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

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monooxygenase pathways involve addition of a hydroxyl g roup to the ring in three dif-

ferent positions (ortho-, meta-, and para-), whereas the fourth involves oxidation of the

methyl group. For all of the pathways, the next step will be cle avage (breaking) of the

ring (usually between the two hydroxyl groups), leading to products (di carboxylic

acids) that after a few more transformations can enter the Krebs cycle. Detailed pathway

information is now available for many degradation reactions (e.g., see the University of

Minnesota Biocatalysis/Biodegradation Database Web site.)

Oxygenases, like most other enzymes, tend to be fairly speciﬁc. However, some degree

of nonspeciﬁcity can be beneﬁcial, in that it can allow an organism to utilize a newly

available substrate, even if at a lower efﬁciency. Over an extended time, it can be expected

that new forms of the enzyme, better adapted to the new substrate, will emerge (through

random variation, genetic exchange, or rearrangement), giving the organism utilizing

them a selective advantage when the substrate is available.

On the other hand, a nonspeciﬁc enzyme may transform a nontarget substrate, yielding

a product that cannot be further metabolized by the cell. For example, a cell growing on

toluene might accidentally cleave an aromatic ring containing a chlorine substitution. The

resulting dicarboxylic acid may not be suitable for further metabolism because of the

large chlorine atom still present. Then the cell has wasted the material and energy it

invested in transforming a compound that provides it with no beneﬁt. This type of activity,

in which a cell growing on one compound ‘‘mistakenly’’ transforms another without any

beneﬁt, is referred to as cometabolism (Figure 13.12). In this case, over time it can be

expected that the organism will develop more speciﬁc enzymes to prevent the drain of

Cl Cl

ClH

toluene

biphenyl

trichlorobiphenyl

pyrene

benzo(a)

rene

trichloroethylene

Figure 13.12 Possible cometabolism. The main (growth) substrate in each pair is located on the

left, with areas of similarity indicated by a, b, or c.

408

MICROBIAL TRANSFORMATIONS

cometabolism, or alternatively, it may develop new enzymes and pathways so that the

dead-end product can be further utilized. Although it is not beneﬁcial to the microorgan-

ism, cometabolism may be desirable from a human point of view because it may be pos-

sible to exploit it to degrade otherwise nondegradable compounds.

Even with energy-yielding metabolism, dead-end products may form. In other cases,

intermediates may accumulate because their rate of production is greater than their rate of

further transformation. Occasionally, this accumulating compound will be more toxic than

the parent material, leading to at least a temporary increase in toxicity during biodegrada-

tion. The increased toxicity may have the effect of further slowing degradative activity, as

well as having other undesirable effects on the ecosystem.

In aero bic systems it is common for a single species of bacteria to be able to utilize a

single organic compound as its sole source of carbon and energy. However, occasionally,

organisms of one species can degrade the compound only partially, and those of another

species will further transform, and probably mineralize, the intermediate produced. Such a

combination of organisms of different species ‘‘working together’’ to metabolize sub-

strates is referred to as a consortium (plural, consortia).

The activities of consortia can make detection of cometabolism difﬁcult, as a cometa-

bolic product may be degraded by other organisms before it accumulates sufﬁciently to be

noticed. Also, the fact that a compound can be completely degraded by a single organism

does not necessarily mean that this is the way it will be degraded in a particular

environment—the actual degradation still may be by a consortium.

Under anaerobic conditions, consortia are com mon. A wide variety of species may be

involved in hydrolyzing, or solubilizing, complex organics, followed by fermentations of

the subunits produced. Then sulfate-reducing or methanogenic organisms may utilize the

fermentation products, leading to mineralization.

The organisms in a microbial consortium may be only loosely associated, or they may

be so closely linked that they are difﬁcult to separate. A classic example is the case of

methane production from ethanol. For many years this was attributed to Methanobacillus

omelianskii, an ‘‘organism’’ that could be grown in ‘‘pure’’ culture with ethanol as the

sole carbon and energy source. However, it was later demonstrated that, in fact, the cultur e

contained two species: a bacterium that converted ethanol to acetate, and a methanogen

(Archaea) that utilized the acetate, producing methane and carbon dioxide.

