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Soil Biology & Biochemistry 42 (2010) 2058e2067
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Soil Biology & Biochemistry
journal homepage: www.elsevier.com/locate/soilbio
Review
Pathways of nitrogen utilization by soil microorganisms e A review
Daniel Geisseler a, c, *, William R. Horwath a, Rainer Georg Joergensen b, Bernard Ludwig c
a
Department of Land, Air and Water Resources, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, USA
Department of Soil Biology and Plant Nutrition, University of Kassel, Nordbahnhofstr. 1a, 37213 Witzenhausen, Germany
c
Department of Environmental Chemistry, University of Kassel, Nordbahnhofstr. 1a, 37213 Witzenhausen, Germany
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 March 2010
Received in revised form
10 August 2010
Accepted 18 August 2010
Available online 4 September 2010
Microorganisms are able to utilize nitrogen (N) from a wide range of organic and mineral compounds. In
this paper, we review the current knowledge about the regulation of the enzyme systems involved in the
acquisition of N and propose a conceptual model on the factors affecting the relative importance of
organic and mineral N uptake. Most of the N input into soil is in the form of polymers, which first have to
be broken down into smaller units by extracellular enzymes. The small organic molecules released by the
enzymes can then be taken up directly or degraded further and the N taken up as ammonium (NHþ
4 ).
When NHþ
4 is available at high concentrations, the utilization of alternative N sources, such as nitrate
þ
(NO
3 ) and organic molecules, is generally repressed. In contrast, when the NH4 availability is low,
enzyme systems for the acquisition of alternative N sources are de-repressed and the presence of
a substrate can induce their synthesis. These mechanisms are known as N regulation. It is often assumed
that most organic N is mineralized to NHþ
4 before uptake in soil. This pathway is generally known as the
mineralization-immobilization-turnover (MIT) route. An advantage of the MIT route is that only one
transporter system for N uptake is required. However, organic N uptake has the advantage that, in
addition to N, it supplies energy and carbon (C) to sustain growth. Recent studies have shown that the
direct uptake of organic molecules can significantly contribute to the N nutrition of soil microorganisms.
We hypothesize that the relative importance of the direct and MIT route during the decomposition of
residues is determined by three factors, namely the form of N available, the source of C, and the availability of N relative to C. The regulation system of soil microorganisms controls key steps in the soil N
cycle and is central to determining the outcome of the competition for N between soil microorganisms
and plants. More research is needed to determine the relative importance of the direct and MIT route in
soil as well as the factors affecting the enzyme systems required for these two pathways.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Microbial N uptake
Direct route
Mineralization-immobilization-turnover
N regulation
Extracellular enzymes
1. Introduction
Nitrogen (N) is required by all organisms as an essential
nutrient. In terrestrial ecosystems, N is generally of limiting availability to plants, resulting in strong competition between microorganisms and plants (Vitousek and Howarth, 1991).
Microorganisms have developed different mechanisms for uptake
and assimilation of mineral and organic forms of N, enabling them
to utilize a wide range of organic and mineral compounds (see
reviews by Merrick and Edwards, 1995; Marzluf, 1997).
In terrestrial ecosystems, plant and microbial residues are the
two main sources of organic input, while the relative contribution of
* Corresponding author at: Department of Environmental Chemistry, University
of Kassel, Nordbahnhofstr. 1a, 37213 Witzenhausen, Germany. Tel.: þ49 (0) 5542 98
1635; fax: þ49 (0) 5542 98 1633.
E-mail address: [email protected] (D. Geisseler).
0038-0717/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2010.08.021
animal residues is small (Kögel-Knabner, 2002). In residues, the
quantitatively most important N containing molecules are proteins,
chitin and peptidoglycan. Proteins alone comprise 60% or more of the
N in plant and microbial cells (Cochrane, 1958; Christias et al., 1975;
Fuchs, 1999; Sinha, 2004). Mineral forms of N, such as ammonium
(NHþ
4 ) and nitrate (NO3 ), usually account for a small proportion of
the total N in cells. Most of the N input into soil is therefore in
the form of polymers, which first have to be broken down into
smaller units by extracellular enzymes (Schimel and Bennett, 2004).
The small organic molecules released by the enzymes can then be
taken up directly or degraded further and the N taken up as NHþ
4
(Fig. 1). Extracellular depolymerases therefore constitute the first
step in the degradation of most organic N input to soil.
Ammonium is considered the preferred source of N for bacteria
and fungi (Merrick and Edwards, 1995; Marzluf, 1997). In addition to
NHþ
4 , glutamate and glutamine, which serve as the key N donors for
biosynthetic reactions in virtually all cells, are also used as preferred
D. Geisseler et al. / Soil Biology & Biochemistry 42 (2010) 2058e2067
2059
2. Extracellular depolymerases
Fig. 1. Main steps of the microbial N utilization from organic substrates.
