Autotrophic Nitrification in Bacteria

J . I . PROSSER
Department of Genetics and Microbiology. Marischal College.
University of Aherdeen. Aherdeen AB9 IAS. U.K.

1. Introduction . . . . . . . . . . . . . . . . 125
I1 . Ecological and economic importance of nitrification . . . . . . 126
Ill . Taxonomy and species diversity . . . . . . . . . . . 128
A. Ammonia oxidizers . . . . . . . . . . . . . 128
B. Nitrite oxidizers . . . . . . . . . . . . . . 129
IV . Biochemistry of nitrifying bacteria . . . . . . . . . . . 129
A. Ammonia oxidation . . . . . . . . . . . . . 130
B. Nitrite oxidation . . . . . . . . . . . . . . 133
C. Carbon metabolism . . . . . . . . . . . . . 133
V . Growth of nitrifying bacteria in liquid culture . . . . . . . . 136
A. Maximum specific growth rate . . . . . . . . . . 137
B. Growth yield . . . . . . . . . . . . . . . 139
C. Cell activity . . . . . . . . . . . . . . . 142
D. Saturation constants . . . . . . . . . . . . . 143
E. Problems of biomass production . . . . . . . . . . 146
v1. Surface growth . . . . . . . . . . . . . . . . 146
VII . The effect of oxygen and light on nitrification . . . . . . . . 148
A. Inhibition of nitrification at high oxygen concentration . . . . 148
B. Photo-inhibition . . . . . . . . . . . . . . 149
C. Nitrification at low oxygen concentrations . . . . . . . 150
D . Reduction of nitrite and nitrate by nitrifying bacteria . . . . . 152
VIII . The effect of pH value on nitrification . . . . . . . . . . 157
A. Do acidophilic strains of nitrifying bacteria exist? . . . . . . 160
B. Protection from effects of pH by surface growth . . . . . . 161
C. Micro-environments and urease activity . . . . . . . . 164
D. Heterotrophic nitrification . . . . . . . . . . . 166
IX. Inhibition of nitrification . . . . . . . . . . . . . 169
A. Mechanism of inhibition . . . . . . . . . . . . 170
B. Strain variability . . . . . . . . . . . . . . 171
C. Inhibition of attached cells . . . . . . . . . . . 174
X . Concluding remarks . . . . . . . . . . . . . . 175
XI . Acknowledgements . . . . . . . . . . . . . . . 177
References . . . . . . . . . . . . . . . . . 177

.
I Introduction
Nitrification is traditionally viewed as the oxidation of ammonia to nitrate. via
nitrite. by two groups of chemolithotrophic bacteria. ammonia oxidizers.
ADVANCES IN MICROBIAL PHYSIOLOGY. VOL. 30
ISBN 0-12-027730-1
.
Copyright 0 1989 by Academic Press Limited
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126 J LPROSSER

typified by the genus Nitrosomonus, and nitrite oxidizers, typified by the genus
Nitrohacter. Oxidation of ammonia or nitrite provides the only source of
energy for these organisms and organic carbon is obtained by fixation of
carbon dioxide. Nitrifying bacteria, and the process of nitrification, are
aerobic with optimal activity at mesophilic temperatures and neutral to
alkaline pH values, with no growth or activity at acid pH values. Growth
of these organisms is considered inefficient in comparison to that of hetero-
trophs and they have low specific growth rates and growth yields due to the
small energy gain obtained from oxidation of ammonia or nitrite.
Frequently, advances in a particular field lead to the formulation of
unifying hypotheses or theories explaining contradictory experimental
findings or conflicting views, and in that sense simplify our view of that field.
To an extent, recent advances in our knowledge of the physiology of nitrifying
bacteria have achieved this by explaining many poorly understood aspects of
nitrification. They have also led to the discovery of many new and interesting
properties of these organisms which demonstrate a greatly increased
metabolic versatility. In a sense these findings produce a more complex
situation which challenges our perception of nitrifying bacteria and their riile
within the nitrogen cycle. The traditional view stated above, therefore, must be
considered at best simplistic.
The majority of these advances have arisen at the interface between
microbial physiology and ecology. The driving force for much of the work
discussed in this article has been the desire to explain aspects of nitrification in
natural environments in terms of the biochemistry and physiology of the
organisms responsible. While the essential r61e of physiology in microbial
ecology is now widely recognized, nitrification provides a situation where
ecological observations have stimulated physiological studies, leading to the
discovery of new and important properties of nitrifying bacteria. This article
will, therefore, consider several aspects of the physiology of nitrifiers of
relevance to their growth and activity in natural environments. After
describing the basic features of the biochemistry of ammonia and nitrite
oxidation, the factors limiting growth and activity of these organisms will be
discussed. The influence of oxygen concentration, pH value and inhibitors on
their physiology will then be considered, and throughout these discussions
additional effects due to surface growth will be considered.

11. Ecological and Economic Importance of Nitrification

Nitrification plays a central r6Ie in the nitrogen cycle of terrestrial and aquatic
ecosystems, converting the most reduced form of nitrogen, NH,, to the most
oxidized form, NO;. Ammonia is produced naturally through mineralization
 AUTOTROPHIC NITRIFICATION IN BACTERIA 127
of organic matter and is also supplied to agricultural systems directly, as
inorganic ammonium, e.g. ammonium nitrate, or indirectly, as urea and other
ammonium-based fertilizers.
In marine environments ammonium is present at concentrations less than
0.01 pg NH: - N ml-l, in other aquatic systems and in unfertilized soil at
concentrations in the order of 1-1Opg ml-', with concentrations rising to
50 pg ml- in areas of intensive agriculture with high inputs of ammonium
fertilizer. In sewage treatment systems, concentrations of ammonia are similar
to those in soil but some industrial effluents give rise to concentrations up to
1000pg NH: - N ml- ' (i.e. about 70 mM).
Nitrification of fertilizer nitrogen in agricultural systems is considered
detrimental. While the anionic NH; is bound to cationic components of the
soil, the product of nitrification, NO;, is much more mobile and is readily lost
through leaching. Losses may also occur through denitrification of nitrate.
Leaching can lead to eutrophication of run-off waters and nitrate pollution of
ground-waters. Concern regarding the latter arises from the production of
nitrosamines from nitrite, following consumption of nitrate in drinking water,
and also the occurrence of methaemoglobinaemia in infants and young
animals. Regulations regarding nitrate in drinking waters have consequently
been strengthened and there is renewed interest in the application of
nitrification inhibitors along with ammonium-based fertilizers.
Nitrification is a necessary component of sewage treatment processes where
large amounts of ammonia are produced through decomposition of organic
material. Nitrification prevents discharge of toxic levels of ammonia into
receiving waters, which may cause death of fish populations and may lead to
eutrophication. The latter may also be caused by nitrate, and complete
removal of nitrogen is achieved by processes involving both nitrification and
denitrification. The low specific growth rates of nitrifying bacteria limit the
rate at which material can pass through suspended growth systems, e.g. the
activated sludge process. Faster through-put is possible using fixed film
systems, e.g. rotating biological contactors and trickling filters.
In aquatic environments, the major pool of inorganic nitrogen is nitrate,
formed through nitrification of ammonia released by mineralization.
Concentrations of ammonia available to freely suspended cells within the
water column are low, but cells associated with decomposing organic matter,
either suspended or in sediments, will receive higher concentrations. Nitrate
acts as a substrate for denitrification which, together with nitrification, leads
to production of nitric and nitrous oxides. The production of these gases is
currently causing great concern with regard to their effects on atmospheric
chemistry, in particular on the ozone layer and on acid rain. Recently,
attention has also been directed to the r61e of nitrification in corrosion of
cement and natural stones. Kaltwasser (1976) and Wasserbauer et al. (1988)
128 I. 1. PROSSER

demonstrated the involvement of nitrifying bacteria in production of nitrous

acid, leading to disintegration of asbestos cement. Nitrifying bacteria have
also been isolated from sandstone bridges and historical monuments at a
number of sites (Bock et al., 1988; Wolters et al., 1988; Meincke et al., 1988) and
are frequently the major component of the microbial population isolated from
these environments. These organisms grow and survive in pores within the
sandstone and oxidize ammonia scavenged from the atmosphere.

111. Taxonomy and Species Diversity

Ammonia and nitrite oxidizers constitute the family Nitrobacteraceae. This

family is designated solely on physiological properties, i.e. the use of ammonia
or nitrite oxidation as an energy source and fixation of carbon dioxide for
organic carbon (Watson, 1974). All members are Gram-negative but exhibit a
wide range of morphologies, with regard to cell shape and arrangement of
intracytoplasmic membranes. These features form the basis for classification
within the family. Nitrifying bacteria, therefore, probably arose from a
number of unrelated strains developing independently the ability to use
ammonia or nitrite as an energy source. Full descriptions of all genera and
strains are given by Watson (1974), Watson et al. (1981) and Bock et al. (1986).

A. AMMONIA OXIDIZERS

Ammonia oxidizers are placed in five genera on the basis of cell shape, which is
reflected in their names: Nitrosomonas, Nitrosococcus, Nitrosospira, Nitro-
solobus and Nitrosovibrio. The most extensively studied ammonia oxidizer is
Nitrosomonas europaea, which is the only species recognized within this group.
Recent analysis of per cent guanine plus cytosine (%(G+ C)) values (Koops
and Harms, 1985) and DNA:DNA hybridization studies (Bock et al., 1986)
suggest the existence of seven new species, some of which are correlated with
environments from which isolates were obtained.
Most physiological studies have been carried out using N. europaea, which
is the most frequently isolated strain. This appears to result from its preference
for growth conditions associated with typical batch isolation procedures and
there is increasing evidence of dominance by other genera in particular
environments. Nitrosolobus species are the most common ammonia oxidizers
in soil (Macdonald, 1986), Nitrosospira species dominate in acid soils (see
Section VII1.A) and Nitrosococcus species are important in marine
environments. In addition, significant qualitative and quantitative physiolog-
ical differences exist between strains and genera. While the knowledge of the
properties of N . europaea argue for its continued use in fundamental
 AUTOTROPHIC NITRIFICATION IN BACTERIA 129
investigations into the biochemistry of ammonia oxidation, full realization of
the metabolic potential and diversity of ammonia oxidizers requires more
detailed study of other genera.

B. NITRITE OXIDIZERS
There are four genera of nitrite oxidizers, Nitrobacter, Nitrococcus, Nitrospira
and Nitrospina, determined, as with ammonia oxidizers, by cell shape and
ultrastructure. Only the genus Nitrobacter has been subjected to detailed
taxonomic and physiological studies. It contains three species, N. winograd-
skyi, N. hamburgensis and a third species distinguished on the basis of
%(G + C ) values and DNA hybridization studies (Bock et al., 1986).Analysis
of cytochromes from the genera Nitrosomonas and Nitrobacter suggests they
are distantly related (Wood, 1986)and N . agilis appears most closely related to
Rhodopseudomonas viridis. Both organisms possess complex intracytoplasmic
membrane systems suggesting a common evolutionary background.
One reason for concentration of studies on the genus Nitrobacter is the
inability to isolate members of other genera from soil. The genera Nitrococcus,
Nitrospira and Nitrospina are generally restricted to marine environments and
grow optimally at high salt concentrations. One exception is a Nitrospira
strain isolated from soil (Bock et al., 1986).
The application of modern taxonomic techniques to nitrifying bacteria is
hampered, as in physiological studies, by difficulties in obtaining sufficient
biomass for analysis. Despite this, %(G + C ) values, DNA hybridization and
RNA sequence analyses are yielding important information. Nitro-
bacteraceae is now considered to contain at least three groups: Nitrobacter,
ammonia oxidizers and Nitrococcus (Woese et al., 1984a,b, 1985). The
extension of such studies to a wider range of nitrifier strains will permit a more
complete understanding of the interrelationships between nitrifiers and of
their evolutionary origin. Molecular biological studies should also increase
our knowledge of the extent and significance of species diversity within
natural populations of nitrifying bacteria through the application of modern,
molecular based, detection techniques.

IV. Biochemistry of Nitrifying Bacteria

Wood (1986, 1987, 1988)has recently reviewed the biochemistry of ammonia

oxidation while Bock et al. (1986) and Wood (1986, 1988) provide
comprehensive reviews of nitrite oxidation. Here I will discuss the basic
features of the biochemistry of nitrifying bacteria and readers are referred to
these reviews for detailed references. Certain aspects of their biochemistry will
also be discussed in later sections where appropriate.
130 J. I . PROSSER

A. AMMONIA OXIDATION

The first stage in ammonia oxidation is its endergonic conversion to

hydroxylamine (AGO = + 17 kJ mol-I). This reaction is carried out by a
membrane-bound ammonia mono-oxygenase for which the substrate is
ammonia (NH,) rather than ammonium ions (NH:) (Suzuki et al., 1974).The
reaction requires oxygen, supplied as molecular oxygen, and a source of
reducing power. Wood suggests that the mono-oxygenase accepts electrons
from the ubiquinone-cytochrome b region of the electron-transport chain.
The reaction may therefore be represented by the equation:
NH, + 0, + 2H+ + 2e- + NH,OH + H,O
Ammonia mono-oxygenase has a broad specificity and appears capable of
oxidizing a number of low-molecular-weight organic compounds including
propene, benzene, cyclohexane, phenol and methanol. Attempts to isolate
ammonium mono-oxygenase in soluble form have failed. The enzyme is
irreversibly inhibited by acetylene (Hynes and Knowles, 1978), which it
attempts to oxidize but which acts as a “suicide” substrate (Hyman and Wood,
1985). Incubation with radiolabelled acetylene allows identification of the
enzyme by SDS-PAGE, which indicates a molecular weight of 28,000 Da.
Of particular interest is the similarity between ammonia mono-oxygenase
and the membrane-bound (but not the soluble) methane mono-oxygenase
found in methane-oxidizing bacteria. Both enzymes are associated with
intracytoplasmic membrane systems and both have common inhibitors and
substrates, although the methane mono-oxygenase is unable to hydroxylate
aromatic compounds. Methanotrophs are capable of oxidizing ammonia, but
at a much lower rate than Nitrosomonas species, and methanotrophs will not
grow with ammonia as the sole energy source. Similarly, oxidation of methane
by Nitrosomonas species is much slower than by methanotrophs and does not
affect its growth.
Ward (1987) carried out comparative studies of ammonium and methane
oxidation by the marine nitrifier Nitrosococcus oceanus. Inhibition of
ammonia oxidation by methane was not simple competitive inhibition (Fig. 1)
and suggested multiple active sites for ammonia, each behaving independ-
ently. The enzyme had similar affinities for both substrates, whereas in N.
europaea the affinity for ammonia was much greater than for methane.
Importantly, radiolabelled methane and methanol were incorporated into cell
material in the presence of ammonia at levels at which nitrification was not
inhibited. Cells, therefore, apparently possess two parallel pathways for
methane and ammonia oxidation, which share a common initial enzyme and
similar capabilities for oxidation of methane and ammonia.
Ward (1986) notes the apparent difficulty in isolating methanotrophs from
 AUTOTROPHlC NITRIFICATION IN BACTERIA 131

I/[S] (pM NH,)-'

