobic growth allows the PAOs to outcompete other chemoorganotrophs that can only consume carbon aerobically. Two of the principal PAOs, the appropriately named bacterium Accumulibacter phosphatis (Betaproteobacteria, Section 16.2) and species of the gram-positive bacterium Tetrasphaera (Actinobacteria, Section 16.10), have been used in the laboratory as model systems for the process of enhanced biological phosphorus removal.

Biological Nitrogen Removal: The Conventional Nitrification–Denitrification Process

The strict regulatory limitations on release of nitrogen as ammonia or nitrate/nitrite, also called reactive nitrogen (Nr), from wastewater treatment facilities reflects their adverse health effects on human and aquatic life and contribution to eutrophication of receiving water bodies (Section 20.9). Thus, there has been increasing use of tertiary treatment to remove remaining Nr from wastewater following secondary treatment or anaerobic sludge digestion (see Figure 22.22).

Conventional (“classical”) treatment converts the Nr to its inert atmospheric form (N2) using a combination of nitrification and denitrification (**Figure 22.20*a***). Nitrification (Section 14.9) is first used to convert ammonia to nitrate followed by anaerobic conversion of nitrate to N2 (and some N2O) by denitrification (Section 14.11). In the following reactions for the conversion of 1 mole of ammonia to 0.5 mole of N2, organic carbon is represented by its chemical oxygen demand (COD), the mass of oxygen that is reduced by a specific amount of organic matter; for example, a COD of 4 g reduces 4 g of O2 (0.125 moles) to water: Ammonia oxidation: NH4 ++1.5 O2→NO2 −+H2O+2 H+Nitrite oxidation: NO2 −+0.5 O2→NO3 −Denitrification: NO3 −+40 g COD+H+→0.5 N2(producing 15 g sludge biomass)

Figure 22.20 Alternative treatment processes for nitrogen removal from wastewater.


(a) Classic denitrification process. (b) Advanced treatment process for more efficient and economical removal of nitrogen. Wastewater is first treated anaerobically to remove incoming nitrate by denitrification before being nitrified in a second reactor and recycled back to the receiving tank for full nitrogen removal. SRT, sludge retention time.

Although the primarily autotrophic ammonia- and nitrite-oxidizing microbes require little or no reduced carbon for growth and also generate very little biomass, a carbon source is required for the subsequent microbial reduction of nitrate to N2 by denitrification. This may require supplementation with additional carbon for wastewaters having a low ratio of organic carbon to reactive nitrogen (C/N). Supplementation is commonly accomplished by adding a relatively inexpensive carbon source, such as methanol in low concentrations. However, because a typical treatment plant treats well over a million gallons (3.8 million liters) of wastewater a day, organic carbon addition can add significant costs, and this has fueled the development of less costly processes for advanced treatment.

Biological Nitrogen Removal: The Advanced Process of Nitritation–Denitrification

The cost of Nr removal can be reduced through advanced treatment processes that suppress the activity of nitrite-oxidizing bacteria, a process called nitritation (Figure 22.20b). By stopping the oxidation of ammonia at nitrite, the carbon requirement for denitrification is reduced by ∼40%, oxygen by ∼25%, and biomass by ∼40%. Ammonia oxidation: NH4 ++1.5 O2→NO2 −+H2O+2 H+Denitrification: NO2 −+24 g COD+H+→0.5 N2 (producing 9 g sludge biomass)

This makes nitrogen removal from low C/N wastewater less costly. The only complication to this treatment strategy is the need to suppress nitrite-oxidizing bacteria (NOB), since they generally have higher substrate utilization rates than the ammonia-oxidizing bacteria (AOB). Achieving cost-effective control of partial ammonia oxidation is therefore key to advanced nitrogen removal treatment systems.

Suppression of nitrite oxidation is achieved by adjusting growth conditions that differentially affect AOB and NOB, primarily those of pH and temperature. AOB grow faster than NOB at temperatures above room temperature, having a specific growth rate (Section 4.7) approximately two times that of NOB at 35 °C. For example, one widely used process relies on washing out NOB from a reactor operated at 30–40 °C and a short sludge retention time (SRT) of 1–1.5 days (Figure 22.20b). The SRT can be controlled by the rate the reactor is fed wastewater or by allowing sludge to settle during a period without mixing before withdrawing the treated effluent. Control of reactor pH provides another way to achieve partial nitrification, as an alkaline pH (7.5–8.5) favors the growth of AOB over NOB (Figure 22.20b).

Aerobic Granular Sludge Treatment

Aerobic granular sludge consists of microbial aggregates of average 0.5 to 3.0 mm diameter having settling velocities at least 10 times greater than the loosely structured microbial flocs in flocculent activated sludge (**Figure 22.21b,*c***). Treatment systems are designed to select for granule formation by repeated removal of suspended floc material that does not settle rapidly. The greater microbial density in granules compared to that in flocs results in much greater nutrient removal capacity on a reactor volume basis than can be achieved using systems that produce flocculent sludge. In addition, the granular architecture provides another unique advantage relative to flocs. As was described for oxygen diffusion into soil aggregates (Section 20.3 and Figure 20.3), the rapid consumption of O2 by aerobes near the granule surface results in the formation of an anoxic core in larger granules. Thus, both aerobic and anaerobic processes can be sustained within the same granule, providing spatially resolved niches for different functional guilds.

Figure 22.21 Granular sludge wastewater treatment.


(a) Simultaneous phosphorus removal and denitrification using granular sludge technology (Nereda® process). Stages of phosphate release and uptake by phosphorus-accumulating organisms (PAOs) are shown by treatment stages T1−T4. At anoxic stage T2, glycogen and polyphosphate intracellular reserves are consumed to fuel production of intracellular polyhydroxyalkanoate (PHA) and the release of phosphate. At T3, PHA is consumed to drive phosphate uptake and synthesis of intracellular polyphosphate, using O2 or nitrite/nitrate generated by nitrification to fuel PAO denitrification. (b) The very rapid settling of granular sludge (left) relative to activated sludge (right) after only a few minutes. (c) Granules average 1–3 mm in diameter. (d) Schematic shows how the layered structure of a granule separates aerobic and anaerobic processes in the same granule.