N sources by many microorganisms (see reviews by Magasanik,
1993; Merrick and Edwards, 1995; Wong et al., 2008). All transformation and N uptake processes are mediated by enzymatic
systems that require carbon (C), N and energy for their synthesis and
expression. In soil where N and C are often of low availability, it is
crucial for organisms to tightly regulate the synthesis and activity of
the different enzyme systems. Soil microorganisms, which regulate
enzyme production according to their needs and the availability of
substrates, will have a competitive advantage (Koch, 1985). In fact,
when preferred N sources are available at high concentrations, the
transcription of genes encoding for enzymes required for the utilization of alternative N sources, such as nitrate (NO
3 ) and most
organic molecules, is repressed. This mechanism is known as N
regulation (Magasanik, 1993).
It is often assumed that all organic N is first mineralized to NHþ
4
before it is taken up by microorganisms in soil. This pathway, which
was largely formulated by Jansson (1958), is generally known as the
mineralization-immobilization-turnover (MIT) route. More recent
studies however, have shown that the uptake of small organic
molecules, such as amino acids, can significantly contribute to the N
nutrition of soil microorganisms (Barak et al., 1990; Hadas et al.,
1992; Barraclough, 1997; Luxhøi et al., 2006; Geisseler et al.,
2009). In this review, the term direct route will be used as
a collective term for the different mechanisms responsible for the
uptake of organic molecules. The route of N uptake has implications
for the competition for N between plants and microorganisms.
When the MIT route is dominant, plants and microorganisms
compete for mineral N (Manzoni and Porporato, 2007). On the
contrary, when the direct route is dominant, microorganisms meet
their N requirements with organic N compounds and plants face
reduced competition from microorganisms for mineral N. However,
a number of studies, recently reviewed by Näsholm et al. (2009),
have shown that plants are also capable of acquiring organic N,
either directly through root cell membranes or via mycorrhizal
fungi. The quantitative contribution of organic N uptake to the N
nutrition of plants is likely affected by the N uptake pathway of soil
microorganisms.
Only a few studies have quantified the contribution of the MIT
and direct route to the N nutrition of soil microorganisms and little
is known about the factors affecting the importance of the two
pathways in soil. The objectives of this present paper are: (i) to
summarize the current knowledge about the regulation of the
enzyme systems involved in the acquisition of N by soil microorganisms; and (ii) to propose a conceptual model on the factors
affecting the relative importance of the MIT and direct route.
Extracellular depolymerases are required to degrade complex
organic polymers from plant and microbial residues into soluble
subunits that can be taken up by microorganisms. Based on the
chemical composition of the main sources of organic residues in
soil, the most important extracellular depolymerases involved in
the hydrolysis of N containing molecules are proteases, chitinases
and peptidoglycan hydrolases.
Extracellular proteases hydrolyze large proteins and polypeptides into peptides and amino acids. The ability to produce
extracellular proteases is widespread among bacteria and fungi
(Ahearn et al., 1968; Gupta et al., 2002). Extracellular proteases
usually have wide substrate-specificities and can degrade most
non-structural proteins (Kalisz, 1988). Proteases are also synthesized for intracellular use, where they play a key role in the regulation of metabolic processes and in protein turnover. The latter is
essential for adaptation of cells to new environmental conditions,
especially in response to starvation for nutrients (Kalisz, 1988).
Chitin, an unbranched polymer of N-acetyl-D-glucosamine, is
widely distributed in nature and one of the most abundant polymers on earth (Duo-Chuan, 2006). Chitinases hydrolyze the links
between N-acetyl-D-glucosamine molecules (Felse and Panda,
1999). Chitinases are produced by a wide range of organisms,
including bacteria, fungi and plants, but not by archaea (Gooday,
1990a; Duo-Chuan, 2006). Bacteria produce chitinases mainly to
degrade chitin for use as a N and C source. In fungi, however, chitinases also play an important role in cell wall development and
architecture during active growth (Gooday, 1990a; Adams, 2004;
Bhattacharya et al., 2007).
Peptidoglycan is composed of different amino sugars and amino
acids which are linked together by three different chemical bonds,
namely glycosidic, amide, and peptide, to form a two- or threedimensional net-like polymer (Vollmer et al., 2008). The hydrolysis
of peptidoglycan therefore requires the activity of different
amidases, peptidases and glycosylases, the latter including glucosaminidases and lysozymes (Shockman et al., 1996). Peptidoglycan
hydrolases are common among fungi (Grant et al., 1986). In
bacteria, peptidoglycan hydrolases play an important role in the
modification of the cell wall for growth, cell separation, or sporulation as well as in the turnover and recycling of peptidoglycans
(Vollmer et al., 2008). Maintaining the balance between necessary
modifications of the cell walls and unwanted weakening of it may
therefore be the main purpose of the regulation of peptidoglycan
hydrolases in bacteria.
2.1. Regulation of extracellular depolymerases
Four major mechanisms, namely (i) substrate induction, (ii) endproduct repression, (iii) de-repression due to insufficient nutrient
supply and (iv) constitutive production, have been found to regulate the production (synthesis and secretion) of extracellular
hydrolases.
