Vaccari D.A., Strom P.F., Alleman J.E. Environmental Biology for Engineers and Scientists

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structures called chlorosomes, in which much of their bacteriochlorophyll is concen-

trated. They are mostly autotrophic, but some photoheterotrophy has also been observed.

Chlorobium is the best known genus.

10.5.6 Proteobacte ria

The Proteobacteria is a vast kingdom, including many of the gram-negative species and

many of the metabolic activities known among the bacteria. Based on 16S rRNA, it is

broken into ﬁve classes, but these are more often referred to as the a, b, g, d, and e sub-

divisions. Interestingly, the 16S rRNA of mitochondria (in eukaryotes) places them in this

group also (Figure 10.19), suggesting that this organelle evolved from endosymbiotic

early Proteobacteria. Table 10.5 lists some of the important and interesting genera.

Because the kingdom is so large, and because organisms with similar activities may

belong to different classes, we discuss them based mainly on phenotypic characteristics

(i.e., based more on observable traits rather than on genetic similarity).

Phototrophs: Purple Sulfur and Nonsulfur Bac teria The purple sulfur bacteria, such as

Chromatium, and the purple nonsulfur bacteria, such as Rh odospirillum, are both anoxy-

genic phototrophic Proteobacteria (Table 10.4). Both groups show considerable morpho-

logical variability. They include species that are rods, spheres, and spirals, and may be

ﬂagellated or not. The purple nonsulfur group also includes some budding and appendage

forming bacteria. All of the known purple sulfur bacteria, which deposit sulfur granules

internally (unlike the external deposits of green sulfur bacteria), are g ¼ Proteobacteria.

Purple nonsulfur bacteria are found in both the a and b groups.

How do the different photo trophs all survive—or where are their places (i.e., niches;

see Chapter 14)? The Cyanobacteria occupy the upper, oxygenated regions of a lake, or

other oxic environments, perhaps competing with algae and plants. The purple sulfur bac-

teria are normally found in the anoxic depths of lakes, where hydrogen sulﬁde has been

released from the underlying sediments. The green sulfur bacteria, which typically can

tolerate higher concentrations of hydrogen sulﬁde, and which also can survive with

lower levels of light, would be found in even deeper zones. Both might also occupy

Figure 10.20 Anabaena, a ﬁlamentous cyanobacteria; with heterocyst.

248

MICROBIAL GROUPS

other anaerobic environments where light and reduced sulfur are present, such as some

hot springs. Purple nonsulfur bacteria have an advantage in lighted anaerobic environ-

ments in which organic matter is present, since they are able to grow well heterotrophi-

cally. Green nonsulfur bacteria also grow heterotrophically and in low light, particularly

in thick microbial mats found in hot springs and shallow marin e systems. Because of dif-

ferences in the chlorophylls and other pigments each contain, the groups also have light

absorption proﬁles with optima at different wavelengths (Figure 10.21).

TABLE 10.5 Kingdom Proteobacteria: Many of the

Gram-Negative Bacteria

Class 1. Alphaproteobacteria

Acetobacter

Agrobacterium

Azospirillum

Beijerinckia

Brucella

Caulobacter

Hyphomicrobium

Magnetospirillum

Methylocystis

Nitrobacter

Paracoccus

Rhodospirillum

Rickettsia

Rhizobium

Sphingomonas

Xanthobacter

Class 2. Betaproteobacteria

Achromobacter

Alcaligenes

Bordetella

Burkholderia

Gallionella

Leptothrix

Methylovorus

Neisseria

Nitrosomonas

Nitrosospira

Ralstonia

Sphaerotilus

Spirillum

Thiobacillus

Zoogloea

Class 3. Gammaproteobacteria

Acinetobacter

Aeromonas

Azotobacter

Beggiatoa

Chromatium

Francisella

Haemophilus

Legionella

Methylomonas

Moraxella

Nitrococcus

Nitrosococcus

Pasteurella

Photobacterium

Pseudomonas

Thiothrix

Vibrio

Xanthomonas

Family Enterobacteriaceae

Citrobacter

Enterobacter

Escherichia

Klebsiella

Leminorella

Proteus

Salmonella

Serratia

Shigella

Yersinia

Class 4. Deltaproteobacteria

Bdellovibrio

Desulfovibrio

Myxococcus

Nitrospina

Polyangium

Class 5. Epsilonproteobacteria

Campylobacter

Helicobacter

All ﬁve classes (subdivisions a to e) are listed, but not orders or families (except

Enterobacteriaceae). Only some representative, important, and interesting genera

are included.