In a granular sludge reactor designed for simultaneous nitrogen and phosphate removal, the reactor is cycled between oxic and anoxic conditions by feeding of wastewater sequentially to the bottom of the reactor (Figure 22.21a). The ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) localize near the granule surface (Figure 22.21d). Facultative denitrifying PAOs are enriched throughout the granule (Figure 22.21d). As was shown for the enhanced biological phosphorus removal process (Figure 22.19), during the anoxic phase, the PAOs consume intracellular reserves of glycogen and polyphosphate to release inorganic phosphate to the bulk wastewater, and at the same time synthesize and store PHAs [Figure 22.21a, T(stage)2]. During the oxic phase the AOB and NOB work together to oxidize ammonia to NO3 −. The PAO populations oxidize their intracellular reserves of PHA and take up phosphate from the bulk liquid (Figure 22.21a, T3). The PAOs near the granule surface use oxygen as their electron acceptor for phosphate uptake, whereas those localized to the anoxic interior use nitrate or nitrite originating from ammonia oxidation for concurrent phosphate uptake and denitrification (Figure 22.21d). The granular sludge is then allowed to settle, some of the sludge is removed to recover phosphorus (Figure 22.21a, T4), and the cycle is repeated.

High efficiencies for nitrogen and phosphorus removal have been shown for full-scale aerobic granular sludge facilities treating municipal wastewaters. As application of these treatment systems becomes more widespread, energy savings are significant, and the footprint of the wastewater treatment facility is reduced relative to traditional systems using flocculent activated sludge (Figure 22.17) designed to treat the same wastewater.

Check Your Understanding

What are the advantages of enhanced biological phosphorus removal relative to traditional chemical removal of phosphorus?

What are two important advantages of aerobic granular sludge technology relative to traditional activated sludge treatment?

22.8 Sludge Processing and Contaminants of Emerging Concern

22.8 Sludge Processing and Contaminants of Emerging Concern

22.8 Sludge Processing and Contaminants of Emerging Concern

The generation of significant microbial biomass (sludge) as a product of secondary and tertiary wastewater treatment requires costly landfill disposal. However, sludge volume can be reduced by anaerobic treatment, using the degradative and fermentative reactions of anaerobic microorganisms to lower disposal costs as well as generating methane that can be burned for energy recovery.

Anaerobic Sludge Treatment

As we saw in Section 21.2, under conditions of electron acceptor limitation, fermentation by a community of Bacteria and Archaea converts organic matter into methane and carbon dioxide (**Figure 22.22*b***; Figure 21.7). This requires syntrophic processes in which the fermentative organisms supply electron donors (such as H2 and acetate) for methanogens. The treatment of wastewater sludge is an ideal example of these cooperative processes in action.

Figure 22.22 Sludge processing.


(a) Anaerobic sludge digester. Only the top of the tank is shown; the remainder is underground. (b) Major microbial processes in anaerobic sludge digestion. (c) Inner workings of a sludge digester. Methane (CH4) and carbon dioxide (CO2) are the major products of anaerobic biodegradation. (d) Following separation of solids and liquids, the warm ammonia-rich brine (about 50% each of NH4 + and NO2 −) is treated by the anammox process.

The anaerobic degradation process takes place in large, enclosed tanks called sludge digesters or bioreactors (Figure 22.22a, c). The process requires the collective activities of many different microbes, and the major reactions are summarized in Figure 22.22b. Anaerobes use polysaccharidases, proteases, and lipases to digest suspended solids and large macromolecules into soluble components. These are then fermented to yield a mixture of fatty acids, H2, and CO2; the fatty acids are further fermented by the cooperative actions of syntrophic bacteria (Sections 14.22 and 21.2) to produce acetate, CO2, and H2. These products are then converted to CH4, and the CH4 is either burned off or used as fuel to heat and power the wastewater treatment plant.

Although sludge volume is greatly reduced by anaerobic treatment, significant nitrogen and phosphate remains in the liquid phase, generating a side stream of N- and P-rich brine, with N concentrations up to 100-fold greater than that produced by secondary treatment. The N-rich side stream must be further treated, using either conventional tertiary treatment or the anammox process, which we consider now.

Nitrogen Removal by Partial Nitritation–Anammox

Since anammox bacteria are anaerobic chemolithotrophic autotrophs (Section 14.10), they do not depend on organic carbon as an electron donor or oxygen as an electron acceptor. The anammox process is as follows: Anammox:NH4 ++1.32 NO2 −+0.066 HCO3 −+0.13 H+→1.02 N2+0.26 NO3 −+2.03 H2O+0.066 Cbiomass

The key to using the anammox process for nitrogen removal is an initial oxidation of only half the ammonia in the side stream brine to nitrite, yielding a 50:50 mixture of ammonia and nitrite. This process is called partial nitritation (Figure 22.22d). The effluent of the partial nitritation reactor is then fed to an anammox reactor for conversion of ammonia and nitrite to N2. The partial nitritation reactor exploits the higher growth rate of AOB compared to NOB at the higher temperatures used, and the higher affinity of AOB for oxygen compared to NOB. By keeping reactor temperature warm and oxygen concentration low, ammonia is oxidized to nitrite at a short SRT. Since AOB and NOB are unable to grow in the absence of oxygen, anammox is the only significant process in the anoxic reactor (Figure 22.22d).

This overall process is called partial nitritation–anammox (PNA). Compared with conventional biological nitrogen removal (Figure 22.20a), PNA has no requirement for added organic carbon, uses less electricity, generates less sludge, and has lower emissions of greenhouse gases (primarily CO2 and N2O). Thus, this process greatly reduces the environmental and energy costs of more conventional nitrogen removal technology.

Contaminants of Emerging Concern

A wide variety of chemicals end up in wastewaters besides the usual organic carbon and inorganic nutrients that processing systems are designed to deal with. These include heavily used agricultural products and chemicals that demonstrate acute toxicity or carcinogenicity. However, bioactive pollutants also enter wastewater streams and pose new challenges for microbial bioremediation. These include pharmaceuticals, active ingredients in personal care products, fragrances, household products, sunscreens, prescription and other medications, and many other uncommon or xenobiotic molecules.