Production of extracellular hydrolases is generally induced by
the presence of a substrate. Microbial proteases have been found to
be induced by the presence of proteins in the medium (Kalisz, 1988;
Haab et al., 1990), while chitinases are induced by the presence of
chitin, but also by low concentrations of N-acetyl-D-glucosamine
(Felse and Panda, 1999; Scigelova and Crout, 1999; Duo-Chuan,
2006). The presence of bacterial cells has been found to induce
the production of extracellular muramidase in the basidiomycete
Schizophyhm commune and the cultivated mushroom Agaricus bisporus (Grant et al., 1990; Lincoln et al., 1997)
In contrast, end-products and easily metabolizable C sources
may repress enzyme production (Table 1). Protease production is
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D. Geisseler et al. / Soil Biology & Biochemistry 42 (2010) 2058e2067
Table 1
Ammonium (NHþ
4 ) concentrations found to repress extracellular enzymes involved in microbial N acquisition.
Enzyme system/NHþ
4 concentration
Effect on activity (%)
Species
Environment
Reference
Mixed community
Beauveria bassiana
Soil
Growth medium
Geisseler and Horwath, 2008
Bidochka and Khachatourians, 1988
30 mM NH4eN
30 mM NH4eN
16% to þ7%
49% (in the medium)
6% mg1 biomass
5%
7%
Scleroaum rolfsii
Scleroaum bataticola
Growth medium
Growth medium
Sayed et al., 1994
Sayed et al., 1994
Glycine aminopeptidase
114 mmol NH4eN added g1 soil
approx. 35%
Mixed community
Soil
Allison and Vitousek, 2005
N-acetyl-D-glucosaminidase
37.5 mM NH4eN
50% mg1 biomass
Beauveria bassiana
Growth medium
Bidochka and Khachatourians, 1993
Urease
5 mmol NH4eN added g1 soil
25 mmol NH4eN added g1 soil
50 mmol NH4eN added g1 soil
110e170 mM NH4eN
13
34
76
71
Mixed community
Mixed community
Mixed community
Proteus rettgeri
Soil
Soil
Soil
Growth medium
McCarty et al., 1992
McCarty et al., 1992
McCarty et al., 1992
Magaña-Plaza and Ruiz-Herrera, 1967
Amino Acid Oxidase
10 mM NH4eN
30 mM NH4eN
22%
30%
Chlumydomonas reinhardtii
Chlumydomonas reinhardtii
Enzyme extract
Enzyme extract
Vallon et al., 1993
Vallon et al., 1993
Protease
2.9 mmol NH4eN added g1 soil
37.5 mM NH4eN
to
to
to
to
15%
85%
95%
92%
The microorganisms were cultivated in the presence of the substrates for the different enzyme systems and varying concentrations of
a
Enzymes were extracted from bacteria grown on an N-free medium.
generally repressed by amino acids, NHþ
4 , glucose and other readily
available C compounds (Glenn, 1976; Allison and Macfarlane, 1992).
High levels of N-acetyl-D-glucosamine, however, repress chitinase
synthesis, as do glucose and other easily available C sources (Felse
and Panda, 1999; Scigelova and Crout, 1999; Duo-Chuan, 2006). In
the entomopathogenic fungus Beauveria basssiana, chitinase was
also repressed by NHþ
4 and certain amino acids (Bidochka and
Khachatourians, 1993; Table 1). Different effects of end-products
on muramidases have been reported. While extracellular muramidases of A. bisporus and S. commune were repressed by glucose
(Grant et al., 1990; Lincoln et al., 1997), b-glucosaminidase
production by A. bisporus was highest in the presence of glucose
and fructose (Lincoln et al., 1997). In addition, Fermor et al. (1991)
found that A. bisporus mineralized bacteria even in the presence
of glucose and ammonium sulfate. De-repression of protease
production has been found to take place when C, N or sulfur (S)
were limiting (Kalisz, 1988).
Finally, extracellular enzymes may be secreted constitutively at
low levels regardless of the availability of substrates or endproduct. Constitutive production has been reported for protease
(Kalisz, 1988; Haab et al., 1990), chitinases (Felse and Panda, 1999),
and muramidase (Grant et al., 1990; Lincoln et al., 1997). Constitutive production of extracellular enzyme synthesis in situations
with limited substrate availability may be sufficient to initiate the
degradation of new substrates, resulting in the release of soluble
oligomers, which in turn induces enzyme synthesis (Felse and
Panda, 1999).
2.2. Extracellular depolymerases in soil
The ability to degrade protein, chitin and peptidoglycan is
widespread among soil microorganisms (Grant et al., 1986; Gooday,
1990b; Adesina et al., 2007). Compared to studies with cultured
microorganisms, less work has been done to systematically study
the regulation of extracellular depolymerases by soil microbial
communities in their natural environment.
Substrate induction has been found to be an important mechanism regulating the production of extracellular depolymerases. In
tundra soil, protease activity was increased with the addition of
protein (Weintraub and Schimel, 2005). This result is in line with
a laboratory incubation carried out by Geisseler and Horwath
NHþ
4;
a
a
except.
(2008), where protease activity increased when casein, zein or
gluten were added to soil. Allison and Vitousek (2005) also found
increased glycine aminopeptidase activity in soil samples amended
with collagen. Similarly, chitinase activity in soil has been found to
be induced by the presence of chitin (Rodriguez-Kabana et al., 1983;
Abdel-Fattah and Mohamedin, 2000; Ueno and Miyashita, 2000).