FIG. 1. a, Oxidation of ammonia by Nitrosomonasoceanus and inhibition by methane.
b, Lineweaver-Burk plot of data in part a. Key: A,control; 0,
inhibition by methane.
From Ward (1987) with permission.

such ecosystems, and suggests that Nitrosococcus strains may fulfil a dual r81e
in marine environments, oxidizing whichever substrate is available. Apart
from this possibility, oxidation by ammonia oxidizers of organic compounds
appears to be fortuitous and has no known ecological r8le.
Hydroxylamine is oxidized to nitrite, via uncharacterized intermediates, by
the soluble enzyme hydroxylamine oxidoreductase, which has a complex
132 J. 1. PROSSER

1
,teversed
,’electron f l o w
,,

J monooxygenase
NH20H, H ,)I

periplasm 1 cytoplasmic membrane cytoplasm

FIG. 2. Proposed scheme for electron transport in the genus Nitrosomonus. From
Wood (1986) with permission.

structure and probably resides in the periplasm. This reaction generates

energy (AGO’= + 60mV) and receives oxygen from water. A scheme for
electron transport in Nitrosomonas species is presented in Fig. 2 and involves a
normal electron-transport chain, with electrons fed in from hydroxylamine
oxidoreductase at a point close to ubiquinone. Oxidation of hydroxylamine
releases four electrons:
NH,OH + H,O + NO; + 5H+ + 4e-
Two of these electrons are required for the ammonia mono-oxygenase while
the remaining two pass down the electron-transport chain generating a proton
motive force. Consequently, the respiratory chain is branched with ammonia
mono-oxygenase and the terminal oxidase competing for electrons. Two
mechanisms have been proposed for the control of electron flow. Hooper and
Terry (1973) observed stimulation of added hydroxylamine in the presence of
uncouplers but inhibition of ammonia oxidation. This suggests that electron
flow to the terminal oxidase may be restrained by the proton motive force. The
second control mechanism involves hydroxylamine which inhibits ammonia
mono-oxygenase. This provides a feed-back inhibition mechanism, with
establishment of a low steady state hydroxylamine concentration following
the onset of ammonium oxidation. Wood (1986) considered H+/O ratios,
which are 3.9 and 2.7 for hydroxylamine and ammonium ions, respectively.
 AUTOTROPHIC NITRIFICATION IN BACTERIA 133
Analysis of stoichiometries implies that the branches to ammonia mono-
oxygenase and the terminal oxidase are equally efficient, which he suggests is
not indicated by the action of uncouplers.

B. NITRITE OXIDATION

Nitrite oxidation is carried out by the soluble enzyme nitrite oxidoreductase

with oxygen supplied by water:
NO; + H,O 4NO; + 2H+ + 2e-
Nitrite oxidoreductase has been purified by Tanka et al. (1983) and
Sundermeyer-Klinger et al. (1984) using different extraction techniques which
are discussed by Bock et al. (1986). The enzyme is reversible, carrying out
nitrate reduction, the significance of which is discussed in Section V1I.D.
Coupling of nitrite oxidation to the generation of a proton motive force is
theoretically possible but the detailed mechanism has yet to be elucidated.
Wood (1986) discusses the experimental evidence for mechanisms of energy
generation by nitrite oxidation and proposes the scheme illustrated in Fig. 3.
Bock et al. (1986) propose a more complex electron-transport system for
Nitrobacter hamburgensis, with two linked respiratory chains, one for litho-
autotrophic growth and the other for heterotrophic growth.

C. CARBON METABOLISM

1. Fixation of Carbon Dioxide

The major source of organic carbon for nitrifying bacteria is carbon dioxide
which is fixed via the Calvin cycle. Some, but not all, nitrifiers contain
carboxysomes (see Bock et al., 1986) which are associated with ribulose 1,5-
bisphosphate carboxylase oxygenase (RuBisCO). In some strains only soluble
RuBisCO is found, while others have both soluble and carboxysome-bound
RuBisCO. In N . hamburgensis two different genes and gene products have
been identified for RuBisCO, one residing on the chromosome and the other
on a plasmid. The energy obtained from oxidizing ammonia or nitrite is very
low. The NO;/NH: redox potential is + 340 mV and that for the NO;/NO;
couple is +430mV. This potential is vastly more positive than the
NAD+/NADH couple and, indeed, ammonia and nitrite are at the limits for
provision of energy for growth. As a consequence, NAD must be reduced by
energy-driven reversed electron flow;
+ 2cyt .c(Fe3+)+ NADH + ( N - l)H!;
Zcyt. c(Fe2') + NAD+ + (N)H,,+",

Much of the limited amount of energy generated by ammonia and nitrite

134 J. I. PROSSER

peri plasm
.. . cytoplasmic membrane cyt opl as m

....
a NAD+

..
,

.:-
0

.
4

hversed

0
0
0
, electron f l o w
. yn H+
cytochrome c-556
NY HP
\
?e-
\nitrite
oxidoreductase

2 H+s, di \
\\\\
’cyt oc hrome
oxidase

to
\
\ 4‘ H+

FIG. 3. Proposed scheme for energy conservation in the genus Nitrobaclrr. From
Wood (1986) with permission.

oxidation is therefore used to generate reducing power, for which carbon

dioxide fixation imposes a high requirement. On thermodynamic grounds,
Kelly (1978) estimates that 80% of energy generated by autotrophs is used to
fix carbon dioxide. Glover (1 985) determined thermodynamic efficiencies for
growth of ammonia oxidizers and nitrite oxidizers in the range 4.421.3%.
The consequences of this for growth of both organisms will be discussed in
Section V.
 AUTOTROPHIC NITRIFICATION IN BACTERIA 135
2. Assimilation of Organic Compounds by Ammonia Oxidizers
Ammonia oxidizers are capable of assimilating organic compounds with
varying effects on growth. Clark and Schmidt (1967a,b) demonstrated uptake
and incorporation of 14 amino acids by N. europaea. While some amino acids
had no effect on growth, others either inhibited or stimulated growth by
effecting the length of the lag phase and the final nitrite concentration. There
was no obvious correlation between such effects and the extent of amino-acid
uptake. Rates of incorporation were much less than those of heterotrophs but
were greater in the presence of ammonium.
Krummel and Harms (1982) investigated growth of a variety of ammonia
oxidizers in the presence of formate, acetate, pyruvate, glucose and peptone.
Again results were variable. Growth of Nitrosomonas strains was not affected
while Nitrosococcus oceanus was inhibited by all five compounds. Formate
and acetate inhibited growth of Nitrosococcus mobilis but stimulated one
Nitrosospira strain and had little effect on a second Nitrosospira strain.
Nitrosouibrio tenuis was stimulated by formate and glucose, but inhibited by
acetate and pyruvate. Mixotrophic growth increased yield, measured as
biomass formed per unit ammonium converted. Heterotrophic growth of
ammonia oxidizers (i.e. use of organic compounds for both carbon and
energy) has never been observed despite incubations for 23 months in organic
media (Krummel and Harms, 1982).
The variability described above, both with regard to species diversity and
responses to different compounds, explains the variable effects of hetero-
trophic contaminants on ammonia oxidizers. Clark and Schmidt (1966) found
that stimulation of growth of N. europaea in the presence of a heterotroph
could be reproduced by supplying pyruvate, which had little effect on
exponentially growing cells but accelerated recovery of old cultures. The
heterotroph did not grow and stimulation of ammonia oxidation was believed
to result from assimilation of the products ofcell lysis. Jones and Hood (1980),
however, report a mutualistic interaction between a Nitrosomonas species, a
Pseudomonas species and Nocardia atlantica isolated from an estuarine
environment. Ammonium oxidation was increased by 150% and growth of
the heterotrophs by a factor of 10, but the precise nature of the interaction is
unknown. Other interactions in mixed culture are discussed by Kuenen and
Gottschal (1 982).

3. Heterotrophic Growth of Nitrite Oxidizers

The situation for nitrite oxidizers is different. Heterotrophic growth on
acetate, casein hydrolysate, glycerol or pyruvate has been demonstrated in
Nitrobacter agilis (Smith and Hoare, 1968; Bock, 1976) and Nitrobacter
136 J. 1. PROSSER

winogradskyi (Kalthoff et al., 1979). Mixotrophic growth stimulates both

maximum specific growth rate and cell yield. Steinmuller and Bock (1976)
obtained stimulation of growth of several Nitrobacter strains supplied with
culture filtrates of a number of heterotrophs grown in yeast extract-peptone
solution. Nitrite oxidation rate was increased by as much as 200% and
biomass yield by a maximum of 48%. Growth also continued after exhaustion
of nitrate, with a 30% increase in biomass concentration. The mechanism of
stimulation is not known, and may involve removal of inhibitory compounds
by the heterotroph, but its presence and extent varied with both the strain of
Nitrobacter and the heterotroph.
Heterotrophic growth of Nitrobacter species is slower than that of other
heterotrophs and slower than growth on nitrite. Bock (1976) reported a
minimum generation time of 65 hours for N . agilis growing on pyruvate, which
also gave greater yields than acetate and formate. N. winogradskyi had
generation times of 70 and 96 hours on pyruvate and acetate, respectively.
(Kalthoff et al., 1979). Heterotrophic growth also leads to differences in cell-
wall antigenic structure and to larger cells of irregular morphology.
Heterotrophically grown cells possess lower levels of nitrite oxidoreductase
(Steinmuller and Bock, 1977), requiring up to 3-4 weeks induction before
regaining the ability to grow autotrophically (Bock, 1976).
Kirstein et al. (1986)demonstrated heterotrophic growth of a third strain, N.
hamburgensis and noted differences in b-type cytochromes between auto-
trophically and heterotrophically grown cells. The former possessed
cytochrome hSh2- 564 while the latter contained a cytochrome b which
absorbed in the 558-560 nm region. Further differences in cytochromes are
discussed by Bock rt al. (1986)and led to the proposition that N. hamburgensis
possesses two respiratory chains, one for autotrophic and one for hetero-
trophic growth. Certain cytochromes are components of both chains,
although their levels may vary with growth conditions. The presence of others
depends on whether growth is autotrophic or heterotrophic.

V. Growth of Nitrifying Bacteria in Liquid Culture

Nitrifying bacteria are typically cultivated in defined inorganic mineral salts

media containing ammonium or nitrite. Representative media for both
ammonia and nitrite oxidizers are listed in Watson et al. (1981). Growth in
batch culture is most readily assessed by following rates of substrate
(ammonia or nitrite) utilization or product (nitrite or nitrate) formation.
Ammonia concentrations in the order of 50-2000 pg NH: - N ml -
(3-140m~)and 5G200pg NO; - N m l - ' ( 3 - 1 0 m ~ )are typical. Ammonia
oxidizers are routinely cultured in weakly buffered media containing phenol
 AUTOTROPHIC NITRIFICATION IN BACTERIA 137
red as a pH-value indicator. Qualitative assessment of growth is then achieved
through a colour change resulting from reduction in pH value through
production of nitrous acid. Acid is neutralized by addition of sodium
carbonate or sodium hydroxide, the former providing a source of carbon
dioxide which is otherwise supplied by aeration. Cultures are generally
incubated aerobically with shaking, but high concentrations of oxygen can be
inhibitory (see Section VILA). Estimation of biomass by absorbance
techniques is not normally possible due to the low cell concentrations and
biomass densities involved. Cell concentrations generally increase from
approximately lo5 cells ml-' to 107-108 cells ml-' in batch culture. Turbid
cultures of ammonia oxidizers may be obtained in strongly buffered medium
containing a high ammonium concentration. Continued replenishment of
cultures of nitrite oxidizers with nitrite, following nitrite exhaustion, also
results, ultimately, in turbidity.
Growth parameters for ammonia and nitrite oxidizers have been measured
in both batch and continuous-flow systems involving pure cultures and in
mixed-culture systems with enriched cultures, particularly from soil and
sewage. Estimates from pure-culture studies will be discussed with regard to
factors limiting their size and their relevance both to physiological studies and
to values obtained in natural environments.

A. MAXIMUM SPECIFIC GROWTH RATE

It has been demonstrated (Belser and Schmidt, 1980;Keen and Prosser, 1987a)
that product concentration increases exponentially during batch growth of
nitrifying bacteria. The slope of a semi-logarithmic plot of product
concentration versus time is equivalent to the specific growth rate and
provides similar values to those calculated as specific increases in cell
concentration. Use of this technique is only valid during balanced growth
when cell size and cell activity are constant. Hofman and Lees (1952) and
Koops (1969) have suggested that increases in nitrite concentration will
decrease the efficiency with which ammonia oxidation is coupled to biomass
formation as batch cultures age due to product inhibition.
Glover (1985) showed decreases in energetic efficiency in ageing cultures to
be due to reduced potential for carbon dioxide fixation (see following section).
The implications for the use of product formation as a measure of specific
growth rate are, however, unlikely to be serious as Glover's data indicate that
this imbalance occurs within the deceleration phase when specific growth rate
is decreasing.
The pmax values for both ammonia and nitrite oxidizers are low for pure-
and mixed-culture systems under ideal conditions in comparison to those of
heterotrophs. The highest reported value for pmax is 0.087 h- ',equivalent to a
138 J. 1. PROSSER