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Chemolithotrophs The Proteobacteria include a number (but not all) of the organisms

able to obtain energy from the oxidation of reduced inorganic compounds, including

hydrogen and forms of nitrogen, sulfur, and iron (Table 10.6). Most of these organisms

are also autotrophic (getting their carbon from carbon dioxide), and aerobic, although

some are also able to use nitrate as an electron acceptor. They also tend to be slow grow-

ing, probably because the amount of energy available from oxidizing most of these com-

pounds is relatively small.

The ability to utilize hydrogen is actually quite widely distributed. Examples of hydro-

gen-oxidizing Proteobacteria include some species of Pseudomonas and Alcaligenes.

Most such bacteria are able to utilize organic substrates as well (unlike most other che-

molithotrophs). Some (e.g., Xanthobacter) can ﬁx nitrogen gas. Since hydrogen is typi-

cally produced under anaerobic conditions, and because the enzyme (hydrogenase)

involved in its oxidation is oxygen sensitive, most h ydrogen oxidizers prefer lower oxy-

gen conditions (i.e., 5 to 10% O

rather than the 21% of air). Some hydrogen oxidizers are

also able to grow on carbon monoxide, and such carboxydotrophic bacteria probably

play a major role in elimination of this toxic gas.

Cyanobacteria

Purple bacteria

Green bacteria

400 600 1000800

Figure 10.21 Schematic of light absorption characteristics (absorption vs. wavelength in nm) of

phototrophic bacteria. (Based on Prescott et al., 1999.)

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MICROBIAL GROUPS

Nitrifying bacteria are aerobic autotrophs that oxidize reduced nitrogen in two sepa-

rate steps. Ammonium oxidizers such as Nitrosomonas, Nitrosospira, and Nitrosococcus

convert ammonium to nitrite. (The ﬁrst two are b-Proteobacteria, the third a g-Protoebac-

teria.) Nitrite oxidizers convert nitrite to nitrate; examples are Nitrobacter (a-Proteob ac-

teria) and Nitrococcus (g-Proteobacteria). (Another nitrite oxidizer, Nitrospira,isa

member of the Xenobacteria.) Th ere appear to be relatively few types of nitrifying bac-

teria, but they are widespread (soil, freshwater, and marine systems), common, and play a

crucial role in the nitrogen cycle. They are relied on in most wastewater treatment systems

that control ammoni a, may exert a substantial oxygen demand on streams receiving

ammonia in efﬂuents, and play an important role in nitrate contamination of groundwater.

All known nitriﬁers prefer slightly alkaline, mesophilic conditions; they are inhibited by

even moderately acidic pH, and none appear able to grow at temperatures much above



Nonphototrophic prokaryotes able to oxidize various forms of reduced sulfur can be

found among the Archaea and several bacterial kingdoms. Hydrogen sulﬁde is the most

commonly used form, but elemental sulfur, metal sulﬁdes, and thiosulfate also are used by

many species . Some sulfur-oxidizing Proteobacteria, including members of the genus

Thiobacillus, produce so much sulfate (sulfuric acid) from the oxidation of pyritic

(metal sulﬁde) minerals that the pH may drop to below 2. This is a major cause of the

environmental problem known as acid mine drainage (Section 13.4.3) and can also

occur in clay soils containing pyrites if they are exposed to the air. Some hot springs

and marine thermal vents also are sources of sulﬁdes.