Protease activity is often well correlated with the microbial biomass
and a certain level of residual activity of extracellular depolymerases is generally found in soil (Doran, 1980; Geisseler and
Horwath, 2008). These findings suggest constitutive synthesis.
However, as soil organic matter and residues are constantly
decomposed, and enzymes may be protected by soil colloids, as
discussed below, it is challenging to determine whether the
enzymes are produced at a constitutive level or are induced by the
presence of substrates. Similarly, microbial biomass C and N as well
as b-glucosaminidase increased in a field study at intermediate
poultry litter rates greater than 6.7 Mg ha1 compared to sites with
no applied litter (Acosta-Martínez and Harmel, 2006). In this study,
the specific b-glucosaminidase activity (per unit microbial C and N)
increased in general with increasing poultry litter application in
cultivated soil, while it remained relatively constant in the soil
under pasture. Based on these studies it cannot be clearly distinguished between substrate induced and constitutive enzyme
synthesis at low levels of activity.
De-repression of protease synthesis under N and S limiting
conditions has been reported by Sims and Wander (2002) in sand
cultures. Not only total, but also specific (i.e. per unit biomass)
proteolytic activity was increased under these conditions. In
contrast, Asmar et al. (1992) found that the addition of glucose to an
agar containing medium increased protease activity.
In contrast to cultured species, where NHþ
4 generally represses
extracellular depolymerase activity, the effect of NHþ
4 on the
activity of these enzymes is less consistent in soil. While Allison and
Vitousek (2005) found that glycine aminopeptidase was decreased
by about one third after 28 days in response to NHþ
4 addition,
Geisseler and Horwath (2008) found that the NHþ
4 concentration in
soil had no effect on protease activity. However, the amount of
NH4eN added was much lower in the latter study compared to the
former (Table 1). In a study by Jezierska-Tys and Frac (2009),
increased protease and urease activity were observed together with
elevated NHþ
4 concentrations over a period of several weeks after
D. Geisseler et al. / Soil Biology & Biochemistry 42 (2010) 2058e2067
the application of dairy sewage sludge to soil. Across a chronosequence, chitinase activity decreased significantly with increasing
soil age and N availability. In addition, N fertilization repressed
chitinase activity only at the N limited young site. These results
suggest that N supply in older soil reached levels high enough to
trigger a negative feedback and inhibit enzyme production
(Olander and Vitousek, 2000). In contrast, in samples of the organic
horizon from forest floors, Michel and Matzner (2003) found that
b-glucosaminidase activity responded positively to mineral N
additions, particularly in samples with low internal N concentrations. They attributed the lack of negative effects to the fact that
b-glucosaminidase is related to the actively growing biomass in
soil. Increasing the N supply in these soils may have increased
fungal biomass. This is in line with a cropping system comparison
carried out by Ekenler and Tabatabai (2002) who found that
b-glucosaminidase activity was generally increased in plots with
þ
NHþ
4 applications compared to plots without NH4 additions.
2.3. Regulatory enzyme mechanisms in soils versus culture media
Studies with cultured microorganisms are very helpful in
determining the regulatory mechanisms for different enzyme
systems. However, the conditions in soil differ from those in culture
media in many ways, which may affect the observed response to
specific treatments.
The studies discussed above suggest that extracellular depolymerases are less tightly regulated in soil than in culture media. One
reason why soil microorganisms do not down-regulate their
enzyme synthesis as readily as cultured microbes may be the fact
that soil microbes live in a nutrient poor environment. Oligotroph
organisms may have adapted over evolutionary time to be very
efficient in growing under low levels of nutrients and the
achievement of high efficiency under low-substrate conditions may
have crippled the cell to not being effective when there are high
concentrations of nutrients. Most organisms in a nutrient-poor
environment cannot be selective and cannot choose to use only
certain organic substrates (Koch, 2005). In fact, it appears that
substrate induction has a stronger effect on enzyme production in
soil than repression by end-products and easily metabolizable C
sources (Asmar et al., 1992; Geisseler and Horwath, 2008). In
contrast to soil microorganisms, cultured microorganisms often
belong to species important for industrial applications. These
species were selected for their capacity to maximize their metabolic activity under constant conditions with a high nutrient
availability. In addition, the microbial population in soil is very
diverse with most of these species being involved in organic matter
turnover. This genetic diversity results in a wide variety of metabolic processes and interactions between organisms.
Another reason of the slow response of soil microorganisms to
an increase in NHþ
4 availability may be that extracellular enzymes
are beyond the control of microorganisms. Extracellular enzymes
have been found to be associated with soil colloids, which may
increase their half-life (Burns, 1982). Several studies found this to
be true for proteases. Asmar et al. (1992) found that in soils
amended with King’s agar and nutrients, the activity of soluble
extracellular protease was about 30% of the total protease activity.
Total protease activity persisted longer than the activity of its
soluble extracellular fraction in these soils. Using different
substrates, Bonmatí et al. (1998, 2009) found that the proteases
induced by these substrates were predominantly associated with
soil organic matter fractions. Only casein-hydrolyzing proteases
were present, at least partially, as glyco-proteins not associated to
humus (Bonmatí et al., 2009).