TABLE I. Maximum specific growth rate (pmaX)

values of pure cultures of nitrifying bacteria

Batch Continuous
culture culture Reference

Ammonia oxidizers
Nilrosomoflus Spp.
Sewage isolate
Sewage isolate
0.016-0.058
0.012-0.043
} Loveless and Painter (1968)
N. europueu 0.088 0.063 Skinner & Walker (1961)
N. ruro~pu~u 0.02-0.03 Drozd ( I 980)
N. i~uro~prrcw
N. cw~~poprrc~u
ATCC
FH 1
0.052-0.066
0.052-0.054
} Belser and Schmidt (1980)
N . iwrop(prrecr 0.036 0.064 Helder and de Vries ( I 983)
N. cwrcpwu 0.017 0.039 Keen and Prosser (l987a)
N. ntrrriprriu 0.018 Glover (1985)
Ni1rosococcic.s ocwnus 0.0I4 Glover (1985)
~ ; ~ Y I ~ . Y 0.032
N ~ I ~ ( J , w JOl'l'LIIIILV
Nilrosospiru AV2
N i l , o s ~ ~ l o h uAV
s 3
0.033-0.035
0.043-0.044
} Belser and Schmidt (1980)
Nitrite oxidizers
Nilrohucli~rspp.
N. sp. 0.058 Could and Lees (1960)
N . N.W.
N. L.
0.039
0.025
0'033
0.0 I8
} Gay and Corman (1984)
N. sp. 0.043 0.035 Keen and Prosser ( 1987a)
Ni1roi~occic.sntohi/i.s 0.033 Glover (1985)

doubling time of 8 h, for N . europaea growing in batch culture, although the

same strain in continuous culture had a pmarof only 0.063 h - ' (Skinner and
Walker, 1961). Values of pmaxusually lie within the range 0.0140.064 h-'
(equivalent to doubling times of 50-1 1 h) (Table 1).
Maximum specific growth rate is limited by the low energy gain from
ammonia and nitrite oxidation. The small amount of energy generated is then
required for production of reducing power for fixation of carbon dioxide.
How, then, could an ammonia or nitrite oxidizer adapt to increase its
maximum specific growth rate. One way would be to increase the amount of
cell material devoted to substrate oxidation. This strategy is employed by
many, if not all, nitrifiers and is seen in the complex invaginations of the cell
membrane believed to be the site of ammonia mono-oxygenase and
respiratory enzymes. Another function for such membranes in ammonia
oxidizers is the compartmentalization of hydroxylamine, which is toxic.
Proliferation of membranes will increase the amount of energy-generating
 AUTOTROPHIC NITRIFICATION IN BACTERIA 139
material within the cell and, potentially, the actual rate of biomass production.
Presumably, a limit will be reached where this imbalance in cellular structure
becomes counter-productive and the amount of other cellular components
required becomes inefficiently small. A complex membranous structure may
also decrease diffusion of ammonia to the sites of oxidation and diffusion of
intermediates and products between reaction centres.
Specific growth rate does not depend solely on the actual rate of biomass
production but rather on the rate at which cell mass may be doubled, i.e. the
specific rate of biomass formation. Specific growth rate can therefore be
increased by decreasing cell size for the same energy-generating capacity. To
an extent this is seen in nitrifiers, which are never longer or wider than 2 pm
and whose cell size is, frequently, in the order of 1 pm. We will also see that cells
of the same specific growth rate can have very different cell activities, again due
to the differences in cell size. The important factor in increasing ammonia-
oxidizing capability is the proportion, rather than the amount, of cell material
occupied by intracytoplasmic membranes.
The benefits of a high maximum specific growth rate in nature are dubious.
Wood (1986) has pointed out the perversity of ammonia oxidizers in their
refusal to generate energy from organic compounds, even though they have
the potential to metabolize such compounds. Assimilation of organic carbon
does not significantly increase maximum specific growth rate or yield. If
heterotrophy presented an advantage, presumably ammonia oxidizers would
have developed to exploit its potential. A high maximum specific growth rate
is considered to provide an ecological competitive advantage where substrate
concentrations are high. Although this may be the case in sewage treatment
processes, in most natural environments bulk concentrations of ammonia are
low and those of nitrite are negligible. The ability of nitrifiers to grow and
survive at low substrate concentrations and to interact closely with the
producers of ammonia and nitrite may, therefore, be more important selective
properties for these organisms than a high specific growth rate.

B. GROWTH YIELD

In the absence of substrate or product inhibition, exponential growth of

ammonia oxidizers usually ceases when the medium becomes acidic, while
that of nitrite oxidizers continues until nitrite is completely utilized. Inhibition
of nitrite oxidation by intact cells and cell-free extracts of Nitrohacter strains
was demonstrated by Boon and Laudelout (1962) to be due to nitrous acid at
low pH values and competitive inhibition by hydroxyl ions at high pH values.
Non-competitive product inhibition by nitrate was also found. Equivalent
studies have not been carried out for ammonia oxidizers, but free ammonia is
inhibitory to both ammonia and nitrite oxidizers and ammonia oxidation is
140 J. I . PROSSER

also subject to inhibition by nitrous acid. Such inhibition generally occurs at

relatively high concentrations (greater than 30 m~ (Focht and Verstraete,
1977)).Keen and Prosser (1987a) found no evidence for substrate inhibition of
ammonia or nitrite oxidation in continuous culture with substrate
concentrations less than 50pg N ml-' (3.5mM). Data from sewage treatment
processes indicate that free ammonia and nitrous acid act as differential
inhibitors of nitrite and ammonia oxidations, respectively (Anthonisen et al.,
1976). Substrate inhibition in sewage treatment processes has been modelled
by Castens and Rozich (1986) using classical enzyme kinetics. In soil, Darrah
et al. (1986) found inhibition by high concentrations of ammonium chloride,
but this appeared to be due to an effect on osmotic pressure or chloride
concentration rather than direct inhibition by high ammonium
concentrations.
There is evidence of strain variability with regard to substrate inhibition of
nitrite oxidizers. Nitrospira gracilis cannot grow at nitrite concentrations
greater than 1 mM and N . winogradskyi dominates in enrichment media
containing high initial nitrite concentrations. The basis for these differences in
tolerance to nitrite inhibition is unknown but has obvious implications for
isolation procedures (see Section VILA). It is also probable that ammonia
oxidizers will show strain variability with regard to substrate inhibition which
may be important in optimal operation of sewage treatment processes.
Values for yield coefficientson ammonia and nitrite are given in Table 2 and
can be seen to be low. Values are similar for both groups of nitrifiers and each
cell must convert in the order of 10 times its own weight of ammonia- or
nitrite-nitrogen to double in mass. Experimental values for true growth yield
(Y,) and maintenance coefficients ( m ) have been determined by Keen and
Prosser (1987a) (Table 2). The maintenance coefficient was calculated from
steady state.continuous culture data assuming m to be independent of specific
growth rate. These values indicate that at an ammonium concentration of 1 jig
NHf - N ml- ', the specific growth rate of Nitrosomonas strains will be 21 YO
of pmax and 76% of substrate will be consumed for maintenance. Similarly,
pmax for Nitrohacter strains will be reduced to 26% of pma,at 1 pg NO; - N
ml-', with 81% of nitrite oxidized for maintenance.
Maintenance requirements are similar for both organisms and are
significant in natural environments where substrate concentrations are low.
Belser (1979) calculated that, on the assumption of a value of m = 0.028
(gcell)-' h-I, an ammonification rate in soil of 6.18pgNHf - N h - ' would
permit establishment of a population of 2.4 x lo3 to 2.4 x lo5 cells (g soil)- '.
This may, therefore, be the major factor limiting population levels in the soil
and the significant feature in limiting cell yields in liquid culture,
The relationship between substrate oxidation and carbon dioxide fixation
has been studied by Glover (1985) who found a decrease in free-energy
 AUTOTROPHIC NITRIFICATION IN BACTERIA 141
TABLE 2. Yield and maintenance coefficients for nitrifying bacteria

Ammonia oxidizers
Biomass and cell yield on ammonia:
Nitrosomonas sp. 0.42-1.40 g biomass mol- Loveless and Painter (1968)
Nitrosomonas europaea 0.6-0.8 g biomass mol- ' Drozd (1980)

I
Nitrosomonas europaeu 1.261.72g biomass mol-' Keen and Prosser (1987a)
Nirrosomonas ATCC 4.61-6.44 x 10l2cells mol- I
Nilr(J.SUm(JnU.7 FH I 1.38-2.44 x 10'2cellsmol-' Belser and Schmidt (1980)
Nitrosospira sp. 8.0410.6 x 1012cellsmol-'
Nitrosolobus sp. 1.85-2.24 x 1012cellsmol-'
True growth yield 5.88 g biomass mol- I
Maintenance coefficient 0.88 mol g biomass-' h-l

Carbon yield on ammonia (ratio of CO, fixed: NO; produced):

Ni~r~J.sOM~J~U.Ssp. 0.090 Wezernak and Gannon (1967)
Nitro.somona.7 sp. 0.033 Helder and de Vries (1983)
Nitrosomona.s spp. 0.08 14.094 Belser ( 1 984)
Nitrosomonas niarinu 0.044.07 Glover (1985)
Nitrosopira spp. 0.0754.096 Belser (1984)
Nitrosocysti.~~ici~anu.~ 0.074. I3 Gunderson ( I 966)
Ni1ro.socysti.s oceanus 0.064.10 Carlucci and Strickland (1968)
Nitroxococcus mohilis 0.014-0.031 Glover (1985)
Nitrite oxidizers
Biomass yield on nitrite:
NirrohoctcJrsp. 1.11-1.51 g biomass mol-' Keen and Prosser (1987a)
Carbon yield on nitrite (ratio of CO, fixed: NO; produced):
Nitrohucrer sp. 0.0I 3 4 . 0I4 Schon (1 965)
Nitrohucti~rsp. 0.0125 Wezernak and Gannon ( 1 967)
Nitrohucter sp. 0.02 Helder and de Vries (1984)
Nirrohacter spp. 0.024.03 Belser (1984)
Nitrococcus mvhilis 0.014-0.031 Glover (1985)
True growth yield 9.8 g biomass mol-
Maintenance coefficient 0.78 mol biomass-I h-l

efficiency of approximately 70% between early and late exponential phases in

cultures of Nitrosomonas marina and Nirrosococcus oceanus. This was
correlated with a decrease in cellular organic carbon and nitrogen content and
chemostat studies demonstrated a decrease in efficiency with specific growth
rate which was independent of nitrite concentration. In continuous culture,
decreasing specific growth rate (dilution rate) was also associated with a
reduction in the activity of RuBisCO (measured as enzyme activity per unit
biomass) and in the yield of organic carbon. Similar behaviour was found for
the nitrite oxidizer, Nitrococcus mobilis. Ageing batch cultures and cells grown
in continuous culture at low specific growth rates therefore had reduced
142 J. I . PROSSER

potential for carbon dioxide fixation and ammonia oxidation was consequent-
ly less efficient in terms of biomass production.
The carbon yield from nitrification (the ratio of carbon fixed to nitrogen
oxidized) varied with substrate concentration and specific growth rate (Table
2) and was greater for ammonia oxidizers than for Nitrococcus species. This is
due to the greater energy gain from ammonia oxidation. Belser (1984)
obtained similar relative values for the ammonia oxidizers N . europaeu and
Nitrosospiru species and a Nitrohacter species (Table 2). The ratio of carbon
fixed to substrate oxidized varied little with specific growth rate and with pH
value in continuous culture, indicating that cellular metabolism is regulated to
maintain an optimal ratio. Short-term changes caused by additions of
substrate to cell suspensions altered the ratio, as did addition of metabolic
inhibitors, particularly for Nitrohucter strains.

C. CELL ACTIVITY

In batch culture, simultaneous estimation of cell concentration and product

concentration allows calculation of cell activity. Values have also been

TABLE 3. Ccll and biomass activities for nitrifying bactcria

20
II Belscr (1979)
23
0.9-5.1 Remacle and DeLeval t 1978)
23 Belser and Schmidt ( 1 980)
0.9-4.9 Glover (1985)
1 .O-7.0 Keen and Prosscr (19874
13.7-31.3 Glover (1985)
Nirrosospirtr hricwi.r
Nirrosolohu.~niultiforniis
4
23 } Belser ( 1979)

Biomass activity (nmol NO; produced g biomass- h - l ) :

Nirr(~.soriionrr.siwrupueu 4 Skinner and Walker (1961)
Nilrosomot7ir.v 1’u~OpUc’U 30-200 Drozd ( 1980)
NilrfJ.YfJnioltu,seuropueu 7.5-16 Keen and Prosser (1987a)
Nitrite oxidizers
Ccll activity (fmol NO; produccd ce1l-l h K 1 )
Nitrohuctcv sp. 5.1-13.6 Remaclc and DeLeval (1978)
Nilrohuctar spp. 9-42 Belser ( 1979)
Nirrohucror sp. 5.1-1 3.6 Keen and Prosscr (1987a)
Ni/roi,ctccus f)i’i~fft7US’ 6.7- 1 1.4 Glover ( I 985)
Biomass activity (nmol NO,; produced g biomass-’ h K 1 )
Ni!rohuctar sp. 15.1-25.2 Keen and Prosser (l987a)
 AUTOTROPHIC NlTRlFlCATlON IN BACTERIA 143
calculated in continuous culture, which also enables measurement of activity
per unit biomass. Typical values are presented in Table 3.
Both Keen and Prosser (1987a) and Glover (1985) found cell activity to
increase with specific growth rate in continuous culture, as might be expected,
and Keen and Prosser's data indicate a Michaelis-Menten relationship. Cell
activities have also been calculated for ammonia oxidizers in sewage treatment
processes and in soil. Enumeration of nitrifiers in samples from natural
environments requires use of the most probable technique which is inaccurate
and leads to underestimation of cell concentrations. This in turn leads to
overestimation of cell activities, but when such errors are taken into account
values are comparable to those in Table 3. The use of cell-activity
measurements for enumeration in natural environments is discussed by Belser
and Mays (1982) and Prosser (1989).
Measured values for biomass yields indicate that large amounts of
ammonia and nitrite must be converted to synthesize biomass. The converse is
that relatively low cell concentrations may be associated with high rates of
substrate conversion and respiration with no perceptible growth. Drozd
(1980)estimates that ammonia oxidizers consume one-third of their weight of
ammonia per hour. The small populations of nitrifiers found in soil and
immobilized cells in attached-growth sewage treatment processes are,
therefore, capable of significant rates of nitrification.

D. SATURATION CONSTANTS

There is much confusion in the literature regarding saturation constants for

ammonia and nitrite oxidation. Many measurements of short-term activity
have been carried out on cell-free extracts or non-growing cell suspensions,
thereby reflecting saturation constants for enzyme activity, K,. Experimental
determinations of saturation constants for growth ( K , ) of growing
populations in continuous culture are rare. Values for both are given in Table
4. Further measurements have been made of nitrifiers in mixed culture systems
from natural environments.
Experimentally measured K, and K , values show some degree of variability
but are generally similar to, or greater than, concentrations of ammonia or
nitrite found in the environment from which the cells were originally isolated.
Only in sewage treatment plants and in fertilized soil will average or bulk
ammonia concentrations exceed 1Opg N ml-' and nitrite rarely accumulates
above 1 p g NO; - N. Substrate affinities are not high in relation to these
concentrations and, like maximum specific growth rates, are therefore unlikely
to give these organisms a competitive advantage in natural environments.
What limits Ks for these nitrifying bacteria? Unfortunately little is known of
the transport mechanisms for ammonia and nitrite. Nitrite can enter microbial
cells by passive diffusion and similarities between K , values for intact cells and
 TABLE 4. Saturation constants for growth and activity of nitrifying bacteria

Ammonia oxidizers Reference Nitrite oxidizers Reference

Satmation cowtaut for activity (&,)