TABLE 10.6 Examples of Chemolithotrophic Proteobacteria

Representative Reduced Electron Oxidized

Group Genera

Substrate Acceptor Product

Hydrogen- Alcaligenes H

(hydrogen) O

(or NO



oxidizing Pseudomonas (nitrate)

Ralstonia

Carboxydo- Xanthobacter

trophic Alcaligenes

Nitrifying Pseudomonas CO (carbon monoxide) O

Step 1 Nitrosomonas NH

/NH



Nitrosospira (ammonia/ammonium)

Step 2 Nitrobacter NO



(nitrite) O



Nitrococcus

Nitrospina

Sulfur- Beggiatoa H

S (hydrogen O

(or NO



and

oxidizing Thiobacillus sulﬁde), S

(elemental SO

2

Thiothrix sulfur), and/or (sulfate)

2

(thiosulfate)

Iron-oxidizing Gallionella Fe

2þ

(ferrous) O

3þ

(ferric)

Leptothrix

Thiobacillus

Manganese- Leptothrix Mn

2þ

(manganous) O

4þ

oxidizing (manganic)

Not necessarily all strains can carry out the indicated reaction.

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Other sulfur oxidizers prefer or require neutral pH. Many of these rely on the produc-

tion of sulﬁde by the anaerobic sulfate-reducing bacteria (see later) growing on organic

material (rather than release from minerals). Since they need oxygen (or in some cases

nitrate), such bacteria tend to grow at the interface between aerobic and anaerobic envir-

onments (e.g., the surface sediments in salt marshes). One of these is the ﬁlamentous form

Thiothrix, which may occasionally cause settling problems in activated sludge wastewater

treatment plants if sufﬁcient sulﬁde is present in the inﬂuent or generated in the process.

Another, the gliding bacteria Beggiatoa (Figure 10.22), is the most common ﬁlamentous

organism observed in rotating biological contactor (RBC) treatment plants, sometimes

producing such heavy growths that the steel shafts collapse. Deeper layers of the bioﬁlm

become anaerobic in many such plants, so that hydrogen sulﬁde is then produced and

released to the overlying aerobic portion of the ﬁlm. It is thought that Beggiatoa’s ability

to glide helps it in remaining at the interface of the aerobic and anaerobic zones. Unlike

many other lithotrophs, Thiothrix and Beggiatoa are also able to use small organic mole-

cules as their carbon source and are thus referred to as mixotrophs.

Some bacteria are able to utilize the oxidation of ferrous (II) iron as a source of energy.

For example, some of the acidophilic Thiobacillus (especially T. ferrooxidans) that grow

on iron pyrites also are iron oxidizers. Ferrous iron is stable at low pH, allowing time for

biochemical activity, but at neutral pH it is chemically oxidized to the ferric (III) form

fairly rapidly. Interestingly, some bacteria, such as Gallionella and Leptothrix, can

grow at the interface b etween oxic and anoxic zones and oxidize the iron before the che-

mical reactions occur. This is sometimes a problem when anaerobic well waters contain-

ing soluble ferrous iron are brought to the surface, as the growths (containing large

amounts of insoluble ferric iron) can cause clogging. Similarly, some manganese-oxidiz-

ing bacteria can oxidize the manganous (II) to the manganic (IV) form.

Figure 10.22 Two species of Beggiatoa in samples from RBC wastewater treatment plants; a

gliding ﬁlamentous sulfur-oxidizing proteobacteria. Note internally deposited sulfur granules.

252

MICROBIAL GROUPS

Methanotrophs Methane (CH

) is a major product of the anaerobic degradation of

organic material, especially in the absence of large amounts of sulfate. Methanotrophs

(e.g., Methylomonas and Methylocystis) are a relatively specialized but environmentally

widespread group of obligately aerobic Proteobacteria (mainly a and g groups) able to

oxidize this methane. They are autotrophic, and some are also able to ﬁx nitrogen.

Most species produce a resting stage resistant to desiccation, either a cyst or an exospore

(which is also quite heat resistant) produced by budding.

Most methanotrophs are also able to utilize methanol (CH

OH) and many can utilize at

least some other one-carbon compounds , such as formaldehyde (CH

O), formic acid

(CHOOH), methyl amine (CH

), or carbon monoxide (CO), or other compounds

without carbon–carbon bonds such as dimethylamine [(CH

)

NH], dimethyl sulﬁde

[(CH

)

S], or dimethyl ether [(CH

)

O]. The ability to utilize such compounds is referred

to as methylotrophy and is more widespread (e.g., including some Bacillus, a gram-

positive bacteria) than growth on methane.