Unlike pesticides, these pollutants are more or less continuously discharged into the environment through the release of treated sewage, which means they do not need to persist to have environmental effects. For example, it is known that low levels of synthetic estrogen compounds, excreted in the urine of women taking birth control pills and eventually discharged in active form from wastewater treatment plants, can activate estrogen response genes in aquatic animals such as fish and contribute to the feminization of males.

The environmental threat from these emerging contaminants has created a need for newly designed wastewater treatment facilities to stimulate bioremediation of these wastes. However, because these substances are often present in very low concentrations and in some cases may be new classes of xenobiotics, they may not actually support microbial growth but rather only be degraded by cometabolism (Section 22.5) or by highly specialized species. Moreover, because wastewaters contain such high concentrations of readily degradable organic materials, structurally complex emerging contaminants may be ignored by microbes until the COD of the wastewater is greatly reduced. We can therefore expect that the bioremediation of emerging contaminants will be an active area of microbiological research in coming years.

Check Your Understanding

What are the advantages of further treating activated sludge by anaerobic digestion?

Why is the incomplete oxidation of ammonia essential for effective treatment of sludge brine using the anammox process?

What advantages does the anammox process have over classical wastewater treatments for nitrogen removal?

22.9 Drinking Water Purification and Stabilization

22.9 Drinking Water Purification and Stabilization

22.9 Drinking Water Purification and Stabilization

Wastewater treated by secondary methods can usually be discharged into rivers and streams. However, such water is not potable (safe for human consumption). The production of potable water requires further treatment to remove potential pathogens, eliminate taste and odor, reduce nuisance metals such as iron and manganese, and decrease turbidity, which is a measure of suspended solids. Suspended solids are small particles of solid pollutants that resist separation by ordinary physical means.

In 1900, water purification in the United States was limited to filtration to reduce turbidity. Although filtration significantly decreased the microbial load of water, many microbes still passed through the filters and thus cases of waterborne diseases such as cholera and typhoid (Section 33.3 and Figure 30.7) were common. Not until the practice of chlorination became widespread were waterborne diseases controlled. Indeed, major improvements in public health in the United States and other developed countries were largely due to the adoption of water filtration and disinfection treatment procedures. Today, waterborne diseases still occur, but are rarely linked to well-run and properly maintained municipal water facilities.

Physical and Chemical Purification

A typical municipal installation for drinking water treatment is shown in **Figure 22.23*a***. Figure 22.23b shows the process that purifies raw water (also called untreated water) that flows through the treatment plant. Raw water is first pumped from the source, in this case a river, to a sedimentation basin where anionic polymers, alum (aluminum sulfate), and chlorine are added. Sediment, including soil, sand, mineral particles, and other large particles, settles out. The sediment-free water is then pumped to a clarifier or coagulation basin, which is a large holding tank where coagulation takes place. The alum and anionic polymers form large particles from the much smaller suspended solids. After mixing, the particles continue to interact, forming large, aggregated masses, a process called flocculation. The large, aggregated particles (flocs) settle out by gravity, trapping microorganisms and adsorbing suspended organic matter and sediment.

Figure 22.23 Water purification plant.


(a) Aerial view of a drinking water treatment plant in Louisville, Kentucky, USA; source water is from the Ohio River. The arrows indicate direction of flow of water through the plant. (b) Schematic overview of a typical community water purification system.

Mastering Microbiology

Art Activity: Figure 22.23b Water purification plant

After coagulation, flocculation, and sedimentation, the clarified water undergoes filtration through a series of filters designed to remove organic and inorganic solutes, as well as remaining suspended particles and microorganisms. The filters typically consist of thick layers of sand, activated charcoal, and ion exchangers. After this step and the previous purification steps, the filtered water is free of particulate matter, most organic and inorganic chemicals, and nearly all microorganisms.

Disinfection

Clarified, filtered water must be disinfected before it is released to the supply system as potable finished water. Primary disinfection is the introduction of sufficient disinfectant into clarified, filtered water to kill existing microorganisms and inhibit further microbial growth. Chlorination is the most common method of primary disinfection. In sufficient doses, chlorine kills most microorganisms within 30 minutes by physically oxidizing and otherwise destroying the cell and its contents. A few pathogenic protists such as Cryptosporidium, however, are not easily killed by chlorine treatment (Section 34.4). In addition to killing microorganisms, chlorine oxidizes and effectively neutralizes many organic compounds. Since most taste- and odor-producing chemicals are organic compounds, chlorination thus improves water taste and smell as well as disinfecting.

Chlorine is added to water from either a concentrated solution of sodium or calcium hypochlorite or as chlorine gas from pressurized tanks. Chlorine gas (Cl2) is commonly used in large water treatment plants because it is most amenable to automatic control. When dissolved in water, chlorine gas forms hypochlorous acid (HOCl), which dissipates within hours from treated water. To maintain adequate levels of chlorine for primary disinfection, many municipal water treatment plants introduce ammonia gas with the chlorine to stabilize the chlorine by forming chloramine (NH2Cl): HOCl+NH3→NH2Cl+H2O.

Chlorine is consumed when it reacts with organic materials. Therefore, sufficient quantities of chlorine must be added to finished water containing organic materials so that a small amount, called the chlorine residual, remains. The water plant operator performs chlorine analyses on the treated water to determine the level of chlorine to be added for secondary disinfection, the maintenance of sufficient chlorine residual in the water distribution system to inhibit microbial growth. A chlorine residual level of 0.2–0.6 mg/liter is the level achieved in most water supplies. After chlorine treatment, the now potable water is pumped to storage tanks from which it flows by gravity or pumps through a distribution system of storage tanks and supply lines to the consumer. Although residual chlorine inhibits growth of bacteria in the finished water prior to the water reaching the consumer, it does not protect against catastrophic system failures, such as a broken pipe in the distribution system.

Ultraviolet (UV) radiation is also used as an effective means of disinfection. As we discussed in Section 4.18, UV radiation is used to treat secondarily treated effluent from water treatment plants. In Europe, UV irradiation is commonly used for drinking water applications, and it is increasingly used in the United States. For disinfection, UV light is generated from mercury vapor lamps. Their major energy output is near 260 nm, a wavelength that is strongly absorbed by DNA and is mutagenic (Section 9.4) and thus bactericidal; irradiation may also kill cysts and oocysts of protists such as Giardia and Cryptosporidium, important eukaryotic pathogens in water (Section 34.4). Viruses, however, are more resistant.