Nifrosvnionas sp.
l4m~NHi Loveless and Painter ( 1968) Nirrohacrer spp. 1.6-3.6m~NO; Boon and Laudelout (1962)
3 . 6 m ~0,
Nitrosonionas rurvpaeo
0.4 mM NH; Suzuki (1974)
Nitrosonionas europaeu
0.12-1Om~(NH: +NH,) Suzuki el a/. (1974) Nifrohacferspp. 0.256 m~ 0,
0.018-0.058 mM NH, 0.062 mM 0, Peeters e f a/. (1 969)
Nitrosonionas sp.
1.1-3.8m~NH: Laudelout et af.(1976)
Nifrosomonus europaea
0.5-7.0m~NH: Drozd (1976)
Saturation constant for growth (KJ
Niirvsomonus sp.
0.07 mM NH: Remacle and D e k v a l (1978) Nifrvbacttv spp. 0.I78 m~ NO; Could and Lees (1960)
Nitrosvmonas sp.
0.055 mM NH: Helder and de Vries (1983) Nirrohacfer spp. 0.045 mM NO; Remacle and DeLeval(l978)
Nitrosomonas europaea
0.051 mM NH: Keen and Prosser ( 1 987a) Nirrohacfer spp. 0.267 m~ NO; Helder and de Vries (1983)
Nifrvhocferspp. 0.039 mM NO; Gay and Corman (1984)
0.015 mM NO;
 AUTOTROPHIC NITRIFICATION IN BACTERIA 145
cell-free extracts and between K , and K , values suggest that uptake
mechanisms do not limit nitrite-oxidizing activity. The evidence for ammonia
transport in ammonia-oxidizing bacteria is conflicting. Suzuki et af. (1974)
found that the K , value for both suspended cells and cell-free extracts of N .
europaea did not vary with pH value if measured in terms of ammonia (NH,)
rather than total ammonium (NH, + NH:) concentration. This was taken as
evidence that ammonia was the substrate for the ammonium mono-oxygenase
and that the saturation constant did not reflect a K , value for transport. This is
also implied by similarities between K, and K , values. Drozd (1980) suggests
that substrate may be transported into the cell as ammonia leaving a H +
behind. This will contribute to acidification of the medium, which
accompanies ammonia oxidation, and could lead to alkalinization of the
cytoplasm with implications for regulation of internal pH value.
On the other hand, Ward (1987) found an increase in K , value with pH
value for ammonia oxidation by Nitrosococcus oceanus and suggested that
low pH values may affect enzyme activity in addition to ammonia availability.
Glover (1982) studied uptake by the same organism of radiolabelled
methylamine, an analogue of ammonium, which she found to inhibit
ammonia oxidation and, to a lesser extent, carbon dioxide assimilation and
growth. Glover suggests that methylamine may affect cells (a) as a transport
analogue of NHf, (b) as an internal analogue of ammonium, or (c) as an
uncoupler of phosphorylation. Further experimentation is therefore necessary
to determine whether ammonia or ammonium, or both, are transported and, if
so, how.
Ammonia, like nitrite, can enter microbial cells by passive diffusion, when
its rate of transport will depend on the concentration gradient across the cell
membrane and, consequently, on the rate at which the cell can use ammonia
and deplete intracellular pools. As the internal concentration decreases below
the K , value for the ammonium mono-oxygenase, the rate of ammonia
oxidation will decrease, reducing the concentration gradient and rate of entry.
The Ks value may therefore reflect the K , value of the ammonium mono-
oxygenase enzyme, rather than any transport process.
On the other hand, a high affinity NH: transport process, giving a low K,,
would drain energy from the cell, which at low ammonia concentrations it
would not be able to replenish. Using values in Table 3, at approximately
1.4pg N H f - N ml-', the specific growth rate for N . europaea is 28% of its
maximum and cell activity will only equal that required for maintenance. Such
a cell will have little energy to spare for scavenging of ammonium from the
environment. However, ammonia oxidizers in marine environments expe-
rience only submicromolar concentrations of ammonia but the K , and K ,
values of marine isolates are much higher (Table 4). It is difficult to understand
how they survive, grow and multiply.
146 J. 1. PROSSER

The effects of substrate concentration on natural populations of marine

nitrifiers indicate much higher substrate affinities than those of pure cultures.
Olson (1981a) found Michaelis-Menten-type kinetics for nitrite oxidation in
sea water in the range 0.1-1 p~ NO; and estimated a K , value of 0 . 0 7 ~ ~ .
Similar behaviour is reported by Ward ( 1 986), and both workers found
constant rates of ammonia oxidation in concentration ranges 0.1-21 p~ and
0.02-2p~.Hashimoto et af. (1983) estimated a K,,, value of 0.1 p~ for natural
populations of marine ammonia oxidizers.
Ward (1986) argues on the basis of immunofluorescence studies that
cultures isolated from marine environments are truly representative of natural
populations. She suggests that differences in substrate affinities between pure
cultures and natural samples arise through (a) loss of low-substrate-affinity
systems by pure-culture isolates or (b) the existence of complex enzyme
systems capable of efficient use of substrates over concentration ranges of
several orders of magnitude. Certainly, complete explanations of the ability of
marine nitrifiers to grow suspended within the water column require further
physiological study.

E. PROBLEMS OF BIOMASS PRODUCTION

Features of nitrifying bacteria discussed above present the physiologist with a

basic problem in experimental work, i.e. that of obtaining sufficient biomass
for analysis. This is partly due to the low maximum specific growth rates and
low growth yields on ammonia and nitrite but also to substrate and product
inhibition and high maintenance requirements. For example, a one-litre
continuous culture growing at near-maximum specific growth rate will
produce only 0.2mg dry weight biomass per hour.

V1. Surface Growth

While nitrifiers present disadvantages for studying growth in suspended

culture, the ease with which substrate and product concentrations may be
measured presents distinct advantages for studying growth on surfaces, where
measurement of biomass or cell concentration is difficult for all organisms. In
addition, the opposite charges carried by the respective substrates for
ammonia and nitrite oxidation (i.e. NH,f and NO;) provide a means for
testing hypotheses regarding the effects of surface charge on attachment and
growth.
The study of nitrification in soil suspensions and the use of calcium
carbonate to buffer defined inorganic liquid media led to the early belief that
particulate material and surface growth were necessary for nitrification.
Goldberg and Gainey (1955) were the first to demonstrate nitrification in the
 AUTOTROPHIC NITRIFICATION IN BACTERIA 147
absence of surfaces, greatly improving the potential for physiological studies.
They also determined the effects of clay minerals on nitrification in batch
culture (see Section V1II.B) but most early studies on surface-attached
populations were restricted to batch or, more usually, continuous-flow
reactors packed with either soil or glass beads. These were based on the re-
perfusion system of Lees and Quastel (1946) with subsequent development of
continuous-flow systems with a high degree of sophisticated control of
moisture content and other environmental factors and facilities for
measurement of soluble and gaseous substrates and products. Such
experimental systems are reviewed by Prosser and Bazin (1988) and have
provided values for cell activities of natural populations of attached cells, but
suffer from difficulties in enumeration. This is normally only possible using the
most-probable-number technique, following extraction of cells, and leads to
significant under-estimation of viable cell concentrations. The use of glass-
bead columns inoculated with pure cultures of nitrifying bacteria alleviates
some of these problems, as cells may be enumerated microscopically following
removal by ultrasonication. This provides more reliable estimates of cell
activity. Importantly, glass-bead columns have demonstrated the production
of large quantities of extracellular polymeric substances binding cells to
surfaces, stabilizing such communities and permitting high rates of ammonia
and nitrite oxidation. More recently, the physiology of attached populations
of nitrifiers has been studied in batch and continuous-flow systems using glass
slides or cover-slips and ion-exchange resin beads as substrata for attachment
(Underhill and Prosser, 1987a; Keen and Prosser, 1988; Powell, 1985).Effects
of surface growth on the pH response of nitrifiers and on inhibition are
described in Sections VIII and IX. In the absence of inhibitors the major
findings are as summarized below.
In the absence of substrate, both N . europaea and Nitrobacter sp. colonize
anion- and cation-exchange resins to a similar but limited extent. In the
presence of ammonia or nitrite N . europaea and Nitrobacter sp. extensively
colonize cation- and anion-exchange resin beads, respectively, i.e. each species
colonizes the resin to which its substrate is adsorbed. This does not represent
passive attachment but actual growth on the substratum and cell-surface
charge therefore appears to be unimportant in initial attachment and growth.
In batch culture the presence of glass slides stimulates growth of Nitrobacter
sp. but reduces the maximum specific growth rate for N . europaea. The reasons
for this are unclear. Glass possesses a small negative charge and clay minerals
with stronger negative surface charges reduce the maximum specific growth
rate of N . europaea (Armstrong and Prosser, 1988), possibly by localized
competition for adsorption sites between ammonium ions and adsorbed cells.
This does not explain stimulation of growth of Nitrobacter. Initial attachment
is not correlated with production of extracellular polymeric substances (EPS)
148 J . I. PROSSER

but reversible permanent attachment is mediated by formation of a slime layer

which stabilizes the biofilm and allows faster response to changing conditions.
The nitri te-oxidizing activity of cells from Nitrohacter sp. attached to anion-
exchange resins in a continuous-flow air-lift column fermenter is found to be
less than that of freely suspended cells under the same substrate
concentrations. This may be caused by nutrient limitation due to diffusion
through the biofilm or to the effect of abrasion on cell viability. Although
statistically significant, the effect of surface attachment on growth parameters
is not dramatic but has important effects on the response of nitrifiers to low pH
values and to inhibitors, as will be seen in later sections.
It is remarkable, however, that organisms growing under energy limitations
should direct efforts to production of large quantities of EPS material. Clearly,
production of this material must provide significant advantages. The
particular continuous-flow systems studied provide very selective conditions,
in that cells without the ability to remain attached will be washed out quickly.
Attachment therefore provides stability and maintenance of the community
and the ability to respond quickly to changing conditions. These will be
important factors in natural environments but in the soil a major function of
EPS may lie in providing resistance to desiccation stress, thus increasing
survival. Cultures recently isolated from soil frequently produce capsular
material but this material, and the ability to produce it, appear to be lost with
continued laboratory subculturing. Production of EPS is therefore an area
requiring study both to determine its function and to determine the
mechanisms for regulation and control of its production in organisms for
whom fixation of carbon dioxide is so expensive in terms of energy.

VII. The Effect of Oxygen and Light on Nitrification

A. INHIBITION OF NITRIFICATION AT HIGH OXYGEN CONCENTRATION

Nitrification is classically described as an aerobic process with molecular

oxygen required for oxidation of ammonia and for respiration in both
ammonia and nitrite oxidizers. At high concentrations, oxygen is inhibitory.
Gunderson (1966) found inhibition of colony growth of Nitrosocystis oceanus
on solid medium in the presence of pure oxygen, poor growth in the presence
of air and rapid colony formation only at low partial pressures of oxygen.
Growth was optimal at a partial pressure 10% that of air. Although growth
was inhibited by high oxygen concentrations, the respiration rate was
increased. High oxygen partial pressure also inhibits growth of Nitrohacter
species and leads to an increase in polyphosphate pools. Growth on solid
media requires low oxygen partial pressures, and free-radical formation is
suggested as the mechanism of oxygen inhibition.
 AUTOTROPHIC NITRIFICATION IN BACTERIA 149
Oxygen inhibition is of practical significance in activated sludge systems
supplied with pure oxygen rather than with air. Inhibitory effects in
laboratory-scale activated-sludge units are manifest at higher oxygen
concentrations (Painter and King, 1976; Charley et al., 1980; Jones and
Paskins, 1982).This appears, however, to be due to reduced removal of carbon
dioxide leading to reduction in pH value. Jones and Paskins (1982)also found
that nitrifiers could become acclimatized to high concentrations of oxygen but
not to sudden changes from normal to high levels, which could lead to
washout of these organisms.

B. PHOTO-INHIBITION

Oxygen is also implicated in the sensitivity of nitrifying bacteria to light. The

bactericidal effects of visible blue, and long-wavelength ultraviolet, light were
first noted by Muller-Neugluck and Engel (1961).Schon and Engel (1962)and
Bock (1965)found strains of Nitrohacter to be much more sensitive than those
of Nitrosomonas and suggested that photo-oxidation of cytochrome c was the
mechanism for inhibition by light. Hooper and Terry (1974) found reduced
sensitivity to photo-inhibition in the absence of oxygen, when ammonia
oxidation was completely inhibited, or in the presence of high concentrations
of ammonia or hydroxylamine. Sensitivity was greatest when flux through the
ammonia hydroxylamine-nitrite pathway was low, e.g. due to low ammonia
concentration, endogenous metabolism, decreased temperature or partial
inhibition of ammonia oxidation. Shears and Wood (1 985) presented
spectroscopic evidence for the existence, in resting cells, of an oxygenated state
of the ammonia mono-oxygenase with a broad ultraviolet absorbance band.
They suggest that ultraviolet absorption results in an excited state giving rise
to an active form of oxygen which destroys the enzyme.
Light inhibition is significant in surface waters and Olson (1981b) found
50% inhibition of ammonia and nitrite oxidizers at light intensities
approximately three orders of magnitude less than the intensity of full
sunlight. This has important implications for nitrification in such environ-
ments. Firstly, it reduces competition for ammonium in surface waters
between nitrification and assimilation by phytoplankton. Secondly, in aquatic
environments the differences in the degree of inhibition of ammonia and
nitrite oxidation have been proposed to result in spatial separation of the two
processes and accumulation of intermediates leading to a primary nitrite
maximum (Olson, 1981b; Ward, 1986).Nitrite maxima of this type have not
been observed in lake environments (Hall, 1986), but an alternative
explanation for their occurrence in estuarine environments based on
differences in affinity for oxygen is presented below.
An important consideration in natural environments is the ability to
150 J. I. PROSSER

recover from inhibition during dark periods. Hooper and Terry (1974)
reported recovery of N . europaea from 90% photo-activation within a
minimum period of 10h and Alleman et ul. (1987) found faster recovery
(2.5-3 h) in a mixed culture system. Yoshioka and Saijo (1985) subjected
ammonia and nitrite oxidizers to 12 h light/dark cycles and found reduced
recovery from photo-inhibition in the absence of ammonia and differences in
sensitivity between ammonia and nitrite oxidizers, with the latter more
sensitive during short-term experiments. They also found that following
illumination for 7 days, recovery from photo-inhibition in the absence of
ammonia did not occur until after 120 days. The extent of photo-inhibition
and the length of the recovery period depend on the intensity of the
illumination, the attenuation properties of the water and circulation of water
within the hypolimnion. The combined effects of these factors are discussed by
Hall (1986).

C. NITRIFICATION AT LOW OXYGEN CONCENTRATIONS

Low concentrations of oxygen reduce rates of nitrification and the dual effects
of substrate and oxygen limitation have been modelled using double-
substrate-limiting kinetics (Sharma and Ahlert, 1977). Saturation constants
for oxygen for pure cultures of ammonia and nitrite oxidizers lie in the range
0.25-2.5 mg dissolved oxygen 1 - (Painter, 1986) (Table 4), with similar values
reported for mixed-culture-activated sludge systems. The Ksvalues are higher
than those for heterotrophs, and nitrifiers are therefore likely to be poor
competitors for oxygen at low oxygen concentrations. This is significant in
two situations. Firstly, heterotrophic nitrifiers have a lower K , value for
oxygen, providing them with a competitive advantage in low-oxygen
environments. Secondly, competition for oxygen may be important where
nitrifiers are a component of mixed biofilms. In addition, Megraw and
Knowles (1987) found that the co-existence of nitrifiers and methanotrophs in
soil was only possible at high levels of oxygen and ammonia. At reduced
oxygen levels, nitrifiers were outcompeted due to a combination of higher K,,,
value for oxygen, lower maximum specificgrowth rate and lower growth yield.
Under ammonium limitation, the high assimilation requirement of the
methanotrophs again led to their dominance.
The effect of oxygen on the marine nitrifier Nifrosococcus oceunus varies
with ammonia concentration and is not represented by simple double-
substrate-limitation kinetics (Ward, 1987), with inhibition of ammonia
oxidation by oxygen at high ammonia concentrations (greater than 2 0 ~and ~ )
activation at low concentrations (Fig. 4). Ammonia oxidation in marine
environments, where substrate concentrations will be low, may therefore be
enhanced by reduced oxygen levels.
 AUTOTROPHIC NITRIFICATION IN BACTERIA 151

12 I"

I I I I
0 .02 .04 .06 .00
I/ IS1 (wM NHf)-'
FIG. 4. a, Effect of reduced oxygen concentration on ammonia oxidation by
Nitrosococcus oceanus. b, Lineweaver-Burk plot of data in part a. Key: 0 , control
(oxygen concentration =4.88 ml I-'); 0,reduced oxygen concentration (1.23ml I-').
From Ward (1987) with permission.