In contrast to culture media, soil is a spatially and temporally
heterogeneous environment. The spatial arrangement of soil
2061
particles creates a large number of microenvironments differing in
their physical, chemical and biological properties (Ranjard and
Richaume, 2001). In addition, the input of organic material in soil
is highly heterogeneous in space and time, and temporal changes in
the soil moisture content affect solute diffusion and nutrient fluxes
(Or et al., 2007). In unsaturated soil, water films are thin and pores
may be disconnected, resulting in restricted diffusion of substrates
towards microorganisms and of waste products and extracellular
enzymes away from cells. Compared to culture media, molecules
must follow a more tortuous path to diffuse from one point to
another, reducing the substrate flux to the cell surface (Griffin,
1981; Moldrup et al., 2001).
Due to these differences between culture media and soil, the
response of soil microbial communities may not be as pronounced
as found with cultured species and processes, which occur separately in culture media, may take place concurrently in soil. It is
therefore important to verify results from cultured species in soil.
The differences between culture media and soil may also affect the
regulation of extracellular N mineralizing enzymes, which will be
discussed in the following section.
3. Extracellular N mineralization
The small organic molecules released by extracellular depolymerases can be taken up directly or be subjected to extracellular
mineralization. The MIT route includes the mineralization of
organic molecules, followed by the uptake and assimilation of the
released NHþ
4 . With the exception of urease, which has been
extensively studied due to the importance of urea as fertilizer, the
enzymes responsible for the extracellular ammonification of
organic compounds in soil and their regulation have received little
attention.
3.1. Urease
Urease catalyzes the hydrolysis of urea to yield NH3 and carbamate. The latter compound spontaneously decomposes to yield
another molecule of ammonia and carbonic acid (Mobley et al.,
1995). Urea is continuously released into the environment
through biological processes such as urine excretion from
mammals. In addition, urea is a product of the degradation of the
amino acid arginine, of uric acid, which is excreted by birds,
reptiles, and most terrestrial insects as their primary detoxification
product, as well as of purines and pyrimidines, which are building
blocks of nucleic acids (Cunin et al., 1986; Mobley and Hausinger,
1989; Vogels and van der Drift, 1976).
Urease can be produced by bacteria, yeasts, filamentous fungi,
and algae (Mobley and Hausinger, 1989), as well as plants (Follmer,
2008). Urease may be synthesized constitutively in some organisms; however, most commonly urease expression is under N
regulation (Mobley et al., 1995). Urease synthesis is repressed when
the cells are grown in the presence of a preferred N source such as
NHþ
4 (Table 1). In contrast, urease synthesis is activated in the
presence of urea and alternative N sources, as well as under
conditions of N starvation (Mobley et al., 1995).
Considerable evidence indicates that urease is cytoplasmatic in
both yeasts and bacteria (Mobley and Hausinger, 1989). However,
a large proportion of the urease activity in soil has been found to be
extracellular and associated with soil particles. Pettit et al. (1976)
calculated that in the soil examined approximately 60% of the
total urease activity was extracellular-bound and that the
remainder was composed of intracellular and extracellularunbound enzyme. Klose and Tabatabai (1999) estimated the
extracellular proportion of urease activity to be 46%. Soil urease has
been found to be primarily associated with soil organic matter and
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D. Geisseler et al. / Soil Biology & Biochemistry 42 (2010) 2058e2067
clay minerals. In surface soil samples of cultivated field, Reynolds
et al. (1985) found a strong positive correlation between urease
activity and soil organic C and total soil N, while the correlation
between urease activity and clay content was weaker. In addition,
Kandeler et al. (1999) reported that organic amendments increased
the capacity of a soil to protect urease in the clay-sized fraction. This
association protects the enzyme from attack by proteolytic
enzymes, does not prevent urease-urea activity and allows diffusion of substrate molecules to, and product molecules from sites of
active enzymes (Burns et al., 1972). The bonds between soil particles and urease appear to be strong. Zanuta and Bremner (1977)
found that leaching a wide range of soils with water, drying for
24 h at temperatures ranging from 30 to 60 C, storage for six
months at temperatures from 20 to 40 C, and incubation under
aerobic or waterlogged conditions did not decrease urease activity
significantly. Therefore, extracellular urease activity in soil seems to
be mainly the result of its release from decaying microbial and
plant cells and subsequent protection from proteolysis, rather than
excretion by living cells.
In contrast to studies with cultured microorganisms, several
studies in soil found that addition of urea did not increase urease
activity (Lloyd and Sheaffe, 1973; Zanuta and Bremner, 1976), which
may be due to the high proportion of protected extracellular urease.
However, repression by NHþ
4 may also be responsible for these
results. In fact, when C in the form of glucose or other readily
available C sources was added together with urea to soil, urease
activity increased. These results suggest that the addition of a C
source increased the N demand of microorganisms above its
availability, so that N became limiting (Lloyd and Sheaffe, 1973;
Zanuta and Bremner, 1976). In response, the genes responsible for
urease synthesis were de-repressed resulting in an increase of
urease activity. This is in line with McCarty et al. (1992) who found
evidence that microbial production of urease in soil was repressed
1
soil resulted
by NHþ
4 and NO3 . The addition of 50 mmol NH4eN g
in a decrease in urease activity of 76e95%, while the same addition
of NO3eN decreased urease activity by about two thirds (McCarty
et al., 1992).
subsequent intracellular deamination when C sources other than
amino acids are available.