Occasionally, the balance among organisms in an anaerobic consortium is disturbed

and mineralization does not occur. In the absence of sulfate, anaerobic mineralization

is dependent on methanogens. Compared to the wide variety of organisms that are able

to hydrolyze and ferment organics anaer obically, methanogens are a relatively limited

group of strictly anaerobic archaea. Although most methanogens utilize acetic acid as a

substrate, as a group they are sensitive to low pH. If the acetic acid is produced too

quickly, the pH of the system will drop, inhibiting methanogenic activity. In turn, this

leads to a further buildup of acid, a greater drop in pH, and even lower rates of methano-

genesis and acid destruction.

Some human foods, such as pickles and sauerkraut, are preserved in this way. Ensi-

lage, the process of making silage, is a means of animal feed preservation utilizing

acid anaerobic conditions that has been employed in agriculture for many years. Fresh

corn, hay, or other feed crops are placed in a silo, where they quickly ferment. The

acid anaerobic conditions then prevent substantial further degradation. However, if oxy-

gen is introduced in large amounts, the organic acids are quickly mineralized, pH rises,

and biodegradation resumes.

CARBON 409

Acid anaerobic conditions are also responsible for the preservation of the large masses

of spongy rich organic material in bogs. This waterlogged peat soil, composed mainly of

dead sphagnum moss (an acid-loving plant), accumulates over hundreds or even thou-

sands of years. If the water is drained from such systems, aerobic conditions develop

and the organics are quickly oxidized.

During waste treatment, acid anaerobic conditions are undesirable because they slow

degradation, leading to preservation of the waste. In poorly run leaf ‘‘composting’’

(Section 16.2.3) operations (really, leaf dumps), large piles of leaves may be formed

and simply left unattended. Acid anaerobic conditions develop quickly, so that on opening

such piles 10 years later, the tree species of individual leaves can still be determin ed.

Similarly, in anaerobic digesters for sludge treatment (Section 16.2.1), overloading or

toxic shocks may lead to acid anaerobic conditions that prevent methane formation.

The digester under such conditions is referred to as having gone ‘‘sour.’’

Another problem with acid anaerobic conditions in waste treatment is the potential for

odors. The organic acids themselves may be volatilized, leading to a sour smell. Often,

some alcohols are also formed, and apparently react with the acids to give esters, which

may have a sweet smell. Thus, such systems often have a sweet–sour smell. This is not

necessarily unpleasant for silage in an agricultural setting, but it is often not appreci ated

near wastewater treatment, composting, and landﬁll sites.

If sufﬁcient proteinaceous material is present in the waste, more severe odors may also

develop. Under most conditions the amino acids released by protein hydrolysis are dea-

minated; that is, the amino group is removed, releasing ammonium (Figure 13.13). This

can lead to an ammonia odor at high pHs where volatilization is favored. However, under

acid anaerobic conditions, amino acids may instead be decarboxylated. This leaves an

amine, some of which are highly odorous, with names such as putrescene and cadavarene.

Another odor concern with anaerobic conditions is hydrogen sulﬁde. This can be

formed through the reduction of sulfate during anaerobic respiration (Section 13.3.1) or

through the releas e of reduced sulfur from organic compounds such as the amino acids

methionine and cysteine.

In some cases, under both aerobic and anaerobic conditions, rather than b eing miner-

alized, organics are polymerized to form products with long-term stability. One example

is with nitroaromatic compounds such as trinitrotoluene (TNT). Because the aromatic ring

amino acid

amine

anic acid

deam

ination

decarboxylation

Figure 13.13 Deamination and decarboxylation of an amino acid. (R represents the speciﬁc amino

acid side-chain.)

410

MICROBIAL TRANSFORMATIONS

is stabiliz ed by the presence of the nitro groups, TNT is recalcitrant. Although it can be

mineralized, biodegradation usually involves reduction of one or more of the nitro groups

to an amine, which then reacts with a nitro group on an adjacent molecule (Figure 13.14).

A series of such reactions leads to the disappearance of TNT through the formation of a

polymer. The environmental signiﬁcance of such polymers is still not thoroughly under-

stood, as there is concern that the monomers may be released under some conditions.