There has been recent interest in the use of methanotrophs for soil bioremediation sys-

tems. Their activity can be promoted by pumping methane into the subsurface environ-

ment, leading to increased generation of the enzyme methane monooxygenase (MM O).

This enzyme can cometabolically convert a number of industrial contaminants, such as

trichloroethylene, to less hazardous or harmless products (Section 16.7.2).

Nitrogen-Fixing Proteobacteria Nitrogen ﬁxation, the conversion of elemental nitro-

gen (N

) to a more readily utilizable form, is an ability that is found scattered among

many bacterial kingdoms (e.g., many of the Cyanobacteria and the gram-positives

Clostridium and Frankia). Among the Proteobacteria this includes som e methan otrophs,

and most strains of the enteric bacteria Klebsiella. Rhizobium, and some simi lar species

are of special importance because of the mutualistic (beneﬁcial to both organisms) rela-

tionship they have with legumes (plants such as beans, peas, soybeans, alfalfa, clover,

vetch, and mimosa). They are able to infect the plant roots to form special nodules in

which they grow. Thereafter, they ﬁx nitrogen (which is often in limited supply in

soils), to the beneﬁt of the plant, while they utilize organic substrates produced by the

plant. Other important nitrogen ﬁxers in soils (and water) are free-living, such as Azoto-

bacter. Both the symbiot ic (having a close relationship with another organism) and the

free-living nitrogen ﬁxers are aerobic, even though the required nitrogenase enzyme is

sensitive to oxygen.

Proteobacteria with Special Morphologies: Appendaged, Sheathed, and Spiral

Forms A number of the Proteobacteria are known for their specialized morphologies,

although these are not always indicative of phylogenetic relationships. Hyphomicrobium

is a common aerobic soil and aquatic Proteobacteria whose prosthecae take the form of

short hyphae from which buds are produced. Caulobacter is another common aerobic

prosthecate bacteria; it uses its stalk for attachment. The chemolithotrophic iron oxidizer

Gallionella also produces a stalk, but it consists of twisted exocellular ﬁbrils coated with

ferric hydroxide.

Sphaerotilus is a ﬁlamentous bacteria that produces a sheath (Figure 10.23). As part of

its life cycle, single cells with polar ﬂagella are formed that swim away to start new ﬁla-

ments. It is aerobic, but can still grow well at low dissolved oxygen concentrations (e.g.,

0.5 mg/L). As a result, it is a common inhabitant of polluted streams and biological waste-

water treatment systems. Large masses, commonly referred to as sewage fungus (although

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they are not fungi), sometimes form on rocks or other aquatic surfaces in such systems.

Heavy growths in activated slud ge wastewater treatment systems are one cause of the set-

tling problem known as bulking. Leptothrix, mentioned above as an iron- and manganese-

oxidizing organism, is a related species.

Spirillum is an example of a spiral Proteobacteria (not related to the spirochetes,

Section 10.5.9). It is motile by tufts of ﬂagella at both ends and is aerobic or microaer-

ophilic.

Myxobacteria The fruiting myxobacteria do not appear to be particularly important in

environmental engineering and science but are of great interest to some microbiologists

because of their life cycle, the most complex of any known prokaryote. The single,

usually rod-shaped vegetative cells have gliding motility and often leave a slime trail

as they move about on solid surfaces. When nutrients become limiting, the cells

‘‘swar m,’’ coming together to form a visible, often brightly colored fruiting body. Cells

within the fruiting body develop into myxospores, which are resistant to drying and some

other environmental stresses. These spores can later germinate to form new vegetative

cells.

Figure 10.23 Sphaerotilus natans:(a) pure culture showing sheath and PHB granules;

(b) branching ﬁlament in activated sludge sample.