UV radiation has several advantages over chemical disinfection procedures like chlorination. First, UV irradiation is a physical process that introduces no chemicals into the water. Second, UV radiation–generating equipment can be used in existing flow systems. Third, no disinfection byproducts (such as chloroform, a possible human carcinogen) are formed with UV disinfection. Especially in smaller systems where finished water is not pumped long distances or held for long periods (reducing the need for residual chlorine), UV disinfection may be preferable to reduce dependence on chlorination.

Check Your Understanding

What specific purposes do sedimentation, coagulation, filtration, and disinfection accomplish in the drinking water treatment process?

What general procedures are used to reduce microbial numbers in water supplies?

What are the advantages of disinfection with ultraviolet radiation versus, or as a complement to, chemical disinfection with chlorine?

22.10 Water Distribution Systems

Once drinking water leaves the treatment facility, the water often travels long distances through municipal and premise distribution pipes from the facility to the consumer (Figure 22.24). In addition to taste and odor problems that may be present in the source water, the long transit and residence times may also contribute to undesirable taste, odors, and color from biological and chemical processes. Although undesirable, taste and odor alone usually do not signal a health threat. However, water distribution systems may also promote the growth of obligate or opportunistic pathogens (Section 25.4), sequester and protect pathogens, or select for more pathogenic and resistant forms of microorganisms. Even though drinking-water-associated disease often goes unreported, disease outbreaks do occur and are occasionally fatal (Chapter 33).

Figure 22.24 Drinking water distribution system.


A municipal distribution system includes a surface reservoir, water purification plant, distribution mains, and domestic lines that encompass many miles of pipes in a typical community.

Mastering Microbiology

Art Activity: Figure 22.24 Drinking water distribution system

The Microbiology of Municipal Water Distribution Systems

Microbial growth in drinking water distribution systems can be eliminated only through complete nutrient removal (elimination of growth substrates originating from the source water and from distribution system structural materials) or by maintaining appropriate residual chlorine levels throughout the distribution system. Neither of these is realistically attainable. Growth is unavoidable as a consequence of reduction in chlorine concentration with increasing distance from the point of production together with the tendency for microorganisms to form biofilms on the pipe walls. Microorganisms in biofilms are more resistant to disinfection (Sections 4.9, 8.10, and 20.4), and significant microbial accumulation is found in all distribution systems, over 90% of which is in the form of biofilms that coat the pipe walls.

Culture-independent molecular techniques, in particular 16S rRNA sequence analysis (Section 19.6), have been used to explore biofilms in water distribution pipes. Although these studies are showing that pathogenic species are rare, some opportunistic pathogens (Section 25.4) are present and can infect susceptible humans, including infants, the elderly, and individuals with compromised immune systems. Opportunistic pathogens found in water distribution systems include (1) nontuberculous mycobacteria (including Mycobacterium avium, M. intracellulare, M. kansasii, and M. fortuitum) associated with many thousands of clinical cases each year in the United States; (2) Legionella pneumophila (the causative agent of Legionnaires’ disease, Section 33.4); (3) Pseudomonas aeruginosa (which can infect the eyes, ears, skin, and lungs); and (4) opportunistic protozoan pathogens such as Naegleria and Acanthamoeba (Section 34.3) that can cause keratitis and encephalitis.

Water distribution systems also support numerous grazing protists that subsist by consuming bacteria. Bacteria that survive and replicate following ingestion by these protists are potentially also less susceptible to clearance by the mammalian immune system. An example is Legionella, which is able to establish residence and replicate in protists inhabiting water-handling systems (Figure 22.25), including premise plumbing, shower heads, and air conditioning systems. The basic mechanisms Legionella uses to gain entry and replicate in a broad variety of protists (including Acanthamoeba, Hartmannella, Naegleria, and Tetrahymena) also allow it to more easily infect human cells.

Figure 22.25 Protists as reservoirs of *Legionella*.


Two cells of the protist Tetrahymena contain chains of the rod-shaped pathogen Legionella pneumophila (arrows). In premise water systems, protists can persist and be reservoirs of bacterial pathogens.

In addition to Legionella, opportunistic pathogens that can survive or grow within protists include Pseudomonas and Mycobacterium species. Nontuberculous mycobacteria (M. avium, M. intracellulare, M. kansasii, and M. fortuitum), also called “environmental mycobacteria,” cause a variety of lung infections distinct from that of tuberculosis. These bacteria are more resistant to chlorine disinfection and protozoal grazing and have been found to be enriched in showerheads receiving municipal water that still maintains a chlorine residual. It is possible that aerosols generated in the showering process could transmit these pathogens to humans. Thus, although water purification has made great strides in eliminating waterborne infections on a grand scale, the architecture of water distribution systems coupled with the aging condition of many systems can still compromise human health (see Figure 22.26a).

We transition now to the final part of this chapter where we examine the microbiology and microbial activities that occur in buildings, homes, and other structures.

Check Your Understanding

Trace the treatment of water through a drinking water treatment plant, from the inlet to the final distribution point (faucet).

How could your household water system transmit disease?

IV: Indoor Microbiology and Microbially Influenced Corrosion

IV: Indoor Microbiology and Microbially Influenced Corrosion

IV Indoor Microbiology and Microbially Influenced Corrosion

Indoor air, both in private dwellings and public places, as well as surfaces in the structures themselves, can be teeming with microbes. In addition, costly losses occur yearly from corrosion catalyzed by microbial activities on metal, stone, and concrete infrastructure.

Although one might think that being indoors protects a person from the microbial world, nothing could be farther from the truth. Microbes are everywhere life can be found. Moreover, microbial metabolism accelerates corrosion through the changes in pH or redox that accompany the metabolism, production of corrosive metabolites, and creation of corrosive microenvironments in biofilms. Here we examine the microbial ecosystems of the built environment and a few cases in which the microbial contribution to corrosion is well understood. Recall that we have previously seen how surfaces are favorable microbial habitats (Section 20.4); virtually any surface that provides nutrients will be colonized by microorganisms.