There is some evidence that ammonia oxidizers have greater saturation

constants for oxygen than nitrite oxidizers. Helder and de Vries (1983) suggest
that ammonium oxidation is inhibited below 30pmol 0, 1 - ' while nitrite
',
oxidation is inhibited below 125pmol0,l- These differencescan give rise to
spatial separation of these two groups in attached biofilms limited by supply
152 J . 1 PROSSER

of oxygen by diffusion. Tanaka and Dunn (1982) modelled this situation using
multiple substrate kinetics and predicted accumulation of Nitrobacter species
below Nitrosomonas species in biofilms carrying out nitrification. Limited
experimental evidence was presented, based on respirometric measurements
in the presence of ammonium or nitrite, for greater nitrite-oxidizing activity
and lower ammonia oxidizing with increasing biofilm depth in a mixed-culture
fluidized-sand-bed reactor.
Helder and de Vries (1983) found a similar effect in an estuarine
environment. Strains of Nitrosomonas and Nitrohacler isolated from an
estuarine sediment were grown together in continuous culture at a range of
oxygen concentrations. At concentrations greater than 95 mmol 0, 1 - ',
ammonium and nitrite concentrations were negligible. As oxygen con-
centration was reduced below this level, firstly nitrite and then ammonia
appeared in steady state culture fluids. This implies a higher K , value for
oxygen for nitrite oxidizers and explains the occurrence of a nitrite maximum
in the surface waters of the estuary from which these organisms were isolated.
It may also explain the occurrence of primary nitrite maxima in marine surface
waters discussed above.

D. REDUCTION OF NITRITE A N D NITRATE BY NITRIFYING BACTERIA

The physiological and metabolic activities of both ammonia and nitrite

oxidizers change significantly at low oxygen concentrations, with important
environmental consequences. Both ammonia and nitrite oxidizers are capable
of reversing what are considered to be their natural processes by reducing
nitrite and nitrate.

1. Reduction of Nitrite by Ammonia Oxidizers

Production of nitrous oxide has been observed in the genera Nitrosomonas,
Nitrosolobus, Nitrosospiru and Nitrosocystis (Goreau et al., 1980) and is
presumably a general property of ammonia oxidizers. Ritchie and Nicholas
(1972) observed that N. europaea produced labelled 1 5 N 2 0from either lSN-
labelled ammonia, nitrite o r hydroxylamine. Subsequently, the presence of a
nitrite reductase was demonstrated in ammonia oxidizers (Ritchie and
Nicholas, 1974; Dispirit0 et al., 1985; Miller and Nicholas, 1985). The
production of nitrous oxide is correlated with nitrification (Hynes and
Knowles, 1984), constituting approximately 0.1YOof nitrogen oxidized under
aerobic conditions, and its production is inhibited by specific inhibitors of
nitrification, e.g. nitrapyrin. In addition, the proportion of nitrous oxide
increases with decreasing oxygen concentration.
Goreau et al. (1980) found yields of 0.1-O.5% total N as N,O for the four
 AUTOTROPHlC NITRIFICATION IN BACTERIA 153
genera of ammonia oxidizers listed above. A reduction in oxygen
concentration from 7 to 0.18 mg I-' greatly increased nitrous oxide yield,
which reached 2.5% of total N in N. europaea. Yields as high as 10% have been
obtained for a marine strain of Nitrosomonas. Cellular rates of production of
N,O and NO; also varied with oxygen concentration from 1-5 x lo-'' nmol
N,O cell-' day-' and 0.47-3.7 x lo-'' nmol NO; cell-' day-'. Lipschultz
et al. (1981) found a correlation between reduced oxygen concentration and
production of both nitrous and nitric oxides and suggested nitrification as a
significant global source of atmospheric nitrogen oxides.
Hynes and Knowles (1984) observed variation in the proportion of N,O to
NO; - N production during growth of N. europaea on a range of initial
ammonium concentrations, and production increased five-fold under
anaerobic conditions. Acetylene, an inhibitor of ammonia oxidation, inhibited
N,O production from ammonium but not from hydroxylamine.
The correlation of nitrous oxide production with nitrification and
production of I5N,O from labelled ammonia and hydroxylamine led to the
belief that nitrous oxide was a byproduct of nitrification, produced by reaction
of intermediates involved in ammonia oxidation, as well as nitrite reduction by
nitrate reductase. Poth and Focht (1985), using isotopic techniques and kinetic
analysis of labelled substrates and products, showed nitrite reduction to be the
sole source of nitrous oxide. Ammonia oxidizers can, therefore, carry out
denitrification, but simultaneous oxidation of ammonia is required as a
source of electrons. Nitrous oxide production occurred only under conditions
of oxygen stress. The authors suggest that the process functions to (a) conserve
oxygen for use by the ammonia mono-oxygenase, (b) reduce production of
nitrite (which may accumulate to toxic levels), and (c) decrease competition
for oxygen by nitrite oxidizers, by denying them their source of substrate.
Wood (1986) suggests that this process may also function to maintain an
optimal redox poise. Additional evidence for nitrous oxide production from
nitrite has come from work on 15Nz0abundance studies by Yoshida (1988).
Poth (1986) has also isolated a strain of N. europaea which, under oxygen
stress, denitrifies nitrite to gaseous nitrogen with little production of nitrous
oxide. Nitrogen gas constituted approximately 0.85% of added labelled nitrite
and this process is similar to that carried out by many aerobic denitrifiers.

2. Nitrate Reduction by Nitrite Oxidizers

In the major nitrite oxidizer genus Nitrobacter, but not in the genus Nitrospira
(Watson et al., 1986), the key enzyme is a nitrite oxidoreductase which is
capable of oxidizing nitrite to nitrate and of reducing nitrate to nitrite in the
presence of reduced methylviologen/benzylviologen or NADH as electron
donor (Tanaka et al., 1983; Sundermeyer-Klinger et al., 1984). During
154 J. I . PROSSER

heterotrophic growth, the enzyme is at high levels in the presence of nitrate but
is repressed when nitrate is absent. It has an apparent K,,, value of 0.9 mM for
nitrate. Bock et al. (1986) reported anaerobic growth of N . winogradskyi with
glycerol as electron donor and nitrate as electron acceptor. Growth, however,
was incomplete, with only 10% glycerol utilized, and was unbalanced in that
80-90% of cell mass consisted of poly-P-hydroxybutyrate (PHB). Growth
limitation was thought to be due to nitrite accumulation and inhibition, but
the authors suggested prolonged anaerobic growth would be possible if nitrite
could be removed.

dissolved
oxygen pyruvate protein
tension Iglll [mgAI

30

20

10

NO; 10 20 30 LO d
NO; NH;
[mM1 ImMl
FIG. 5. Growth of Nitrohacrer species in a gas-tight culture chamber. Liquid medium
contained nitrite, nitrate and pyruvate, and a biofilm was formed on silicone tubing
within the liquid medium. Key: 0, cell protein in the biofilm; 0.
cell protein in the
liquid medium; A,pyruvate; 0 ,nitrite; +, nitrate; A,ammonia; --, oxygen. From
Freitag e/ ul. (1987) with pcrmission.
 AUTOTROPH IC NlTRl FICATION IN BACTERIA 155
This has now been achieved in a biofilm system which allows both
production of nitrite, from nitrate, and its removal by nitrite oxidation
(Freitag et al., 1987; Bock et al., 1988). The biofilm was formed on the outside
of silicone tubing placed within gas-tight bottles containing mineral salts
medium plus nitrite and pyruvate. Air pumped through the tubing provided
oxygen by diffusion and a Nitrobacter-species biofilm developed initially due
to mixotrophic growth on nitrite and pyruvate (Fig. 5). This reduced the
oxygen concentration in the liquid medium, giving rise to an oxygen gradient
across the biofilm and subsequent development of a heterogeneous
population. Cells adjacent to the silicone tubing continued to be supplied with
oxygen and oxidized nitrite, and were seen, in electron-micrograph ultrathin
sections, to contain many carboxysomes. Cells adjacent to the anaerobic
liquid medium were morphologically distinct, containing many large PHB
granules, and were similar to cells suspended in the liquid medium. The growth
of these cells was correlated with production of nitrate and ammonia and
reduction in nitrite. In addition, up to 40% of nitrogen was lost as nitrous
oxide. This is strong evidence, therefore, for growth via reduction of nitrate,
produced by nitrite oxidation, with further reduction to ammonia. It also, of
course, provides an alternative explanation for the accumulation of nitrite
oxidizers beneath ammonia oxidizers in trickling filter biofilms discussed
above.

3. Ecological Implications
The metabolic diversity of nitrifiers under conditions of low oxygen
concentration can have great significance for their r81e within the nitrogen
cycle and their interactions with other groups involved in this cycle. We now
see that in the presence of organic carbon, Nitrobacter species can carry out
nitrification and dissimilatory nitrate reduction, providing ammonia for
ammonia oxidizers under the correct conditions, thereby reversing the whole
process of nitrification and converting commensalism into a form of
mutualism. The production of nitrous oxides is of particular ecological
significance in terms of acid rain and their effect on the ozone layer.
Unfortunately, it is often difficult to determine the organisms responsible, as
denitrification and nitrification may occur in close proximity and denitrifiers
require nitrification as a source of nitrate. There is also evidence for nitrous
oxide production by other microbial groups (Knowles, 1986) and by non-
biological means. Metabolic inhibitors, in particular acetylene, have been used
to distinguish sources of N,O production but the conditions for use are critical
and vary between soils (Davidson et al., 1986; Klemedtsson et al., 1988).
Despite these difficulties, nitrification is believed to be a significant source of
nitrogen oxides in fertilized agricultural soils (Bremner and Blackmer, 1979;
156 J. 1 PROSSER

Blackmer et al., 1980),marine environments (Lipschultz et al., 1981; Ward and

Zafiriou, 1988)and freshwater ecosystems (Downes, 1988).In non-agricultural
forest soils the evidence indicates nitrifiers to be of less significance
(Robertson and Tiedje, 1987).

4. Interactions Between Nitrijiers and Denitrijiers

The interaction between nitrification and denitrification must also be assessed
in the light of advances of our knowledge of nitrifier physiology. These
processes are traditionally considered to be separated spatially as nitrification
is aerobic and denitrification anaerobic. As we have seen, however, ammonia
and nitrite oxidizers can be active at low or zero oxygen concentrations and
produce gaseous intermediates similar to those of denitrification. In addition,
aerobic denitrification is now seen to be feasible (Robertson and Kuenen,
1983).There is strong evidence to show spatial separation of these processes in
natural environments such as estuarine sediments (Keith et al., 1987) and the
sediments of streams(Cookeand White, 1987).Macfarlane and Herbert (1985)
studied the interaction between nitrifiers and denitrifiers in a series of three
chemostat vessels interlinked via membranes which allowed diffusion of
soluble substrates and products between vessels. The first vessel was
maintained anaerobic and was inoculated with a denitrifying Vihrio strain,
supplied with glycerol and nitrate. The second and third vessels were aerobic
and were inoculated with strains of Nitrohacter and Nitrosomonas,
respectively, and were supplied only with mineral salts medium. Under
nitrogen limitation, the Vibrio strain produced ammonium which stimulated
growth of the Nitrosomonas strain and subsequently that of the Nitrohacter
strain, although nitrite production was insufficient for significant growth of
the Nitrohacter strain. Under carbon limitation, nitrite was the major product
leading to growth of the Nitrobacter strain but washout of the .Nirrosomonas
strain. This sort of experiment demonstrates the ability of denitrifiers to
generate substrates for nitrification with diffusion of products across an
oxygen gradient and also the effect of nutritional conditions on denitrification
and on the relative numbers of ammonia and nitrite oxidizers.
The coupling of nitrification and denitrification is of value in sewage
treatment processes requiring complete removal of nitrogen. This may be
achieved in two-stage systems, the first being aerobic and the second
anaerobic, but Kokufuta et al. (1988) devised a single-stage system for both
processes. This involved co-immobilization of N . eurupaea and Paracoccus
denitriJicans using a pol yelectrolyte complex. They obtained simultaneous
nitrification and denitrification, due to the existence of aerobic and anaerobic
regions within the support system. Ammonia was converted through to
gaseous material enabling complete removal of nitrogen. In addition,
 AUTOTROPHIC NITRIFICATION I N BACTERIA 157
ammonia oxidation was faster in the presence of the Paracoccus strain because
of immediate removal of nitrite, thereby preventing inhibition.

VIII. The Effect of pH Value on Nitrification

Both ammonia and nitrite oxidation are considered to be optimal at neutral-

alkaline pH values. Watson (1974) quotes a pH range for pure cultures of
ammonia oxidizers of 5.8-8.5 and a range of 6.5-8.5 for nitrite oxidizers. Most
nitrifiers have pH optima for growth in the range 7.5-8 and grow within a pH
range of approximately 2 pH units. N. europaea is exceptional with a pH range
for growth of 5.8-8.5, reflecting the diversity of strains within this species.
Similar properties are reported for mixed cultures such as those found in
activated sludge processes. Below the optimum pH value, specific growth rate
falls off sharply. Figure 6 shows the effect of pH value on the maximum specific
growth rate of Nitrobacter sp. in pure-batch culture(Keen and Prosser, 1987b).
A maximum specific growth rate of 0.0048 h- was found at pH 7.5. At pH 6
this fell to 59% of the maximum and no growth occurred at pH 5.5. We have
obtained similar data for N . europaea with a maximum specific growth rate at
pH 8, a 54% reduction in pmaxat pH 7 and no growth at pH 6.5.

FIG. 6. Effect of pH value on maximum specific growth rate of Nitrobacter species.