254

MICROBIAL GROUPS

Myxobacteria are aerobic chemoorganotrophs found in soil. Many, such as Myxococ-

cus, are bacteriolytic (they lyse, or rupture, bacteria to feed on their cellular constitu-

ents—particularly proteins), and commonly occur on animal dung (which includes a

high percentage of bacteria). However, a number (including some species of Polyangium)

are instead cellulolytic (digest cellulose), and grow on decaying vegetation.

Pseudomonads The pseudomonads are a large group of aerobic (although a few can uti-

lize nitrate or nitrite when oxygen is unavailable), nonfermentative, chemoorganotrophic

(except that a few can grow on hydrogen or carbon monoxide), heterotrophic, non-spore-

forming gram-negative rods with polar ﬂagella. Pseudomonas (Figure 10.8) is the major

genus; however, when it was realized that bacteria called Pseudomonas were members of

the a- and b- as well as the g-proteobacteria (based on 16S rRNA), it was split into several

genera, including Sphingomonas, Burkholderia, and Ralstonia. Still, there is much diver-

sity in this group and disagreement among characterization methods as to where the nat-

ural groupings lie.

Pseudomonads are common soil and water bacteria, and becau se of their metabolic

diversity, many are important in biodegradation of a very wide variety of natural and

human-made organic compounds (including for bioremediation applications). A few,

such as Ps eudomonas aeruginosa, are opportunistic path ogens of humans. Some Pseudo-

monas and most Xanthomonas are plant pathogens . Another pseudomonad, Zoogloea

ramigera, is found in some activated sludge wastewater treatment systems, where it

forms a special type of zoogloeal ﬂoc (randomly organized aggregations) that can lead

to poor settling conditions (Figure 10.24).

Other Aerobic Proteoba cteria Other aerobic proteobacteria include the nonﬂagellated

species Neisseria, Moraxella, and Acinetob acter and the ﬂagellated Acetobacter. Several

Neisseria (including N. gonorrhoeae, the cause of gonorrhea; Section 12.6.2) and

Moraxella (e.g., Section 12.7.4) are pathogenic to humans or other animals. Neisseria

are cocci, whereas Moraxella are plump rods. Moraxella, which gives negative results

for many classic biochemical tests, is of interest for another reason as well: Bacteria

Figure 10.24 Characteristic Zoogloea ramigera ﬂoc from activated sludge.

BACTE RIA 255

isolated from the environment are also often negative for these tests, and hence have been

misidentiﬁed as Moraxella. Acinetobacter is unusual in that it is an aerobe that is oxidase

negative. These rods are common in soil and water. It sometimes shows twitching moti-

lity, as do some Moraxella.

Some Acinetobacte r strains have been reported as phosphate accumulating organisms

(PAOs) in activated sludge systems that achieve biological phosphorus removal (BPR).

(This identiﬁcation has since been challenged, and other aerobic proteobacteria using

the same mechanism are now believed to be the major PAOs in these systems.) They con-

tain metachromatic (volutin) granules composed of polyphosphate, which is used as a

method of energy storage. This type of BPR is induced by having an anaerobic zone fol-

lowed by an aerobic zone in the reactor. In the anaerobic zone, the PAOs utilize the stored

polyphosphate for energy (releasi ng phosphate) to accumulate and store fermentation pro-

ducts. Subsequently, in the aerobic zone, they utilize the stored organics and remove phos-

phorus to very low levels (storing it again as polyphosphate). The low-P water is then

separated and discharged, and the PAOs are recycled to the anaerobic zone to repeat

the reaction. This is a fascinating application in which favoring a strictly aerobic organism

in an ecosystem is accomplished by including a periodic anaerobic condi tion!

Acetic acid bacteria such as Acetobacter, although obligate aerobes, produce acetic

acid from ethanol. This can lead to spoilage of alcoholic beverages but is also used com-

mercially for vinegar production. Some Acetobacter are also able to produce a very pure

form of cellulose.