22.11 The Microbiology of Homes and Public Spaces

Humans in urban environments spend the majority of their life indoors. They share this indoor environment with a microbiota that inhabit the air, dust, surfaces, and ventilation and water systems (Figure 22.26). The health effects of indoor microbial exposure may be positive or negative (Figure 22.26a). Pathogens, such as antibiotic-resistant Staphylococcus aureus and other Staphylococcus species, may be elevated in the indoor environment as a consequence of shedding from human skin. In contrast, the increased incidence of allergies and autoimmune disorders in children in developed countries has been attributed in part to the “too-clean” indoor environments that reduce exposure to microbes important for “training” the immune system early in life. Thus, a microbially depleted indoor environment may actually be detrimental to human health.

Figure 22.26 Sources of airborne and surface-associated microorganisms in the built environment.


(a) Sources of microorganisms in a typical household, including surfaces, humans, pets, plumbing systems, and outdoor air. The colors correspond to microorganisms or spores that may be beneficial or not harmful (green) or potentially detrimental (red) to human health. (b) Air particle collector (arrow) deployed in the New York City subway system for surveying the diversity of airborne bacteria by 16S rRNA gene sequencing.

In addition to living with microbes in our home environment, what microbial exposures does a person experience in major public spaces, such as subway systems, the supermarket, or even the classroom? These questions can now be addressed using 16S (or 18S) rRNA gene sequencing of DNA isolated from samples collected from different parts of the indoor environment (Figure 22.26b; Section 19.6).

Microbiology of the Indoor Air in Private Dwellings

Studies of the microbiology of indoor air and surfaces have become increasingly popular. For example, dust collected from upper trims of inside and outside doors of over a thousand homes in the United States revealed distinct indoor and outdoor microbial communities composed of fungi and bacteria. The outdoor fungi closely resemble the outdoor fungal populations found in the same geographical region, whereas the indoor bacterial communities more strongly reflect the type and number of occupants, including pets. Local geography also influences the indoor microbiome. For example, rural and farmland households harbor a greater abundance of microorganisms associated with the guts of farm animals, including the opportunistic pathogens Bartonella, Enterococcus, and Ruminococcus, than do urban dwellings.

In general, the overall indoor diversity of bacteria and fungi is greater than that found outdoors, reflecting a mixing of indoor and outdoor sources. A small number of fungal species are more abundant inside the house, including common molds such as Aspergillus, Penicillium, Alternaria, and Fusarium. In addition, the incidence of these fungal populations increases with the age of the dwelling and whether or not a basement is present (basements are often damp, which increases mold abundance).

Bacteria that are characteristic of the human skin (gram-positive bacteria such as Staphylococcus, Streptococcus, Corynebacterium, and Propionibacterium), feces (Bacteroides, Faecalibacterium, Ruminococcus), or the vagina (Lactobacillus, Bifidobacterium, Lactococcus) are much more commonly found inside a house than outdoors. The increased incidence of species of the bacteria Prevotella, Porphyromonas, Moraxella, and Bacteroides is typically associated with the presence of domestic animals—in particular dogs or cats—in a home. In fact, it is possible to predict with near certainty whether a home has these pets based only on a molecular analysis of household microbiota.

In addition to the surveys of dust described above, molecular surveys of home surfaces show that the microbial communities of human hands, noses, and feet (Section 10.10 and Figure 10.28; Section 24.5) closely resemble the organisms found on household surfaces, including floors, light switches, countertops, and door knobs (Figure 22.26a). Moreover, the surface-associated microbiota of a home is highly predictive of a specific family, and the composition of the microbial community has been found to shift within days of a change in home occupants.

Diagnostic microbial populations in an individual are also highly correlated with the objects they touch, such as desks, computers, phones, and chairs. For example, in a study that combined 16S rRNA gene sequencing and metabolomics (Sections 10.10 and 19.8) of the surfaces of commonly touched objects, residues from medicines and personal care products such as shampoos, deodorants, and lotions were detected. The presence or absence of theophylline, a by-product of caffeine, identified coffee drinkers or nondrinkers. When information of microbial species composition and chemical profiles were combined, all the individuals working in the same office could be linked to specific office furniture and equipment at high confidence. Studies such as this point to the likely development in the not-too-distant future of microbiology-based forensics.

The common use of antimicrobials in cleansers and soaps is now known to significantly alter the indoor microbiome. Although now banned in the United States, the chlorinated antimicrobials triclosan and triclocarban were widely used and can still be found in older household products such as soaps, cosmetics, deodorants, and kitchen cutting boards. Because gram-positive bacteria are less susceptible to these substances than gram-negative bacteria, gram-positives, including some drug-resistant species, become predictably enriched in households using these products. Thus, practices once thought to be a measure of a healthy household and good personal hygiene may actually select for, rather than eliminate, potentially harmful organisms.

Microbiology of Public Places

Public buildings, office spaces, and transit systems are another important component of the built environment. The heavily trafficked New York City municipal subway system (Figure 22.26b) alone moves over 1.5 billion passengers a year. Similar to homes, subways and offices contain a mixture of airborne microorganisms sourced from humans and the outdoor air. Because air exchange in a subway system must be extensive, most airborne microorganisms in subway systems are typical of those found outdoors. In addition, however, about 5% of the microbial population in subway air is composed of microbes that normally reside on the feet, hands, arms, and heads of humans; these are most likely shed from the more exposed areas of subway riders.

Indoor plumbing of public and private buildings is another well-recognized point of microbial exposure. Each flush of a toilet generates over 100,000 small (<5-μm) aerosol particles. Since aerosolized bacteria and viruses can remain viable for hours after they deposit on bathroom surfaces, flushing is a potential mechanism of enteric pathogen transmission as well as a means of transmitting harmless saprophytes from person to person. Transmission of human diseases caused by direct contact (such as sexually transmitted diseases) is not a major issue with bathroom fixtures because pathogens such as Neisseria gonorrhoeae (gonorrhea) and Treponema pallidum (syphilis) are very sensitive to drying. However, because of the enormous numbers of bacteria shed in feces, transmission of enteric bacteria by bathroom aerosols is a distinct possibility.