Polynuclear aromatic hy drocarbons (PAHs or PNAs) are compounds containing two or

more condensed (fused together) aromatic rings (Figure 13.15). They are common con-

stituents of coal, oil, and creosote (a wood preservative) and are also products of incom-

plete combustion. They include the ﬁrst known human carcinogen, benzo[a]pyrene

−

trinitrotoluene

2-methyl-3,5-dinitroaniline

Figure 13.14 Polymerization of trinitrotoluene.

CARBON 411

(BAP), which was found to be a cause (because of its presence in soot) of testicular cancer

in English chimney sweeps.

Generally, the PAHs with two or three rings are readily biodegradable, at least under

aerobic conditions. The four-ring compounds pyrene and chrysene are also usually miner-

alized, although chrysene more slowly. However, there usually seems to be little miner-

alization (with occasional exceptions) of the higher-molecular-weight PAHs. BAP does

appear to be cometabolized to some extent. Probably as a result of initial cometabolic

activity, it also appears to be incorporated into the soil humic materials. These complex,

irregular soil organic polymers, such as humic acid (Figure 13.16), include phenolics, het-

erocyclics, sugars, and amino acid residues as building blocks, some probably derived

from lignin degradation. Once a BAP or other PAH residue is incorporated into the poly-

mer, it probably cannot be distinguished from other subunits, and thus is no longer

expected to be of concern.

Over long periods of time, humic materials buried in anaerobic layers may be reduced

further. Eventually, after millions of years, a combination of microbial degradation and

abiotic processes at elevated temperatures and pressures has led to coal and oil formation.

These organic materials have thus been held out of the active cycling of carbon for

naphthalene

anthracene

phenanthrene

fluorene

dibenzofuran dibenzodioxin

biphenyl

pyrene

chrysene

benzo(a)pyrene

fluoranthene

Figure 13.15 Polynuclear aromatic hydrocarbons and related compounds.

412

MICROBIAL TRANSFORMATIONS

100 million years or more. The rapidly increasing use of these fossil fuels over the last

century is responsible for an increase in atmospheric CO

as this carbon reenters active

cycling.

Note that although humic materials are not usually referred to as recalc itrant, they do

not biodegrade rapidly. Rather, they are considered to be stable or to change only slowly.

Stabilization of wastes thus refers to elimination of the readily degradable components

through both mineralization and conversion to high molecular weight products such as

humics.

Greenhouse Gases In most cases, a desired major product of biodegradation is carbon

dioxide. However, it is now recognized that atmosp heric CO

concentrations have

increased dramatically during the industr ial age, from around 280 ppm prior to the late

eighteenth century to 380 ppmv today. Most of this increase is from the combustion of

fossil fuels, although some other human activities, such as deforestation, loss of soil orga-

nic matter, and draining of wetlands, may also have had a small role. The highe r concen-

tration of CO

acts to capture more of the infrared energy radiated from Earth’s surface.

This phenomenon, often referred to as the greenhouse effect, has contributed to an

increase in average temperature worldwide, or global warming. Thus, some thought is

now being given to slowing mineralization of certain organic materials and perhaps to

HOH

Figure 13.16 Example of a possible humic acid chemical structure.

CARBON 413

rebuilding some stores of humic materials in soil, for example. This may be an area in

which environmental engineers and scientists can make contributions in the future.

Carbon dioxide is the most abundant, but it is not the only greenhouse gas. In fact,

methane has about 26 times the heat-trapping capacity of CO

. Additionally, its concen-

tration in the atmosphere has increased even more (proportionally) than that of carbon

dioxide, from about 0.7 to almost 1.8 ppm. Much of this increase is from fossil-fuel

extraction and use, but other major anthropogenic sources include cattle herds (digestive

tracts), rice ﬁelds (ﬂooded soils), and solid waste landﬁlls. Even landﬁlls with gas collec-

tion systems produce fugitive emissions. This is another area in which environmental pro-

fessionals can expect to be called upon to help control greenhouse gas releases.

13.2 NITROGEN

Nitrogen is probably second only to carbon in the complexity of its cycling. Its roles in

living things are likewise diverse, its organic compounds contributing to physical form

(e.g., cells walls, exoskeletons, ﬁngernails, hair), genetics (nucleic acids), and metabolism

(enzymes).