158 J. 1. PROSSER

The response of specific growth rate of nitrifiers to pH value reflects the pK

values for their respective substrates. For NH:/NH, the pK value is 9.25
(Wood, 1988). Evidence has been presented in Section V that ammonia is the
substrate for ammonia oxidation and reduction in pH value will decrease the
concentration of free ammonia by one order of magnitude for each unit
decrease in pH value. For a typical ammonia-oxidizer medium containing
1OOpg NH: ml-' the ammonia concentration will be 5.6,0.56 and 0.056pg
ml-' at pH values of 8, 7 and 6, respectively, explaining the sharp cut-off
effects discussed above. Reduced ammonia availability will only affect specific
growth rate if the cell must expend increasing amounts of energy on
maintenance of internal pH value and/or if ammonia is the form in which
substrate is transported into the cell. If ammonia is transported by passive
diffusion, the rate of uptake will be reduced at low pH values due to a
decreased concentration gradient across the cell membrane. If an energy-
dependent ammonium uptake mechanism exists, the increased maintenance
energy demand required to maintain internal pH, in addition to that required
for uptake, may leave insufficient energy for growth. There is also evidence
that reduction in pH value affects enzyme activity and not just ammonia
availability (Glover, 1982).
Above the optimum pH value for growth, the advantages of increased
availability of free ammonia are counterbalanced by the need to maintain an
internal pH value below that of the external medium (Wood, 1987).
The above discussion implies that the optimum pH value for growth will
depend on ammonium Concentration. Quinlan ( 1984)presented a quantitative
theoretical model relating optimum pH value and ammonium concentration
based on the assumptions that (a) ammonia was the substrate for ammonia

+
kl ,

11. Ilr(
EH S
' k2

Sd EH+S

FIG. 7. Proposed mechanisms for the effect of pH value on the kinetics of ammonia
oxidation. Redrawn from Quinlan (1984).
 AUTOTROPHIC NITRIFICATION IN BACTERIA 159
oxidation, (b) substrate ionization occurred, and (c) ammonia oxidation was
controlled by ampholytic ionization of the rate-limiting enzyme substrate
(EHS) complex, but not of the free enzyme (EH) (See Fig. 7).
The values k , and k , represent forward and reverse rate constants for the
formation of enzyme substrate complex and k , is the rate constant for
irreversible product formation. The constant K3 represents reversible and pH-
dependent ionization of NH, to NH:, and is consequently related to pK,
while K, and K, represent ionization of the ES complex. A mathematical
model based on this hypothesized mechanism was used to describe the effects
of pH value on enzyme velocity and on the saturation constant. Predictions
provided a good fit to experimental data of Suzuki et al. ( 1 974) and Laudelout
et al. (1976) on the effectsof pH value on whole cells and cell-free extracts and
allowed calculations of values for pK,, pK, and pK,. Values of K, derived
from these experimental data compared well with independent estimates of
K,, providing support for the proposed scheme. Further analysis of the model
allowed prediction of the effect of ammoniaconcentration on the optimum pH
value for growth (pH,) which is described by the equation:

where N is the total nitrogen concentration and K$ = (K,/K,)K,. Experi-

mental data described above indicated values for K: sufficiently large to
predict a linear decrease in pH, with a logarithmic increase in ammonia
concentration over the pH range 1-50pgml-'.
The pK, value of nitrite is 3.15 and inhibition at high pH values results from
competitive inhibition by hydroxyl ions (Boon and Laudelout, 1962). At acid
pH values, inhibition occurs through increased formation of nitrous acid (see
Section V.B.). In addition, as pH values decrease below 4, significant chemical
decomposition of nitrite occurs with formation of nitrogen oxides. Laudelout
et al. ( 1 976) incorporated kinetics associated with these proposed mechanisms
for pH effects, and those of oxygen concentration, into a mathematical model
for nitrification and found good agreement between predicted and
experimental results. In particular, the model correctly predicted the effect of
aeration on transient increases in nitrite concentration.
Despite the neutrophilic/alkalophilic growth of pure cultures of nitrifying
bacteria in liquid medium, nitrification in acid soils is well documented (Weber
and Gainey, 1962; Vitousek et al., 1982; Wickramasinghe et al., 1985; Klein et
al., 1983; Federer, 1983; Adams, 1986).Four explanations have been proposed
for this phenomenon: (a) the existence of acidophilic strains, (b) surface
growth, (c) the existence of micro-environments of neutral pH value, and (d)
heterotrophic nitrification. Each of these will now be discussed in turn.
1 60 J. I. PROSSER

A. DO ACIDOPHILIC STRAINS OF NITRIFYING BACTERIA EXIST?

1. Ammonia Oxidizers
On the basis of energetic considerations discussed above, and on the effects of
pH value on ammonia availability, the existence of acidophilic ammonia
oxidizers would appear unlikely. However, ammonia oxidizers are frequently
isolated from acid soils in which nitrification takes place. Strains of Nitro-
sospira, Nitrosomonas and Nitrohacter were isolated by Bhuiya and Walker
(1977) from acid tea soils (pH 5.0-6.2) and Walker and Wickramasinghe
(1979) found a Nitrosospira strain to be the only isolate from acid tea
soils in Bangladesh (pH4.0-4.5). Sri Lankan soils of similar pH values also
contained strains of Nitrosolobus and Nitrosouibrio. Martikainen and
Nurmiaho-Lassila (1985) and Hankinson and Schmidt (1984) also found
Nitrosospira to be the dominant genus in coniferous forest soils. Other strains
including Nitrosouibrio have been isolated by Vitousek et al. (1982) from an
acid forest soil. In our laboratory we have isolated a similar broad range of
ammonia-oxidizing organisms from soils of pH value down to 2.5 and from
one soil maintained at pH 4.0-4.5 for 2-5 years (Allison, 1989).
In addition, nitrification in some acid soils is inhibited by inhibitors of
autotrophic nitrification. For example, Wickramasinghe et ul. (1985) found
inhibition of nitrification in acid tea soils (pH 4.0-4.3) by nitrapyrin and
dicyandiamide (DCD)and Killham (1986) reported inhibition by acetylene of
nitrification in arable soils of pH 4.5.
All of the strains isolated from acid soils are neutrophilic or alkalophilic with
pH ranges and optima for growth typical of ammonia oxidizers from other
environments. Acidophilic strains of ammonia oxidizers have, therefore, not
been isolated in pure culture and none have been reported in enriched cultures.
Many of these studies may be criticized for use of isolation media of neutral pH
value, but we have been unable to isolate ammonia oxidizers on media of low
pH value even after incubation for several months. It is, however, possible that
specific, as yet undiscovered conditions are required for successful isolation.
The inability to isolate acidophilic ammonia oxidizers, coupled with plausible
explanations for their lack of existence, indicate that they are not responsible
for nitrification in acid soils.

2. Nitrite Oxidizers
The same would have been said with regard to nitrite oxidizers until isolation
by Hankinson and Schmidt (1984) of acidophilic and acid-tolerant strains of
Nitrobarter dominant in acid forest soils of pH 4.3-5. Isolation was achieved
by carrying out most-probable-number counts in liquid media of pH 7 and 5,
enabling simultaneous isolation and enumeration and subsequent assessment
 AUTOTROPHIC NITRIFICATION IN BACTERIA 161
of the importance of each isolated strain. In addition, nitrite concentration
was maintained below 0.1 PM to prevent nitrite toxicity at low pH values. The
acid-tolerant strain grew at pH values down to 5.5 with an optimum of 7.2,
while the acidophilic strain grew between pH values of 4.6 and 7, but not at pH
8, with optimal growth at pH 5.5. At low pH values the acidophile exhibited
signs of stress, including cell lengthening and branching, but nitrite oxidase
activity was demonstrated at pH values down to 3.5. In terms of nitrite
oxidation, therefore, acidophilic autotrophic strains may be responsible for
the second stage of nitrification in acid soils.

B. PROTECTION FROM EFFECTS OF pH BY SURFACE GROWTH

The effect of surface attachment on the growth and activity of nitrifiers has
been discussed in Section 1V.C. Lees and Quastel (1946)provided evidence for
the association of nitrifiers with the surface of particles in soil, but McLaren
and Skujins (1963) were the first to consider the significance of surface
attachment for pH optima for nitrification. They compared the rate of nitrite
oxidation by a pure culture of Nitrobacter sp. in liquid culture with that in
either soil or negatively charged ion-exchange resins perfused with nitrite.
Relative rates of nitrification by attached populations were found to be less
than those in liquid culture over the pH range 5.5-7 and the pH value for half
the maximum rate of nitrite oxidation was 0.5 pH units higher in soil. This
difference was explained by inhibition caused by localized increases in pH
value due to adsorption of H + ions or reduced substrate availability due to
repulsion of NO; ions by the negatively charged particles. The situation for
ammonia oxidation is more complex as both substrate (NHf) and Hf ions
will be attracted to soil particles (see below).
Keen and Prosser (1987b) studied the combined effects of three
physiological factors, relevant to soil nitrification, on nitrite oxidation by a
Nitrobacter species: (a) low substrate concentration, (b) surface growth, and (c)
whether cells were actively growing or in stationary phase. In liquid batch
culture the optimum pH for growth was 7.5 but cells did not grow below pH 6
in standard liquid culture media containing 50pg NO; - N ml-'. At lower
initial nitrite concentrations (5-40pg NO, ml- '), however, growth occurred
down to pH 5.5, but not below this value. This can be explained by reduced
toxicity through nitrous acid and is relevant to the methodology used by
Hankinson and Schmidt (1985) for isolation of the acidophilic Nitrobacter
strain. A similar effect was found in nitrite-limited chemostat cultures with
growth down to pH 5.5 at a dilution rate of 0.016 h-' (33% of the maximum
specific growth rate in batch culture) but not at pH 5.
Unlike Mclaren and Skujins (1963), Keen and Prosser found no differences
between the pH profiles for growth of suspended cells and colonized glass
162 1.1 PROSSER

coverslips, possibly due to differences in the degree of negative charge on the

surface. Specific growth rate was, however, stimulated by the presence of glass
coverslips.
Growth of a Nitrohacier strain was also studied on positively charged
anion-exchange resin beads which, as discussed earlier, provide a better
substratum for growth due to attraction of nitrite and, possibly, repulsion of
H f ions. Initial attachment to glass was unaffected by pH value but
attachment to anion-exchange resin beads increased sharply with increased
pH. This is thought to be due to an increase in the negative cell surface charge
with pH.
The effect of pH on attached cell populations was studied further in a
heterogeneous continuous flow system in which cells were present as a biofilm
on ion-exchange resin beads suspended and circulated within liquid medium.
Under these conditions, nitrite oxidation was possible down to a pH value of
4.5, that is 1.5 units lower than in batch culture. Colonized beads removed
from this continuous flow culture at pH 5 were unable to carry out nitrite
oxidation in batch culture at pH values less than 6. Attached cells were
contained within a glycocalyx, confirming earlier work of Cox et al. (1980) on
growth of nitrifying bacteria on glass beads in a continuous-flow packed-
column reactor.
Thus, it is possible for actively growing, surface-attached cells, supplied
continuously with a low concentration of nitrite to exhibit high rates of nitrite
oxidation at pH 4.5. Experiments with glass slides indicate that localized pH
effects, i.e. concentration of Hf by the resin beads, may not be important and
that attraction of substrate (NO;) is a more significant factor than tolerance
to low pH values. In soil, where the majority of particles are negatively
charged, this effect will be reversed and there is in fact some evidence that cells
of the Niirohacter strain may be less firmly attached than those of the
Niirosumonas strain to such surfaces.
In the soil, ammonia will be adsorbed to clay minerals and humic material.
This concentration of substrate offers the potential for increased rates of
ammonia oxidation, but this effect may be counteracted by localized increases
in H + concentration a t the same anionic sites. In fact, comparison of specific
growth rates of pure cultures of N . eurupaea inoculated into sterile soil with
specific growth rates in liquid medium indicate little stimulating or inhibitory
effects caused by surface properties (Powell and Prosser, 1986a). Surface
properties are, however, important when considering pH effects, and work on
nitrification in the presence of purified clay minerals is of relevance.
Goldberg and Gainey (1955) studied the effect of several clays, of differing
bufferingcapacity and saturated with K or NHZ, on nitrification by enriched
+

cultures of nitrifiers. They demonstrated that oxidation of ammonia only

occurred after release of NH; into the liquid medium. They also found
 AUTOTROPHIC NITRIFICATION IN BACTERIA 163
incomplete oxidation of adsorbed ammonia, a proportion of which appeared
to be “fixed” by the clay minerals. Powell (1985) studied the kinetics of nitrite
formation by N . europaea in the presence of clay minerals. A single phase of
nitrification was observed in the presence of illite while vermiculite and
montmorillonite (both expanding clays) gave rise to a second phase of nitrite
production at a lower specific rate. This may be explained by the existence of
two “pools” of ammonia, one of which is less available, producing lower
concentrations of dissolved ammonia.
Although the available evidence indicates dissolved rather than fixed
ammonia as substrate for ammonia oxidation, adsorption of clay minerals
may provide localized buffering of surface-associated ammonia oxidation,
reducing limitation due to acid production. Armstrong and Prosser (1988)
investigated growth of N . europaeu in batch culture in the presence and
absence of ammonia-treated vermiculite(ATV),prepared by passing anhydrous
ammonia through vermiculite (Ahlrichs et al., 1972). In complete inorganic
’
medium containing 50 pg NH: - N ml- and inoculated with N . europaea, a
’
yield of 5.6 pg NO; - N ml- was obtained before growth ceased due to acid
production (Table 5). The presence of ATV did not significantly change this
final nitrite yield and did not buffer the medium, which again became acidified.
The ATV did, however, reduce the maximum specific growth rate and
abolished the lag phase before ammonia oxidation. In liquid medium in which
ATV provided the only source of ammonia, the final nitrite concentration
increased significantly to I6 pg NO; - N ml- and medium was not acidified.
Limitation here is thought to have been due to exhaustion of freely available
ammonia, as some will be unavailable in the internal layers of the clay
particles. An alternative possible limiting factor is localized reduction in pH
value.
These data imply that ammonia derived from adsorption sites on the clay
mineral, probably following diffusion into liquid medium, is exchanged for
H + ions produced during ammonia oxidation. Further, in terms of ammonia

TABLE 5 . Growth parameters of Nitr0.WJm~JnU.Seuropuc.a in the presence and absence of

ammonia-treated vermiculite (ATV) (Armstrong and Prosser. 1988)

Final nitrite
Specific growth Duration of concentration Final pH
rate (h-’) lag phase (h) (pgNO; - N ml-’) value

Complete medium minus ATV 0.060 10 5.6 < 7.0

Complete medium plus ATV 0.045 0 6.0 < 7.0
Incomplete medium plus ATV 0.038 0 16.0 > 7.0

Complete and incomplete media contained 50 and Opg NH: - N ml-’, respectively. The pH
values of the media were assessed by change in colour of the indicator phenol red at pH 7.
164 J. I. PROSSER

oxidation, these H ions were effectively immobilized and the only noticeable
+

effect was a decrease in the maximum specific growth rate to approximately

50% of that in liquid culture. This may merely reflect the low concentration of
ammonia in the liquid medium, released by the clay mineral, which is the direct
source of substrate. This, therefore, provides a mechanism for ammonia
oxidation in acid soils on a localized scale and links with the consideration of
microsites, below.
Protection may also be provided by EPS material described here and in
other studies, but the precise mechanism of this protection is unclear and
much more detailed and critical studies are required before this can be
considered seriously. Virtually nothing is known of the chemical composition
of EPS material nor of the means by which it might give protection. It is
therefore easy to provide it with any properties which are considered desir-
able. Biofilms do, however, increase stability by preventing removal of
cells which is particularly important in sewage treatment processes and may
permit high rates of activity not necessarily associated with growth.
There is now evidence (Allison, 1989) that the pH minima for growth and
activity of ammonia oxidizers may be significantly lower in soil than in liquid
batch culture. A continuous-flow sand column, inoculated with N . europuea,
and supplied continuously with ammonium, was capable of significant
nitrification at pH 5.7. The same strain in liquid culture did not grow below pH
7. Ammonia oxidation was not possible at pH 5.5 or lower and surface growth
and protection does not provide a complete explanation for autotrophic
nitrification in soils of pH 4. It does, however, significantly lower the pH
minimum for ammonia-oxidizing activity.