Studies of indoor microbiology continue to reveal the types and origins of microorganisms we spend much of our day in contact with, providing information that has several applications. Besides identifying potential hot spots of infectious disease, knowledge of indoor microbiology can inform the design of future construction such that new dwellings enhance beneficial exposures and limit detrimental exposures to microorganisms. Although microbe-free dwellings are impossible because humans and pets carry microbes, controlling the microbial content of recirculated air transmitted through dwellings and the development of environmentally friendly yet still effective antimicrobials for indoor use are worthwhile goals.

Check Your Understanding

How can a microbial inventory reveal information about the presence or absence of household pets?

Which room(s) in a private dwelling are potentially the most dangerous from a microbiology perspective, and why?

How might microbiology be used to develop a new type of forensic science?

22.12 Microbially Influenced Corrosion of Metals

Iron is the most commonly used metal in the built environment. Water, gas, and oil distribution pipelines—especially those laid decades ago—are made of metal, and their corrosion contributes to the greatest loss of infrastructure in the built environment. Corrosion of iron by oxygen in air is thought to be solely an electrochemical process. However, much critical iron-containing infrastructure is buried or submerged, restricting exposure to oxygen, and these are subject to microbial attack. At near-neutral pH and anoxic conditions, corrosion of iron and steel is significantly accelerated by microbially influenced corrosion (MIC). Microbial groups implicated in MIC include sulfate-reducing bacteria (Sections 14.12 and 15.11), ferric-iron-reducing bacteria (Sections 15.13 and 21.5), ferrous-iron-oxidizing bacteria (Sections 14.8, 15.14, and 21.6), and methanogens (Sections 14.15, 17.2, and 21.2).

Metal Corrosion by Sulfate-Reducing Bacteria

Metal structures submerged in the marine environment and pipelines used for transmission of low-grade oil are particularly subject to MIC through the activities of sulfate-reducing bacteria. Corrosion by sulfate-reducing bacteria is partly attributable to the chemically corrosive nature of hydrogen sulfide (H2S), the product of their metabolism. Crude oils containing more than about 0.5% sulfur by weight are called “sour” and may be naturally corrosive because of the H2S that is present. In oil fields near the ocean, such as in the Middle East and Alaska, seawater is injected to maintain reservoir pressure and force oil into the producing well. Since seawater contains nearly 30 mM sulfate, an undesirable consequence of injection is further souring by stimulating the growth of sulfate-reducing bacteria, some species of which can oxidize hydrocarbons anaerobically using sulfate as electron acceptor (Section 22.4; Section 14.24).

A strategy now used by the petroleum industry to control souring is the addition of nitrate (NO3 −) in the injection water, stimulating the growth of nitrate-reducing bacteria. Since nitrate respiration is energetically more favorable than sulfate respiration (Sections 14.11 and 14.12), the nitrate reducers outcompete sulfate reducers for usable organic electron donors in the oil. Nitrate also stimulates the growth of sulfide-oxidizing, nitrate-reducing chemolithotrophs (Sections 14.7 and 15.12), thereby reversing souring by oxidizing the sulfide.

Mechanisms of Metal Corrosion

At least two mechanisms have been proposed for how sulfate reducers corrode iron. In the first mechanism, H2 consumption by the sulfate reducer accelerates electrochemical pitting of the iron surface (**Figure 22.27*a***). This model is based on the capacity of many sulfate reducers to use hydrogen (H2) as an electron donor, thereby accelerating the energetically favorable but kinetically slow H2 production originating from the chemical oxidation of elemental iron (Fe0+2 H+→Fe2++H2). The overall stoichiometry of this reaction shows that Fe2+ formed from pitting reacts with sulfide from sulfate reduction in an energetically favorable reaction.

Figure 22.27 Corrosion of iron by sulfate-reducing bacteria.


Two models for the activities of sulfate-reducing bacteria in metal corrosion. (a) Oxidation of metallic iron is accelerated by bacterial consumption of H2 produced abiotically by proton reduction at the metal surface. (b) Direct electron transfer from the metal occurs via electron-conductive outer cell wall structures connecting to an electron transfer system spanning the periplasm. (c) Top: photo of a model iron surface undergoing sulfidic corrosion. Bottom: scan of a side view of the metal surface in the photo shows in dark green the areas where corrosion and pitting of the metal surface is greatest.

A second mechanism, such as that carried out by the bacterium Desulfopila corrodens, has the capacity to take up electrons directly from Fe0 (Figure 22.27b). In this mechanism, cells attached to the metal surface engage in direct (cathodic) electron uptake from Fe0 through an electroconductive sulfidic corrosion layer (Figure 22.27b), likely facilitated by conductive cellular structures like those used in the oxidation or reduction of insoluble electron donors or acceptors (Sections 15.13 and 21.5). A similar ability to take up electrons directly from Fe0 has been observed for a Methanobacterium species that produces methane (CH4) from CO2 reduction rather than sulfide from sulfate reduction during growth on Fe0. The Fe2+ resulting from methanogenic Fe0 oxidation then complexes with HCO3 − to form FeCO3 (Figure 22.27b). However, regardless of mechanism and the organism oxidizing the Fe0, the end result is metal corrosion (Figure 22.27c).

Check Your Understanding

How does a nitrate addition prevent sulfide souring of crude oil?

Why is accelerated microbial corrosion of iron metal thought to require a direct interaction between the sulfate reducers and the metal surface?

22.13 Biodeterioration of Stone and Concrete

In the same way that microorganisms contribute to soil formation through the dissolution of mineral and rock surfaces by their physical and metabolic activities (Section 20.6), buildings or other structures composed of natural stone or concrete are also subject to microbial colonization and may undergo a slow loss of structural integrity through microbial metabolic activities. This degradative process is called biodeterioration.

Biodeterioration of Stone Building Materials

Microbial colonization of natural and structural stone building material is ubiquitous. Microbes can colonize the surface and penetrate several millimeters into rocky material depending on its physical characteristics (e.g., surface roughness, porosity, light penetration). Microbes can also grow on and within the facades of buildings constructed of limestone, sandstone, granite, basalt, and soapstone. These “within stone,” or endolithic, communities are phylogenetically diverse, comprised of chemoorganotrophic and chemolithotrophic Bacteria and Archaea, microbial eukaryotes including fungi and algae, and cyanobacteria (Figure 18.35). The cyanobacteria and algae primarily nourish the community, living in close or symbiotic association with other microbial members.