With AN ¼ 7, nitrogen has seven electrons, ﬁve of them in its outer shell. This results

in a range of oxidation states from 3toþ5 (Table 13.1). These extremes are represented

by ammonia- and nitrate-nitrogen, respectively (Figure 13.17). Many of the intermediate

forms exist naturally, but 3 (ammonia and organic nitrogen), 0 (elemental N

, also

called dinitrogen), þ3(nitrite), and þ5 (nitrate) are most common in soil, water, and

biomass. Other fairly common states include 1(hydroxylamine,NH

OH, an intermedi-

ate in the oxida tion of ammonia), þ1(nitrous oxide,N

O, also known as laughing gas,

which is an intermed iate in nitrite reduction), þ2(nitric oxide, NO, another intermediate

in nitrite reduction and also an important air contaminant), and þ4(nitrogen dioxide,

, another air contaminant). In Figure 13.18 these forms are shown as part of the nitro-

gen cycle schematic.

The major accessible source of nitrogen in the biosphere is the elemental nitrogen gas

) that makes up 78% (by volume) of the atmosphere. However, the ability to utilize

elemental nitrogen is limited to a few groups of bacteria and archaea. Thus, nitrogen is

-3

Nitrate

Ammonia

NITROGEN

Figure 13.17 Nitrogen oxidation state extremes.

414

MICROBIAL TRANSFORMATIONS

a limiting nutrient in many environments, especially for photoautotrophs. Combined

nitrogen, whether organic or inorganic, is referred to as ﬁxed-N. In addition to microbial

activity, nitrogen is naturally ﬁxed in small amounts through photochemical reactions and

electrical discharges (lightning) in the atmosphere. Beginning in the early twentieth cen-

tury, large amounts of nitrogen have also been ﬁxed through fertilizer production, and to a

lesser extent, through high-temperature combustion (automobiles, electric power genera-

tion, and industrial processes) and explosives manufacturing. These human activities now

account for almost 4  10

metric tons per year, more than 20% of all ﬁxation. As of yet,

this large increase in nitrogen ﬁxation on a global scale does not seem to have ‘‘over-

loaded’’ other parts of the nitrogen cycle.

In addition to the essential role of microorganisms in nitrogen ﬁxation, there are only a

limited number of prokaryotes and a few fungi that can oxidize nitrogen. And although

many prokaryotic and eukaryotic microorganisms and plants can utilize inorganic nitro-

gen in the form of ammonium and/or nitrate, many other microorganisms and all animals

can obtain their nitrogen only from organic forms.

When referring to the concentrations of nitrogen-containing compounds, it is common

to report them on the basis of the amount of N present rather than the amount of the com-

pound. This makes it easier to keep track of the nitrogen as it is converted from one form

to another. Thus, if 1.0 mg/L of NH

-N is completely converted to nitrate, it yields 1.0

mg/L of NO



N. If this same conversion was reported as the concentration of the species

themselves, it would instead be

1:21 mg=LNH

! 4:43 mg=LNO



Ammonium

-3

+4+3

-1

-2 +5

-3

+4+3

-1

-2 +5

-3

+4+3

-1

-2 +5

-3

+4+3

-1

-2 +5

Hydroxyl-

amine

Nitrogen

Nitroxyl

NOH

Nitrite

Nitrate

Nitrification Nitrification

Denitrification Denitrification

Ammonia

Nitrogen Oxidation States

Nitrous

Oxide

Nitric

Oxide

Organic-N

Assimilation

Ammonification

Nitrogen

Dioxide

Abiotic

Nitrate Reduction

Nitrate Assimilation

Nitrogen Fixation

Figure 13.18 Biochemical nitrogen transformations.

NITROGEN 415

since for ammonia,

ð1:0mg=LÞ¼1:21 mg=L

and for nitrate,

ð1:0mg=LÞ¼4:43 mg=L

where the atomic weight of nitrogen ¼14, the molecular weight of NH

¼ 17, and the

formula weight of NO



¼ 62.

Example 13.3 The drinking water standard for nitrate-N is 10 mg/L. How much is this

when it is expressed as nitrate?

Answer The formula weight for nitrate ¼ 1ð14Þþ3ð16Þ¼62 g/mol:

62 g=mol nitrate

14 g=mol N

ð10 mg=LNÞ¼44:3mg=L nitrate

In addition to the oxidation and reduction reactions discussed in more detail below,

several other reactions are important in the nitrogen cycle. One of these is the con-

version of organic nitrogen to ammonia. As with any change from an organic form

to an inorganic one, this process can be referred to as mineralization. However, a

more speciﬁc name, ammoniﬁcation, is often used in this case. The reverse react ion,

assimilation, refers to the uptake of an element for incorporation in cell material.