C. MICRO-ENVIRONMENTS AND UREASE ACTIVITY

The existence of micro-environments of alkaline or neutral pH value has been

suggested as a possible explanation for autotrophic nitrification in acid soils.
Unfortunately, as when invoking protection by surface growth, it is easy to
postulate the existence of micro-environments with any required property but
difficult to determine their existence and more importantly their significance.
To an extent, a biofilm provides a micro-environment where organisms may
be protected or buffered from the effects of low pH value. A major feature of
biofilms is their spatial heterogeneity and organization which can lead to
development of communities with distinctive properties not found in spatially
homogeneous environments.
Molina (1985) provided a theoretical basis for the study of soil micro-
aggregates and the effect of microsites on the kinetics of nitrification. Soil was
considered to consist of an infinite population of micro-aggregates, each
containing a cluster of ammonia oxidizers. Ammonia oxidation within a
 AUTOTROPHIC NITRIFICATION IN BACTERIA 165
cluster continued until limited by acid production. Nitrification therefore
consisted of numerous asynchronous pulses of ammonia oxidation, the
asynchrony arising through variability in the length of lag phases before
oxidation commenced. The kinetics of nitrification, as measured by increases
in nitrite concentration, represented accumulation of nitrite produced by the
whole population of micro-aggregates, and clusters, and was related to the
distribution in length of lag phases. Virtually nothing is known of the
physiological factors determining the lengths of lag phases for ammonia or
nitrite oxidizers.
Molina (1985) tested this theory by sieving soil, to obtain micro-aggregates,
which were then inoculated individually into ammonia-oxidizer liquid
medium, with growth assessed by testing for acid production. His data
explained observed “gross” kinetics for nitrification in soil and suggested that
the soil contained 56 clusters of ammonia oxidizers per gram. In addition, the
specific rates of nitrite production calculated from his data were lower than
those observed in liquid culture. A similar approach has been adopted by
Darrah et al. (1987) to explain variation in the observed kinetics of nitrate
formation by soils of different pH value.
Overrein (1967) and Hankinson and Schmidt (1985) suggested that close
association between nitrifiers and heterotrophic organisms mineralizing
organic nitrogen may allow nitrification in acid soil microsites. Ammonia
released by mineralization would raise the local pH value with subsequent
reduction in pH value through nitrification. An increase in the numbers of
autotrophic nitrifying bacteria in an acid forest soil following addition of
nitrogen, phosphorous and potassium fertilizers, even when soil pH remained
at 4.5, was observed by de Boer et al. (1989a). Inhibitor studies showed
nitrification to be autotrophic and organisms isolated were not acidophilic. In
addition, nitrification appeared to be directly coupled to mineralization, as in
soil suspensions of both acid and neutral pH values, the onset of nitrate
production corresponded with the onset of net ammonia production when
nitrification was inhibited by the addition of nitrapyrin. The delay in
nitrification was not due to substrate limitation, as sufficient ammonia was
present, nor due to an increase in pH value, but appeared to involve the close
proximity of mineralizing and nitrifying organisms.
Further evidence came from the observed stimulation of nitrification in soil
suspensions supplemented with urea. Again, this stimulation did not result
from an increase in pH value but was related to the previously undiscovered
presence of urease activity in ammonia oxidizers. An ammonia-oxidizing
strain isolated from acid soil, when incubated at pH 4.3 in the presence of
Nitrobacter (to reduce nitrite toxicity), resulted in complete conversion of
1 mM urea to nitrate. During this process, pH rose to 6.6 as urea hydrolysis
occurred and then returned to pH 4.5 as nitrification became dominant.
166 J ILPROSSER

Although ATCC strains of Nitrosomonas and Nitrosospira showed no

urease activity under the conditions tested, strains isolated from other acid
soils (Allison, 1989) have since been shown to have similar properties.
Consequently, de Boer et al. (1989b) suggested that urease activity may
provide cells with an alternative source of ammonia which is particularly
important at low pH values. Urea may enter the cell by passive diffusion and
may accumulate to higher intracellular levels than ammonia at low pH values.
Urease activity, producing intracellular or extracellular ammonia, will then
provide a source of ammonia, simultaneously and locally increasing the pH
value.

D. HETEROTROPHIC NITRIFICATION

Nitrification is now defined as the oxidation of reduced N compounds rather

than restricting the definition to oxidation of ammonium. This broader
definition encompasses heterotrophic nitrification which is reviewed
extensively by Verstraete (19759, Focht and Verstraete (1977) and Killham
(1986). Heterotrophic nitrifiers include a wide range of fungi, actinomycetes
and bacteria, with most experimental work being carried out on aspergilli,
streptomycetes, Alcaligenes species and arthrobacters. Fungi are generally
considered to be the most efficient and most numerous of these groups in soil.
Substrates include ammonium, hydroxylamine, hydroxamic acids, amino or
oxime nitrogen, aliphatic and aromatic nitro compounds, and nitrite. The
products are nitrite, nitrate and a wide range of nitrogenous organic
compounds. In no case has heterotrophic nitrification been shown to be
associated with energy production or growth and the process is described as
endogenous or secondary metabolism.
The products of heterotrophic nitrification provide the only clues to its
function in these organisms (Focht and Verstraete, 1977). They include
hydroxaminic acids which can act as growth factors and have been implicated
in iron uptake. On the other hand, many of the products are toxic and may
provide a competitive advantage in natural environments. Killham (1989)also
suggests heterotrophic nitrification may enable oligotrophic growth of fungi
in coniferous forest soils.
Two biochemical pathways have been proposed and may act in
combination (Fig. 8). The inorganic pathway (Aleem, 1975) is essentially a
combination of those carried out by individual autotrophic organisms, with
oxidation of organic N to hydroxylamine, nitroxyl, nitrite and nitrate. The
organic pathway (Doxtader, 1965) is believed to begin with oxidation of an
amine or amide to a substituted hydroxylamine, with subsequent oxidation to
nitroso and nitro compounds and finally to nitrate. At each stage this pathway
can link with the inorganic pathway, e.g. by release of free hydroxylamine. A
 AUTOTROPHIC NITRIFICATION IN BACTERIA 167
NR, A NH,OH --D (HNO) NO;

t t t
R N I 3 . d R N H O H A RNO 4 RNO,
FIG. 8. Proposed inorganic and organic pathways for heterotrophic nitrification.

third mechanism, involving OH- radicals arising from hydrogen peroxide

production and lignin breakdown, has been suggested by Wood (1987).
Complete nitrification may require the involvement of autotrophic nitrite
oxidizers. Castignetti and Gunner (1980, 1982) studied the production of
nitrite from pyruvic oxime and hydroxylamine by an Alcaligenes species, with
subsequent nitrite oxidation to nitrate by Nitrobacter sp. In monoculture,
however, Nitrobacter sp. did not oxidize pyruvic oxime, the presence of which
also inhibited nitrite oxidation. In addition, Nitrobacter sp. was very sensitive
to levels of hydroxylamine which were tolerated by Alcaligenes strains. In co-
culture, despite the presence of pyruvic oxime and high levels of hydroxlamine,
the Nitrobacter strain remained viable and oxidized nitrite to nitrate. In
addition to providing an explanation for the relatively high populations of the
genus Nitrobacter in comparison to those of Nitrosomonas found in the soil,
this work raises important questions regarding interactions of nitrifiers with
other organisms involved in the nitrogen cycle. This close interaction is
similar in some respects to that between ammonifiers and nitrifiers discussed
above and requires new experimental approaches for more detailed study.
Rho ( 1 986) also reported an interaction between a heterotrophic nitrifier,
Arthrohacter sp., and a Corynehacterium species, isolated from an estuarine
environment. In medium containing ammonium, acetate and inorganic salts,
the Arthrohacter species produced low, and the Corynehacterium species
negligible, amounts of nitrite and nitrate. In co-culture, production of nitrite
and nitrate was an order of magnitude greater than by Arthrobacter species
alone. This stimulation occurred for both resting and actively growing cells. In
monoculture, neither organism was capable of growth on media containing
1 mg ml-' of NO; - N or acetaldoxime but both grew in co-culture and
nitrification of acetaldoxime occurred. As with many of the interactions
discussed here, the physiological basis of this particular interaction is
unknown and needs to be determined before its ecological significance can be
assessed.
Rates of nitrification by heterotrophic organisms, measured as nitrite
producer per cell or per unit biomass, are traditionally considered low in
comparison to those of autotrophic organisms. For example, Castignetti and
Hollocher (1984) quote specific activities of 0.066-0.003 pmol N min-' mg
protein- for nitrification of pyruvic oxime and hydroxylamine by
168 J. 1. PROSSER

Alcaligenes species. Although this is the highest rate of heterotrophic

nitrification reported, it is still one order of magnitude less than that carried
out by Nitrosomonas species. The biomass concentrations of heterotrophic
nitrifiers may, however, be several orders of magnitude greater than those of
autotrophs and many, particularly the fungi, can grow at low pH values. Such
organisms have been isolated from acid soils (Remade, 1977; Johnsrud, 1978).
The majority of heterotrophic nitrifiers isolated are not acidophilic, but
Stroo et al. (1986) isolated a fungal nitrifier, Ahsidia cyfindrospora, from an
acid forest soil in which nitrate was produced at pH 3.2-6.1. N o autotrophic
nitrifiers could be isolated but A . cylindrospora produced nitrite and nitrate
from p-alanine at pH values of 4 . W . 8 . Hydroxylamine, hydroxamic acids and
primary aliphatic nitro compounds did not accumulate during nitrite
formation, and addition of sterile soil was required for complete oxidation to
nitrate. This final step may therefore have been non-enzymic. Lang and
Jagnow (1986) also reported isolation of fungi from a forest soil with
capability for heterotrophic nitrification at low pH values.
The significance of heterotrophs appears to depend on a number of factors.
The process of heterotrophic nitrification has been identified in a heath soil
(pH 4.3) (van de Dijk and Troelstra, 1980),a mature coniferous forest soil (pH
5.8) (Schimel et al., 1984) and a larch humus (pH 3.5-4.0)(Adams, 1986).
Heterotrophic nitrification in these studies was indicated by a lack ofeffect of
inhibitors of autotrophic nitrification, lack of stimulation by addition of
ammonium, increased nitrate production after amendment with peptone or
identification of the fate of 'N pools following addition of radio-labelled
ammonium. The discovery of urease activity in ammonia oxidizers
necessitates the reassessment of some of these assumptions and in some acid
soils there are strong indications that nitrification is not due to heterotrophs.
Killham (1987), using acetylene as an inhibitor of autotrophic nitrification,
found nitrification potentials of acid coniferous forests soils to be dominantly
of heterotrophic origin while those of agricultural soils of pH 4.5-7.5 were all
dominantly autotrophic. Killham (1986) discusses this topic more fully and
suggests that factors other than pH value (e.g. the form and mineralization
rate of organic nitrogen) may be significant in determining the relative
significance of autotrophic and heterotrophic processes.
Kreitinger et al. (1985) found stimulation of nitrate production by peptone
in an acid forest soil of pH 3.6-4.0. Nitrapyrin did not inhibit nitrification in
unamended soil, but caused inhibition in soils amended with ammonium,
decreased carbon dioxide fixation and stimulated mineralization. On the basis
of these results, the authors suggest that methylotrophs may have been
responsible for significant levels of ammonium oxidation.
The situation is complicated further by the discovery by Robertson and
Kuenen (1983) of an aerobic denitrifier, Thiosphaera pantotropha, which can
 AUTOTROPHIC NITRIFICATION I N BACTERIA 169
also carry out heterotrophic nitrification. Castignetti and Hollocher (1984)
demonstrated that many common denitrifiers were capable of heterotrophic
nitrification, but some were considered poor nitrifiers. Thiosphaera panro-
tropha produces nitrite from ammonia, hydroxylamine and urea (Robertson
and Kuenen, 1984,1988)with both nitrification and denitrification maximal at
dissolved oxygen concentrations approximately 30% of that of air. Correct
evaluation of the ability of such organisms to nitrify requires full nitrogen
balances to be carried out, as nitrite will only accumulate when denitrification
(i.e. nitrite reduction) is prevented. Kuenen and Robertson (1987) suggest that
all aerobic denitrifiers are heterotrophic nitrifiers, although the level of oxygen
at which denitrification ceases varies between species. The discovery of
organisms such as T.pantotropha which can carry out several processes within
the nitrogen cycle previously considered distinct impacts on the way in which
we measure and perceive nitrogen-cycle processes and the organisms involved.
It highlights the versatility of heterotrophic and autotrophic nitrifiers and
demonstrates their previously unrecognized metabolic potential. Physiolog-
ical studies can provide information on the conditions required for expression
of these different metabolic potentials but assessment of their significance in
natural environments provides a much greater challenge.

IX. Inhibition of Nitrification

Inhibitors of nitrification serve three practical purposes. Firstly, their

application along with ammonia-based agricultural fertilizers can reduce
economic losses and pollution due to denitrification or leaching of nitrate
formed by nitrification. Secondly, they are used as metabolic blocks in
ecological studies, to enable distinction of different processes within the
nitrogen cycle. Thirdly, inhibitors are important in sewage treatment
processes where they may result in washout of nitrifiers from suspended
biomass systems.
Inhibitors are listed by Hauck (1980) and Bremner (1986) and many
pesticides and plant products are also inhibitory (Goring and Laskowski,
1982;Sahrawat and Keeney, 1985).The majority of inhibitors are specific to
ammonia oxidation, although often inhibiting nitrite oxidation at high
concentrations. The most widely used commercial inhibitor is nitrapyrin or N-
Serve (2-chloro-6-(trichloromethyl)pyridine) (Goring, 1962). Its major dis-
advantage is its low solubility in water but it is an effective inhibitor of soil
nitrification and is widely used in ecological studies to inhibit specifically
autotrophic nitrification. Etridiazol (5-ethoxy-3-trichloromethyl-1,2,4-tri-
adiazole) and dicyandiamide (DCD) have also attracted commercial interest.
The former has fungicidal properties while DCD acts as a slow-release
170 J I PROSSER

nitrogenous fertilizer. Inhibition by allylthiourea is occasionally preferred to

nitrapyrin because of its greater water solubility (Hall, 1986).
Two groups of inhibitors are based on gaseous compounds. Xanthates act
by releasing carbon disulphide, the most useful compound being potassium
ethyl xanthate (Ashworth et al., 1979). Their initial discovery arose through
inhibition of nitrification in experiments for which water was collected
through rubber tubing, which releases carbon disulphide (Powlson and
Jenkinson, 197 1). Inhibition of ammonia oxidation by acetylene was discussed
in Section 1V.A and the effectiveness of non-gaseous substituted acetylenes
has been studied (Bremner, 1986). Greatest inhibition of soil nitrification was
produced by 2-ethynylpyridine and phenylacetylene, the former being as
effective as nitrapyrin and etridiazole.
Chlorate is considered an inhibitor of nitrite oxidation, although the true
inhibitor is thought to be chlorite, ClO,. Chlorate is an alternative substrate
and competitive inhibitor of nitrate reductase in Nitrohacter species (Straat
and Nason, 1965) and can act as a terminal electron acceptor for nitrite
oxidation of Nitrohacter species in the absence of oxygen (Hynes and
Knowles, 1983). Nitrohacter species therefore convert chlorate to chlorite
which then inhibits nitrite oxidation. Importantly, Hynes and Knowles
demonstrated that N . europaea is 50 times more sensitive to chlorite than a
Nitrohacter strain. In co-culture, therefore, inhibition was not specific to
Nitrobacter strains, and N . europaea was inhibited by chlorite produced by the
nitrite oxidizer.
Keeney (1986) has recently reviewed the factors affecting inhibition in soil
and Oremland and Capone (1988) discuss the use of inhibitors, particularly
nitrapyrin and acetylene, as metabolic blocks. Suggestions have been made for
a r81e for naturally occurring nitrification inhibitors in allelopathic inhibition
of nitrification in climax ecosystems. Rice and Pancholy (1972, 1973, 1974)
proposed that phenolic compounds and tannins, produced by vegetation in
such ecosystems, inhibited ammonia oxidation thereby reducing nitrite losses.
There is now increasing evidence (see McCarty and Bremner, 1986; Killham,
1989) that inhibition by such compounds is neither widespread nor significant
and that nitrification in such ecosystems is limited rather by the rate of
ammonification of organic nitrogen.