Life on and within stone building materials requires adaptation to multiple extreme conditions, including intense solar radiation, desiccation, temperature and moisture fluctuations, and lack of nutrients. Protection from solar radiation is conferred by production of UV-absorbing pigments (for example, melanin, mycosporines, and carotenoids) by fungi and other microbial community members. The fungi also play a central role in this process of slow biodeterioration through the production of oxalic acid, which dissolves and mobilizes mineral constituents of the stone. Mineral dissolution and mobilization provide the communities with nutrients and increase habitability by enlarging pore spaces within the stone and thereby accelerating deterioration.

Crown Corrosion of Wastewater Distribution Systems

A rapid form of microbial biodeterioration is observed in the crown corrosion of concrete sewer tiles, a process leading ultimately to collapse of the pipe. Crown corrosion is a consequence of interactions between sulfate-reducing bacteria (Sections 14.12 and 15.11) and chemolithotrophic sulfur-oxidizing bacteria (Sections 14.7 and 15.12) in these underground wastewater transmission systems (Figure 22.28).

Figure 22.28 Crown corrosion of concrete sewer pipes.


Corrosion is the result of a microbial sulfur cycle that develops within the transmission pipe. Sulfate-reducing bacteria consume organic material in the anoxic wastewater, producing H2S. The latter is oxidized by sulfur-oxidizing chemolithotrophic bacteria that attach to the oxic upper (crown) pipe surface, accelerating corrosion from the production of H2SO4 (sulfuric acid).

Mastering Microbiology

Art Activity: Figure 22.28 Crown corrosion of concrete sewer pipes

The first step in crown corrosion is the reduction of sulfate in the sewage to H2S by sulfate reducers, using primarily organic electron donors available in the waste stream water. The H2S is then released into the headspace of the pipe where conditions are oxic. The sulfide, or partially oxidized intermediates such as thiosulfate or sulfur, is then oxidized by neutrophilic thiobacilli such as Thiobacillus thioparus. As the pH drops to 4–5 with continued microbial production of sulfuric acid, acidophilic sulfur-oxidizing species such as Acidithiobacillus thiooxidans displace the neutrophilic species. Destruction and ultimate structural failure of the concrete results from the reaction of sulfuric acid with lime in the concrete, producing CaSO4⋅2H2O (gypsum) that penetrates into the concrete. The gypsum subsequently reacts with natural calcium–aluminum minerals present in the concrete, forming a new mineral that increases internal pressure and contributes to cracking and further acceleration of the corrosion process.

A series of steps and microbial participants similar to those of crown corrosion can corrode concrete holding tanks and cooling towers, particularly those in or near the marine environment where sulfate levels are typically high. Such deterioration can cause slow structural damage, such as concrete chipping, or even lead to a catastrophic rupture with threats to both the environment and humans.

Check Your Understanding

How does the production of oxalic acid by fungi contribute to the deterioration of stone building materials?

Which two major microbial physiologies are required to achieve crown corrosion? Which is an aerobic process and which is anaerobic?

Chapter Review

Go to Mastering Microbiology for videos, animations, practice tests, and more.

I Mineral Recovery and Acid Mine Drainage

22.1 The capacity of bacteria to oxidize Fe2+ aerobically at acidic pH is used to mine metals, principally copper-, uranium-, and gold-containing low-grade ores, through the process of microbial leaching. Bacterial oxidation of Fe2+ to Fe3+ is the key reaction in most microbial leaching processes because Fe3+ can extract metals in the ores under either oxic or anoxic conditions.

**Q Which crucial step in the oxidation of copper ores is carried out by Acidithiobacillus ferrooxidans? How is copper recovered from copper solutions produced by leaching?**

22.2 Spontaneous microbial oxidation of ferrous iron in pyritic ore or coal that has been exposed to air and water, such as occurs during some coal-mining operations, causes a type of pollution called acid mine drainage.

**Q Which Bacteria and Archaea play a major role in acid mine drainage? Why do they carry out the reactions that they do? Why is air necessary for this process?**

II Bioremediation

22.3 Although an inorganic pollutant such as uranium cannot be destroyed, containment is possible by reducing its mobility. For example, metal-reducing microorganisms in a region of uranium contamination can be stimulated to reduce U6+ to U4+, forming the immobile mineral uraninite that does not move into the groundwater.

Q What could thwart microbial bioremediation of a site that contains buried nuclear weapons that are leaking uranium?

22.4 Hydrocarbons are excellent carbon sources and electron donors for bacteria and are readily oxidized when O2 is available. Hydrocarbon-oxidizing bacteria bioremediate spilled oil, and their activities can be assisted by addition of inorganic nutrients.

Q What physical and chemical conditions are necessary for the rapid microbial degradation of oil in aquatic environments?

22.5 Some xenobiotics persist, whereas others are readily degraded, depending on their chemistries. Chlorinated xenobiotics can be degraded both aerobically and anaerobically. Highly chlorinated xenobiotics can be detoxified by organohalide-respiring bacteria that reductively remove the chlorides. Some oil-based plastics such as PET can be completely degraded by bacteria such as Ideonella.

**Q Why are some bacterial transformations of xenobiotics possible only through cometabolism? How might Ideonella sakaiensis or its enzymes be used in the recycling industry?**

III Wastewater and Drinking Water Treatment

22.6 Sewage and industrial wastewater treatment reduces the organic and nutrient load of wastewater. Primary and secondary wastewater treatment employs physical, biological, and physicochemical processes. Following treatment, the effluent water has significantly reduced BOD and is suitable for release into the environment.

Q Trace the treatment of wastewater in a typical plant from incoming water to release. What is the overall reduction in the BOD for typical household wastewater? What is the overall reduction in the BOD for typical industrial wastewater?

22.7 Tertiary wastewater treatment for nitrogen and phosphorus removal is used to improve the quality of the wastewater previously subjected to primary and secondary treatment. The increasing use of granular sludge treatment technology offers significant reductions in the energy, space, and chemical requirements of traditional wastewater treatment.