Why would an organism utilize a cell-surface amino acid
oxidase rather than take up the amino acid? An advantage of the
latter, and therefore of the direct route, is that amino acids not only
supply N, but also C. In addition, the ability to incorporate the
amino acid directly into protein saves the energy required to
synthesize a C skeleton. However, the composition of the soil amino
acid pool may not match the needs of the microorganisms.
Furthermore, it might require several different amino acid transporters to take up the different amino acids available while NHþ
4
transporters are most likely already operative (see below). Secretion of a broad-specificity L-amino acid oxidase may provide
a means to release NHþ
4 from diverse amino acids while the toxic
by-products are produced outside the cell (Davis et al., 2005). In
addition, the production of H2O2 may also suppress competing
species (Tong et al., 2008).
Several ectomycorrhizal basidiomycetes and ascomycetes as
well as saprotrophic fungal and bacterial species, some of them
isolated from soil, have been found to produce amino acid oxidases
(Braun et al., 1992; Davis et al., 2005; Nuutinen and Timonen,
2008). The amino acid oxidases of soil microorganisms seem to
have predominantly broad substrate specificities (Nuutinen and
Timonen, 2008). Assays specific to amino acid oxidase activity are
not yet available for soil samples. However, assays for enzymes
deaminating glutamine, asparagine, hystidine, aspartate have been
developed for soil (Frankenberger and Johanson, 1981;
Frankenberger and Tabatabai, 1991a,b; Senwo and Tabatabai,
1996). As these assays measure enzyme activity based on the
release of NHþ
4 , amino acid oxidases may contribute to the activity
measured. Relatively little is known about the location and regulation of these deaminases in soil. The activities of asparagines,
glutamine and histidine deaminating enzymes in soil were found to
originate predominantly from intracellular enzymes (Badalucco
et al., 1996; Deng et al., 2006). However, enzymes protected by
soil colloids may also contribute to the overall activity (Klose and
Tabatabai, 2002).
4. Nitrogen uptake mechanisms by microbial cells
3.2. Amino acid oxidase
More than 60% of the organic N input into soil is in the form of
proteinaceous material (Christias et al., 1975; Fuchs, 1999; Sinha,
2004). The deamination of amino acids and subsequent uptake of
the released NHþ
4 are therefore key reactions of the MIT route.
While amino acid deaminases seem to be mainly intracellular
enzymes (Badalucco et al., 1996; Deng et al., 2006), the production
of cell surface-bound amino acid oxidases has been reported for
fungal and bacterial species (Böhmer et al., 1989; Braun et al., 1992;
Davis et al., 2005; Nuutinen and Timonen, 2008). L-amino acid
oxidases catalyze the oxidative deamination of L-amino acids to
produce the corresponding keto-acids, NH3 and hydrogen peroxide
(H2O2). Some amino acid oxidases have been characterized by strict
substrate specificity, while others exhibit relatively broad substrate
specificity (Braun et al., 1992). Extracellular deamination of amino
acids is mainly associated with enzymes bound to the cell surface
rather than with enzymes liberated into the environment or with
abiotic deamination processes (Pantoja and Lee, 1994).
The expression of the genes encoding for amino acid oxidases
have been found to be repressed by NHþ
4 (Palenik and Morel, 1990;
Vallon et al., 1993; Table 1), de-repressed by N limitation or starvation (Davis et al., 2005) and induced by amino acids in the
presence of a C source (Vallon et al., 1993; Davis et al., 2005). The
requirement for C may indicate that extracellular deamination is
only favorable compared to the direct amino acid uptake and
The products of extracellular depolymerases and N mineralizing
enzymes are N sources for the cells. The enzyme systems responsible for the transport across membranes are generally very
specific. Enzyme systems for mineral N uptake and assimilation,
required for the operation of the MIT route, as well as systems for
the uptake of organic molecules, which play a central role for the
direct route, will be presented in the following sections.
4.1. Uptake of ammonia and ammonium
Ammonia is a gas that can diffuse through biological
membranes (Andrade and Einsle, 2007). Most biological
membranes seem to exhibit a measurable permeability towards
NH3. The rate of unspecific diffusion depends of the concentration
gradient across the membrane and the permeability of the
membrane (Kleiner, 1981). Microorganisms therefore have limited
possibilities to regulate NH3 diffusion into or from cells. However,
they possess membrane proteins for the uptake of NHþ
4 and NH3.
Several fungal and bacterial species have been found to possess
more than one system with distinct uptake properties (Marini et al.,
1997; Meier-Wagner et al., 2001; Monahan et al., 2002; Mitsuzawa,
2006).