A. MECHANISM OF INHIBITION

Despite widespread use and commercial interest in inhibitors of nitrification,

very little is known of their mechanism of action or of their effects on the
physiology of ammonia oxidizers. Many inhibitory compounds act as
chelating agents and chelation of metal components of the ammonia mono-
oxygenase enzyme has been suggested as a general mechanism for inhibition
(Lees, 1952; Hooper and Terry, 1973). The most extensively studied inhibitor is
 AUTOTROPHIC NITRIFICATION IN BACTERIA 171
nitrapyrin and Campbell and Aleem (1965) reported that addition of copper
relieved inhibition of ammonia oxidation by cell suspensions of N . europaea.
Bhandari and Nicholas (1979) reported a similar effect on inhibition by
sodium diethyldithiocarbamate. Chelation of the copper components of
ammonia mono-oxygenase was therefore accepted as the mode of action for
this inhibitor, although inhibition by another chelating agent, allylthiourea,
was not affected by addition ofcopper. The chelating action of nitrapyrin must
be specific to ammonia mono-oxygenase as other copper-containing enzymes,
e.g. hydroxylamine oxidoreductase and nitrite oxidoreductase, are not effected
(Oremland and Capone, 1988). In addition, Powell and Prosser (1986a) found
increased inhibition by nitrapyrin of growing cultures of N . europaea in the
presence of copper. Effects on growing cells and cell suspensions may differ for
several reasons (see below) but these data suggest that the mechanisms of
inhibition may be more complex than simple chelation. The inhibitors based
on carbon disulphide are also chelating agents. Underhill and Prosser (1987b)
investigated inhibition of both N . europaea and Nitrobacter sp. by potassium
ethyl xanthate in continuous culture at a range of inhibitory concentrations.
Chelating compounds would be expected to act as non-competitive inhibitors
but steady state data indicated competitive inhibition of the Nitrobacter
species while data for N . europaea did not indicate non-competitive inhibition.
Nitrapyrin also inhibits methanogenesis (Salvas and Taylor, 1980) and
methane oxidation (Topp and Knowles, 1984). Nitrapyrin hydrolyses
chemically to chloropicolinic acid, which contains no trichloromethyl group.
Salvas and Taylor (1980, 1984) found inhibition of methane oxidation by this
compound but not of nitrification or methanogenesis. They suggested the
involvement of the trichloromethyl group in inhibition of nitrification and
methanogenesis and the potential use of chloropicolinic acid for selective
inhibition of methane oxidation. Powell and Prosser (1985) found inhibition
by both nitrapyrin and chloropicolinic acid of growing cultures of N . europaea,
and trichloromethane (chloroform) at the equivalent concentration had no
effect. Inhibition by chloropicolinic acid occurred after a lag of approximately
8 h, while nitrapyrin caused inhibition immediately after addition. Salvas and
Taylor (1980, 1984) studied effects on cell suspensions, rather than growing
cultures, and differences in response to inhibitors may result from differences
in uptake properties or their conversion to more inhibitory compounds. As
discussed above, extrapolation of data on cell suspensions to growing
populations of cells is dangerous.

B. STRAIN VARIABILITY

The extent and type of inhibition varies markedly between different strains
and different genera of ammonia-oxidizing bacteria. This was first noted by
Belser and Schmidt (1981) who found differences in sensitivity to inhibition by
172 J. I. PROSSER

nitrapyrin between strains of Nitrosospira, Nitrosolobus and five strains of

Nitrosomonas. Differences within Nitrosomonas strains were as great as those
between Nitrosomonas, Nitrosospira and Nitrosolobus and are therefore not
associated with specific genera.
Powell and Prosser (1986b) found differences in sensitivity between two
strains of N. europaea derived from the same parent strain during continued
laboratory subculturing. Nitrapyrin was added to batch cultures either at the
beginning of incubation or during exponential growth and the kinetics of
nitrite production is illustrated in Fig. 9. Growing cells were more sensitive to
inhibition than stationary phase cells. In the example illustrated, maximum
specific growth rate was reduced from 0.047 to 0.0133 h - after addition to
exponentially growing cultures and to 0.029h-' after addition at the
beginning of incubation. Similar behaviour was found for all strains (Table 6)
but quantitative effects on both maximum specific growth rate and the
length of lag periods varied between strains. In addition, detailed examina-
tion of these kinetics, coupled with most-probable-number counts indicated a

2
w

25 50 7s 100 125
TIME <h)
FIG. 9. Production of nitrite by Nitrosomonas europaea in liquid batch culture. Key:
0 ,control; 0 ,addition of O S p g nitrapyrin mi-' before incubation; 0,addition of
O S p g nitrapyrin ml- during exponential growth. From Powell and Prosser (1986a)
with permission.
 AUTOTROPHIC NITRIFICATION IN BACTERIA 173
TABLE 6. Growth characteristics of three strains of Nitrosomonas europaea in the presence and
absence of OSpg nitrapyrin ml-' (from Powell and Prosser, 1986a).

Parent strain Strain 2 Strain 3

Length Specific Length Specific Length Specific

of lag growth oflag growth of lag growth
Tred tment phase (h) rate(h-l) phase (h) rate(h-') phase (h) rate(h-l)

N o nitrapyrin 22 0.0452 9 0.0465 7 0.084

Nitrapyrin added
before incubation 33 0.03I3 9 0.0285 7 0.0342
Nitrapyrin added to
exponentially
growing culture - 0.0076 - 0.0133 - 0.0195

bacteriostatic effect of nitrapyrin on the parent strain and one derived strain,
but a bactericidal effect on the second derived strain.
The basis for marked qualitative and quantitative differences in sensitivity
between such closely related strains is unclear and is an obvious and important
area for study. It has important implications for the commercial use of such
inhibitors and for discovery of new inhibitory compounds. It may also help to
explain difficulties in distinguishing between bacteriostatic and bactericidal
effects of nitrapyrin added to soil (Rodgers and Ashworth, 1982)where strains
of different sensitivity may be selected after inhibitor treatment.
More detailed studies of inhibition of both N. europaea and a Nitrobacter
strain by potassium ethyl xanthate (Underhill and Prosser, 1987b) and
inhibition of N . europaea by nitrapyrin (Powell, 1985) in continuous culture
produced the surprising result that both inhibitors stimulate ammonia
oxidation at low concentrations. Figure 10 shows stimulation of ammonia
oxidation by 0.2 pg potassium ethyl xanthate ml- ',a concentration which in
batch culture had no effect on stationary phase or exponentially growing cells.
A reduction in steady state ammonia concentration was associated with an
increase in cell concentration and yield coefficient but cell activity remained
constant, i.e. cells appeared to grow more efficiently. Cell activity was reduced
at higher, inhibitory concentrations.
Powell (1985)observed stimulation of ammonia oxidation by N. europaea at
concentrations of nitrapyrin (0.5-1.5 p g ml- ') which were inhibitory in batch
culture. This stimulation was associated with increases in cell activity by a
factor of two at a concentration of 1.5pg ml-'. These effects occurred at
substrate concentrations approaching those of the K, value and are, therefore,
particularly significant for inhibition of nitrification in soil, but again the
biochemical mechanism of stimulation and inhibition requires further study.
174 J. I . PROSSER

IS

100 200 300 400 500 600 700

Time (h)
FIG. 10. Stimulation of ammonia oxidation following addition of potassium ethyl
xanthate (0.2pg ml- I ) to a steady state culture of Nitrosomonas ruropaea. The time of
addition is indicated by the arrow. From Underhill and Prosser (1978b) with
permission.

C. INHIBITION OF ATTACHED CELLS

Both Nitrosomonas and Nitrobacter species are more sensitive to inhibition by

potassium ethyl xanthate (Underhill and Prosser, 1985)and nitrapyrin (Powell
and Prosser, 1986b) than when treated with equivalent concentrations after
inoculation into sterile soil. An interesting differential effect was found for
inhibition of potassium ethyl xanthate, with a Nitrohacter strain being more
sensitive than a Nitrosomonas strain in liquid culture and the reverse
behaviour in soil. This can be explained by adsorption of xanthate or its
breakdown products to soil particles, which will also adsorb ammonium ions
and encourage colonization by the Nitrosomonas strain. The Nitrohacter strain
will, therefore, be less affected by the inhibitor if it can exist in the soil solution,
where its substrate will be concentrated.
Much of the protection afforded by soil is believed to result from
immobilization of inhibitors by organic matter but there is also evidence of
protection through surface attachment. Powell (1985) demonstrated that
cells of N. europuea newly attached to glass slides were as sensitive to
inhibition by nitrapyrin as freely suspended cells, while established biofilms
were not affected by nitrapyrin concentrations which gave significant
inhibition in liquid culture. Cells retained this reduced sensitivity after
detachment, suggesting a relatively permanent change in physiology. EPS
 AUTOTROPHIC NITRIFICATION IN BACTERIA 175
material, produced within the biofilm, may be involved in such protection,
being retained by detached cells as capsular material. Protection was also
afforded by colonization of ATV, which again may suggest protective effects
by clay minerals within the soil.
While these physiological studies are of some relevance to ecology, what is
severely lacking is an understanding of the biochemical mechanisms for
inhibition by these compounds. Such an understanding will have important
practical implications for development of new inhibitors and more efficient
use of existing compounds and, importantly, will provide information on the
biochemistry of ammonia and nitrite oxidation and its control and regulation
with regard to activity and growth.

X. Concluding Remarks
In this article I have attempted to highlight the ways in which our traditional
view of nitrification must be modified in the light of recent advances in our
knowledge of the physiology of autotrophic ammonia- and nitrite-oxidizing
bacteria. These advances have arisen through basic physiological and
biochemical studies and also through the need to explain phenomena
associated with nitrification in natural environments.
We can no longer consider N . europaea to be the dominant or even a typical
ammonia oxidizer. Other species and strains are important in a range of
environments, and physiological differences leading to dominance of these
strains requires investigation. In particular, marine strains appear to have
significantly different properties and the mechanisms by which they survive at
low substrate concentrations is a particularly interesting area for study.
Many aspects of basic biochemistry and physiology are poorly understood.
Little is known of the mechanisms of transport of substrate into the cell,
despite their potential importance in determining saturation constants for
growth and activity. Only limited data are available on maintenance energy
requirements which, at environmental substrate concentrations, will be
significant. More work is also required to confirm the proposed mechanisms
for regulation and control of substrate oxidation, energy generation and
carbon dioxide fixation. Such studies are of fundamental value in our
understanding of the autotrophic mode of existence. Practical applications
include a greater understanding of the mechanisms of action of inhibitors,
particularly those of ammonia oxidation.
Nitrifiers are trapped by the mode of existence which they have chosen.
Their substrates provide the bare minimum of energy required for growth.
They must then use much of this energy to form reducing equivalents required
for carbon dioxide fixation. This requirement would be alleviated by
assimilation of organic carbon. Ammonia oxidizers, however, appear to gain
176 J I PROSSER

little benefit from this, although nitrite oxidizers are more versatile and are
capable of mixotrophic and heterotrophic growth. Both groups are inhibited
by high concentrations of their substrates and have little energy to spare for
high-affinity substrate uptake systems which would enable growth at low
substrate concentrations. The range of concentrations over which they can
exist is therefore limited.
Despite these disadvantages, nitrifying bacteria occupy niches in many
ecosystems and must, therefore, compete successfully with faster and more
efficiently growing organisms for oxygen, ammonia and carbon dioxide. One
reason for this may be their previously unrecognized metabolic versatility.
Nitrifiers are capable of reversing the nitrification process, carrying out
denitrification and producing nitrite, ammonia, nitrous and nitric oxides and
gaseous nitrogen. Ammonia oxidizers can metabolize urea and can assimilate
carbon from methane while nitrite oxidizers can grow anaerobically in the
presence of organic compounds and nitrate. In addition, they grow and have
significant activity in environments which data from laboratory studies
indicate are totally unsuitable. The most obvious example is their growth in
environments of low pH value for which several explanations exist. Some
involve interactions with other organisms, particularly heterotrophs releasing
ammonia through mineralization of organic matter. Others involve
modification of their environment to gain protection. Production of
extracellular polymeric substances by attached organisms appears to provide
some protection from low pH values, presumably by altering in some way the
pH value which the cell experiences. This material will also affect diffusion of
oxygen within a biofilm leading, for example, to anaerobic growth of nitrite
oxidizers. Growth on clay minerals also appears to provide a local buffering
effect during ammonia oxidation. The production of EPS material by these
organisms is remarkable and its study is of interest and importance to both
physiologists and ecologists.
Amid these discoveries of new metabolic functions within nitrifying
bacteria, we should not lose sight of the fact that they are primarily aerobic
organisms, oxidizing ammonia or nitrite and fixing carbon dioxide at
neutral/alkaline pH values. Physiological studies may indicate their metabolic
potential but the extent to which this potential is expressed in natural
environments is much more difficult to assess. Interactions with heterotrophs
have been described which significantly affect nitrification, and the
complexities introduced by heterotrophic nitrifiers make study of processes
within the nitrogen cycle in natural environments both difficult and
challenging.
In addition, other factors of environmental importance, in particular
temperature and water stress, have been little studied at the physiological level.
The techniques of modern molecular biology have barely been applied to
 AUTOTROPHIC NITRIFICATION IN BACTERIA 177
nitrifying bacteria. I t is hoped that their application, coupled with sound
physiological studies, will build on the recent work described in this article to
provide a fuller understanding of the mechanisms regulating growth and
activity in this fascinating and unique group of organisms. Similar application
of modern techniques of microbial ecology will enable a fuller assessment of
their r81e within the nitrogen cycle and the extent to which their metabolic
versatility is expressed in nature.

XI. Acknowledgements
I would like to acknowledge Dr Booth and Dr Killham for helpful discussions
and authors who provided copies of papers in press.

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