Q Why is tertiary wastewater treatment desirable from an environmental point of view?

22.8 Sludge processing is used to reduce the amount of sludge for disposal and produce methane for energy. The anammox process is an effective and efficient way to remove nitrogen from high-ammonia sludge brine. Of increasing concern are pharmaceuticals and ingredients in personal care products that are not degraded by conventional treatment systems and that can have adverse environmental effects, even at very low concentrations.

Q Which of the following are aerobic treatment processes and which are anaerobic: nitrification, anammox, denitrification, sludge digestion to methane?

22.9 Drinking water purification plants employ industrial-scale physical and chemical systems that remove or neutralize biological, inorganic, and organic contaminants from water sources. Water purification plants employ clarification, filtration, and chlorination processes to produce potable water.

Q Identify (stepwise) the process of purifying drinking water. What important contaminants are targeted by each step in the process?

22.10 The pipes that carry municipal drinking water and the premise plumbing that provide it to the consumer have created new microbial habitats. By forming biofilms on water pipe walls, microbial communities are more resistant to chlorine, can sustain or sequester opportunistic pathogenic bacteria, and its members can be distributed by household water usage.

Q What features of municipal and premise water distribution systems might contribute to a microbial health hazard? Why might showering increase your exposure to opportunistic pathogens?

IV Indoor Microbiology and Microbially Influenced Corrosion

22.11 The indoor air and surfaces of dwellings and other buildings contain a diversity of mostly harmless saprophytic microbes that are typically a reflection of the humans and animals that reside there. However, the microbiota of certain parts of the indoor built environment, such as bathrooms and toilets, may contain enteric pathogens from aerosols generated there.

Q From the perspective of a child’s health, can a home be “too clean”? Explain.

22.12 Corrosion of metal structures exposed to the environment can be accelerated by microbial activity during microbially influenced corrosion. Structures in or near seawater are particularly prone to corrosion as a consequence of the direct and indirect activities of sulfate-reducing bacteria.

Q What are the two general models for the acceleration of corrosion by sulfate-reducing bacteria?

22.13 Complex microbial communities colonize stone and concrete and produce substances that dissolve and mobilize its mineral constituents. Crown corrosion of concrete sewer lines results from the concerted activities of sulfate-reducing and sulfur-oxidizing bacteria growing within the wastewater and the headspace of sewer pipes, respectively. The resulting sulfuric acid is primarily responsible for the destruction of the concrete.

Q How does the role of sulfate-reducing bacteria differ for metal corrosion and crown corrosion?

Application Questions

Acid mine drainage is in part a chemical process and in part a biological process. Discuss the chemistry and microbiology that lead up to acid mine drainage and point out the key reactions that are biological. What ways can you think of to prevent acid mine drainage? How might you prevent further generation of acid drainage?

Why is reduction of BOD in wastewater a primary goal of wastewater treatment? What are the consequences of releasing wastewater with a high BOD into local water sources such as lakes or streams?

Discuss the microbial ecology contributing to crown corrosion of concrete sewer lines. In consideration of this ecology, what intervention strategies might be useful in reducing or eliminating corrosion?

Chapter Glossary

acidic water containing H2SO4 derived from the microbial and spontaneous oxidation of iron sulfide minerals released by coal mining Anaerobic treatment

degradative and fermentative reactions carried out by microorganisms under anoxic conditions to treat sludge solids or wastewater containing high levels of insoluble organic materials Biochemical oxygen demand (BOD)

the relative amount of dissolved oxygen consumed by microorganisms for complete oxidation of bioavailable organic and inorganic material in a water sample Biodeterioration

microbially catalyzed processes that degrade stone or concrete Bioremediation

the cleanup of oil, toxic chemicals, and other pollutants by organisms, usually microorganisms Chloramine

a disinfectant chemical manufactured on-site by combining chlorine and ammonia at precise ratios Chlorination

disinfection of water with Cl2 at a sufficiently high concentration that a residual level is maintained throughout the distribution system Clarifier

a reservoir in which suspended solids in raw water are coagulated and removed through precipitation Coagulation

the formation of large, insoluble particles from much smaller, colloidal particles by the addition of aluminum sulfate and anionic polymers Crown corrosion

the destruction of the upper half, or crown, of concrete wastewater pipes by sulfuric acid produced through the concerted activities of sulfate-reducing and sulfur-oxidizing bacteria Effluent water

treated wastewater discharged from a wastewater treatment facility Filtration

the removal of suspended particles from water by passing it through one or more permeable media (e.g., sand or activated charcoal) and ion exchangers Finished water

water delivered to the distribution system after treatment Flocculation

the water treatment process after coagulation that uses gentle stirring to cause suspended particles to form larger, aggregated masses (flocs) Microbial leaching

the extraction of valuable metals such as copper from sulfide ores by microbial activities Microbially influenced corrosion (MIC)

the contribution of microbial metabolic activities to accelerating the corrosion of metal structures Potable

drinkable; safe for human consumption Primary disinfection

the introduction of sufficient chlorine or other disinfectant into clarified, filtered water to kill existing microorganisms and inhibit further microbial growth Primary wastewater treatment

physical separation of wastewater contaminants, usually by separation and settling Pyrite

surface water or groundwater that has not been treated in any way (also called untreated water) Reductive dechlorination

an anaerobic respiration in which a chlorinated organic compound is used as an electron acceptor, usually with the release of Cl− Secondary disinfection

the maintenance of sufficient chlorine or other disinfectant residual in the water distribution system to inhibit microbial growth Secondary wastewater treatment

oxidative reactions carried out by microorganisms under aerobic conditions to treat wastewater containing low levels of organic materials Sediment

in treatment of water for drinking, the soil, sand, minerals, and other large particles found in raw water Sewage

liquid effluents contaminated with human or animal fecal material Suspended solid

a small particle of solid pollutant that resists separation by ordinary physical means Tertiary wastewater treatment

any treatment process in which unit operations are added for the further processing of the secondary treatment effluent or solids Turbidity

a measurement of suspended solids in water Untreated water

surface water or groundwater that has not been treated in any way (also called raw water) Wastewater

domestic sewage or liquid industrial waste, which cannot be discarded in untreated form into lakes or streams Xenobiotic