These membrane protein systems are capable of forming a large
NHþ
4 gradient across biomembranes. For this reason they were
generally believed to be transporters, which actively, at the expense
D. Geisseler et al. / Soil Biology & Biochemistry 42 (2010) 2058e2067
of energy, transport NHþ
4 into the cytoplasm (Kleiner, 1981). Some
recent studies, however, suggested that these proteins may serve as
passive NH3 channels (Khademi et al., 2004; Zheng et al., 2004).
Andrade and Einsle (2007) proposed a mechanistic model that links
the transport of NH3 to a symport of Hþ. According to their model,
NHþ
4 is being deprotonated after binding to the transporter, and
NH3 and Hþ are taken up following separate, but coupled, pathways. In the presence of a proton motive force (pH in solution
below cytoplasmatic pH) the transport would be active, while in its
absence passive uptake of NH3 may be observed (Andrade and
Einsle, 2007). Therefore, in alkaline environments, less energy
may be required for NH3/NHþ
4 uptake, as NH3 diffusion may
contribute considerably to the intracellular accumulation of NHþ
4
(Kleiner, 1985). The optimum pH for the active uptake of NH3/NHþ
4
has been found to be between 6 and 7 (Kleiner, 1981).
At external NHþ
4 concentrations greater than 10e20 mM, the
synthesis of the NH3/NHþ
4 uptake proteins is repressed, presumably
because diffusion of NH3 through the membrane is fast enough to
support the cell’s demand for N (Kleiner, 1981; Marini et al., 1997;
Table 2). However, when the NHþ
4 supply is low and the organisms
þ
grow on N sources other than NHþ
4 , the synthesis of the NH3/NH4
uptake systems is de-repressed (Kleiner, 1985).
4.2. Assimilation of ammonium
Once taken up, NHþ
4 is predominantly assimilated into biological
molecules via either the glutamine synthetase/glutamate synthase
pathway (GS/GOGAT) or through glutamate dehydrogenase (GDH).
The energy requirement for the GS/GOGAT pathway has been
estimated to be 18e30% higher than for the GDH pathway (Helling,
1994; Neidhardt et al., 1990). The expenditure of energy is
compensated for by the fact that GS has a much higher affinity for
þ
NHþ
4 than GDH and therefore enables organisms to scavenge NH4 at
low concentrations (Merrick and Edwards, 1995; Schmid et al.,
2000; Silberbach et al., 2005).
2063
In both, bacteria and fungi, GS activity is repressed by high NHþ
4
concentrations (Ertan, 1992; Baars et al., 1995; Table 2) and its
activity is decreased when the organism is stressed for energy
(Gräzer-Lampart et al., 1986; Helling, 1998). GS has also been
reported to be repressed by several amino acids such as alanine and
glycine (Hubbard and Stadtman, 1967; Zofall et al., 1996).
4.3. Uptake and assimilation of nitrate
Nitrate can be used by many microorganisms as N source and is
taken up by specific transporters (Gonzalez et al., 2006). While NHþ
4
can be assimilated directly, NO
3 has to be reduced first in two steps
by assimilatory NO
3 reductase (ANR) and nitrite reductase before it
can be assimilated in the form of NHþ
4 (McCarty, 1995). This
þ
assimilatory reduction of NO
3 to NH4 requires energy. As many
other N utilization enzymes systems, NO
3 uptake is under N
regulation. The assimilation of NO-3 has been found to be induced by
NHþ
4 limitation and by the presence of NO3 and nitrite, while it is
in
the
culture
medium (Table 2;
repressed by the presence of NHþ
4
Stewart, 1994; Gonzalez et al., 2006).
In aerated soil, NHþ
4 is generally rapidly oxidized to NO3,
a reaction known as nitrification (Myrold, 1998). Therefore, the NO
3
concentration often exceeds the NHþ
4 concentration in soil. Several
studies found a strong inhibition of NO-3 uptake by soil microorganisms in the presence of NHþ
4 in soil solution (Rice and Tiedje,
1989; McCarty and Bremner, 1992). In a study using selective
inhibitors, Myrold and Posavatz (2007) found that bacteria had
a greater potential for assimilating NO
3 than fungi in the soils used.
4.4. Uptake of organic nitrogen molecules
For the direct route to be operative, microorganisms have to be
able to take up organic N molecules. In fact, microorganisms
possess a wide range of transport enzymes for the uptake of organic
N molecules and monomers, such as amino acids and amino sugars.
Table 2
Ammonium (NHþ
4 ) concentrations found to repress microbial enzyme systems for N uptake and assimilation.