The Family Enterobacteriaceae The Enterobacteriaceae includes many bacteria of

importance to humans and hence has been studied extensively. They also form a relatively

stable taxonomic grouping; although origina lly based on testing by traditional (phenoty-

pic) methods, there has been little change after genotype testing was employed. These

members of the g-proteobacteria are gram-negative, nonsporeforming rods that are typi-

cally facultatively anaerobic, capable of fermenting sugars. They either have peritrichous

ﬂagella or are nonmotile. Many live within the intestinal tract of warm-blooded animals

(where they may play an important role in digestion) and hence are referred to as enteric

bacteria. Some are important pathogens; others live in soil or water.

Escherichia coli (Figure 10.25) is probably the best known of all bacteria. E. coli is

present in large numbers in the human intestines and is one of the coliforms used as indi-

cator organisms to monitor fecal pollution of water (Section 12.9.2). A few strains are

pathogenic (Section 12.3.2).

Important pathogenic members of this family include Salmonella, the cause of typhoid

fever (S. typhi) and salmonellosis (but also used for testing chemical mutagenicity in the

Ames test; Section 20.1.13); Shigella, which causes bacterial dysentery; and Yersinia

(Y. pestis is the cause of plague). Other common species that may be intestin al or envir-

onmental (soil and water) include Enterobacter (formerly called Aerobacter), Citrobacter,

and Klebsiella. K. pneumoniae , which is able to ﬁx nitrogen, can also occasionally cause

pneumonia. Proteus is well known to microbiologists because of the characteristic

swarming of cells of some strains on the surface of agar plates. Serratia is also widely

recognized in the lab because of the red colonies it often produces on plates.

Other Facultatively Anaerobic Proteobacteria Other facultative bacteria include

Vibrio, Photobacterium, Aeromonas, and Chromobacterium. These species differ from

the Enterobacteriaceae in that they are polarly ﬂagellated (Chromobacterium may have

256 MICROBIAL GROUPS

other ﬂagella as well) and usually oxidase positive. They differ from Pseudomonas in that

they are capable of fermentative metabolism. They are common soil and/o r water organ-

isms but include a few pathogens, such as Vibrio cholerae (cause of cholera) and V. para-

haemolyticus (a marine species that can cause enteritis, usually from eating raw ﬁsh). V.

ﬁscheri and Photobacterium are marine species that are luminescent: They emit blue-

green light (at 490 nm) through reactions similar to those of the ﬁreﬂy. Some live in

the specialized light-emitting organs of certain ﬁsh; others live freely and give some sea-

waters their luminescence. They are also utilized in the Microtox (Azur Environmental,

Strategic Diagnostics Inc., Newark, Delaware, www.azurenv.com) toxicity test. Aeromo-

nas sometimes gives false-positive results in the coliform test. It is an aquatic organism

sometimes associated with diseases of ﬁsh and frogs. Chromobacterium produces a violet

pigment (violacein), so that colonies on agar plates are very noticeable.

Sulfur-Reducing Proteobacteria Some strictly anaerobic proteobacteria, such as

Desulfovibrio (Figure 10.26), are able to utilize oxidized forms of sulfur, especially sul-

fate and elemental sulfur, as the terminal electron acceptor for respiratory metabolism. As

their energy source they use fermentation products (such as hydrogen, lactate, and pyru-

vate) produced by other bacteria in the same ecosystem. Sulfate reducers are very com-

mon in habitats containing organic material and sulfate, such as salt marsh sediments and

animal intestinal tracts. They may also be active in anaerobic digesters used for sewage

sludge treatment, and in the deeper layers of the bioﬁlms in wastewater treatment pro-

cesses such as trickling ﬁlters and rotating biological contactors. The hydrogen sulﬁde

produced may be released, giving the characteristic rotten egg odor, and/or may combine

with iron or some other metals to give the black color characteristic of such anaerobic

systems. In conﬁned spaces, hydrogen sulﬁde can build up to toxic levels. In sewers

with inadequate ﬂushing, the hydrogen sulﬁde released can cause odor problems and

also dissolve in the moist ﬁlms that form on the aerobic, unsubmerged surfaces; there

the sulﬁde may undergo biological oxidation by chemolithotrophic bacteria, resulting

in production of sulfuric acid and crown corrosion of the sewer.

Figure 10.25 Escherichia coli. (SEM image courtesy of the University of Iowa Central

Microscopy Research Facility.)

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