Ammonia assimilation does not represent a change in the oxidation state of the nitro-

gen. However, nitrate assimilation (as discussed below in Section 13.2.1) represents a

reduction and requires energy.

Another im portant nitrogen transformation without oxidation or reduction included

in Figure 13.18 is the acid–base reaction between ammonia (nonionized) and ammonium

(a cation):

, NH

þ H

ð13:9Þ

When speaking of the ammonia-N concentration in water, it is usually the

total ammonia-N, or NH

-N þ NH

-N that is meant, as this is what is measured

by all of the commonly used analytical methods. The pK

value (log K

)at25



for this reaction, at which half of the total ammonia would be in each form, is

about 9.3:

½NH

½H



½NH



¼ 10

9:3

Since acid–base reactions are fairly rapid, at neut ral pH values, only a smal l per-

centage of the total ammonia is expected to be present as NH

(0.50% at pH 7,

4.8% at pH 8).

416 MICROBIAL TRANSFORMATIONS

Example 13.4 The pH of a water sample at 25



C is 7.5. If the measured total ammonia-

N concentration is 3.0 mg/L, what is the concentration of nonionized NH

-N?

Answer Let ½N

¼ NH

½þNH



; then

½NH

½H



½

½H

½

½NH

½

½¼N

½

½þK

-N ¼ N

½þK

¼ N

pK

pH

þ 10

pK



¼ N

pH

þ 1



¼ 3:0

9:37:5

þ 1



1:8

þ 1

¼ 0:047 mg=L

13.2.1 Nitrogen Reduction

Important nitrogen reduction reactions include nitrogen ﬁxation, dissimilatory nitrate

reduction (including denitriﬁcation), and nitrate assimilation.

Nitrogen Fixation Although a gaseous sea of N

blankets Earth, it offers nothing in the

way of nutritional value except to a select few prokaryotes. The trivalent bond (N



N) of

this molecule is simply too strong for normal metabolic cleavage. Indeed, one of the

names that Antonie Lavoisier originally considered in the late eighteenth century for

this recently discovered gas was ‘‘azote,’’ meaning lifeless or inert.

Since the vast majority of life-forms are unable to utilize nitrogen gas, a critical step in

the global cycling of nitrogen is its ﬁxation. Nitrogen ﬁxation is the reduction of N

organic or ammonia nitrogen. As indicated in Figure 13.18, some nitrogen is also ﬁxed

abiotically in natural systems through oxidation reactions involving combustion, photoly-

sis, or electricity (lightning).

A variety of nitrogen ﬁxers are known, scattered among a number of taxa, but all are

prokaryotes. They are often separated into free-living vs. symbiotic forms, although this is

not a phylogenetic approach. Although Azotobacter and Beijerinckia are probably the best

known of the free-living, nitrogen-ﬁxing aerobes among the proteobacteria, other exam-

ples are found among species of Klebsiella (but only when growing under anaerobic con-

ditions) and Citrobacter (members of the Enterobacteriaceae family), Methylomonas (a

methanotroph), and Thiobacillus (a sulfur-oxidizing autotroph). There are also aerobic

gram positives (e.g., a few Bacillus and the actinomycete Streptomyces) and many cyano-

bacteria, such as Anabaena (see Figure 10.20) and Nostoc. Free-living anaerobic nitrogen

ﬁxers include members of the gram-positive spore-formers Clostridium and Desulfotoma-

culum (a sulfate-reducer); the sulfate-reducing proteobacteria Desulfovibrio; several

phototrophs (Table 10.4), such as Chlorobium (green sulfur bacteria), Chr omatium (purple

sulfur), Rhodospirillum (purple nonsulfur), and Heliobacterium (another gram-positive

spore-former); and a few methanogens, such as Methanococcus, which are archaea.

The best known symbiotic nitrogen ﬁxers are rhizobia, proteobacteria such as Rhizo-

bium associated with leguminous plants (e.g., beans, peas, soybeans, peanuts, alfalfa, and

clover). Within the rhizospere (root zone in the soil), these bacteria infect the roots of the

NITROGEN 417