Enzyme system/NHþ
4 concentration
Effect on activity (%)
Species
Environment
Reference
NHþ
4 /NH3 uptake systems
10 mM NH4eN
10 mM NH4eN
50 mM NH4eN
100%
43%
99%
Rhizobium Ieguminosarum MNF3841
Aspergillus nidulans
Aspergillus nidulans
Growth medium
Growth medium
Growth medium
O’Hara et al., 1985
Pateman et al., 1973
Pateman et al., 1973
Glutamine synthetase
10 mM NH4eN
100 mM NH4eN
20 mM NHþ
4 eN
142 mM NH4eN
14%
72%
approx. 70%
83%
Corynebacterium callunae
Corynebacterium callunae
Agaricus biosporus
Klebsiella aerogenes MK53
Growth
Growth
Growth
Growth
Ertan, 1992
Ertan, 1992
Baars et al., 1995
Friedrich and Magasanik, 1977
NO
3 uptake system
8.6 mM NH4eN
714 mM NH4eN
57 mM NH4eN
57 mM NH4eN
57 mM NH4eN
65%
60 to 80%
0%
79%
93%
Mixed community
Mixed community
Acotobacter vinelandii
Saccharomyces cerevisiae
Pseudumonas fluorescens
Soil slurries
Soil slurries
Growth medium
Growth medium
Growth medium
Rice
Rice
Rice
Rice
Rice
Nitrate reductase
48 mM NH4eN
100%
Alcaligenes eutrophus
Growth medium
Warnecke- Eberz and Friedrich, 1993
General amino acid permease
10 mM NH4eN
approx. 90%
Saccharomyces cerevisiae NY13
Growth medium
Springael and André, 1998
Acidic Amino Acid Permease
25 mM NH4eN
10 mM NH4eN
approx. 91%
9%
Aspergillus nidulans (germinating)
Aspergillus nidulans (germinated)
Growth medium
Growth medium
Robinson et al., 1973b
Robinson et al., 1973a
L-Glutamate permease
10 mM NH4eN
93%
Aspergillus nidulans
Growth medium
Pateman et al., 1973
medium
medium
medium
medium
The microorganisms were cultivated in the presence of the substrates for the different enzyme systems and varying concentrations of
a
An N-free medium served as control.
and
and
and
and
and
NHþ
4;
Tiedje,
Tiedje,
Tiedje,
Tiedje,
Tiedje,
a
a
1989
1989
1989
1989
1989
except:.
a
2064
D. Geisseler et al. / Soil Biology & Biochemistry 42 (2010) 2058e2067
Utilization of amino acids as N sources involves transport of the
molecules into cells followed by enzymatic removal of the alpha
amino N through deamination or transamination (DeBusk and
Ogilvie, 1984). The transport of amino acids into cells is catalyzed
by functionally specific transport systems that are located in the
cytoplasmatic membrane. Up to twelve kinetically-defined transport systems for groups of structurally related amino acids have
been found in bacterial cells. Fungi posses fewer amino acid
transport systems than bacteria. However, fungal transport systems
are less specific, acting on groups of amino acids with similar
properties. The transport of amino acids is active (Anraku, 1980;
Booth and Hamilton, 1980; Wolfinbarger, 1980). While some
amino acid transport systems are constitutive, the majority of the
systems are under N regulation. Ammonium and high intracellular
concentrations of amino acids repress amino acid transport
systems (Table 2), whereas C, N, or S starvation activates the
synthesis of amino acid transport systems (Oxender et al., 1980;
Wolfinbarger, 1980).
Like the amino acid transporters, microbial transport systems
for peptides are energy dependent and are located in the cytoplasmatic membrane. The upper size limit of peptide transport
systems across membranes seems to be about 600 Da, which
corresponds to a penta- or hexapeptide (Payne and Smith, 1994).
The uptake of peptides as opposed to corresponding mixtures of
amino acids generally requires less energy and has been shown to
be nutritionally superior (Matthews and Payne, 1980).
Transporters for the uptake of amino sugars originating from
chitin and peptidoglycan have been identified for different bacteria
and fungal species. The transporters have been found to be induced
by the presence of the substrate and repressed by glucose
(Plumbridge, 1990; Brinkkötter et al., 2000; Ezquerro-Sáenz et al.,
2006; Alvarez and Konopka, 2007; Vollmer et al., 2008). Given
the widespread occurrence of the N regulation system, it seems
likely that these uptake systems follow the same regulatory
mechanisms as amino acid transporters.
5. Nitrogen uptake pathways in soil
Our review of the enzyme systems involved in the utilization of
N by microorganisms and their regulation shows that in soils, being
an environment with many different N sources and often limited
supply of N, the conditions favoring the direct as well as the MIT
route may be met.
Several studies determined the relative importance of the two
pathways in soil. In a study with labeled leucine and glycine added
to soil, Barraclough (1997) showed that the conventional MIT route
was not operative and the results were consistent with the direct
route. Drury et al. (1991) came to a similar conclusion, based on the
observation that addition of 15NHþ
4 to soil in which net mineralization was occurring resulted in no incorporation of the added 15N
into the microbial biomass. The authors of two other studies
concluded that both pathways, the direct and MIT route, operated
concurrently (Hadas et al., 1992; Luxhøi et al., 2006). Different
approaches were used in these studies to determine the relative
importance of the two routes. Hadas et al. (1992) used the NCSOIL
model to simulate the CeN turnover and 15N distribution among
soil pools in soils amended with NHþ
4 and alanine. Luxhøi et al.
(2006) compared gross N mineralization rates with gross litter N
decomposition rates in soil amended with litter using a NH4-pool
dilution approach. They estimated that about 70% of the litter N was
directly assimilated by soil microorganisms in organic form. These
studies are in line with Geisseler et al. (2009), who added labeled
amino acids to soil samples. They estimated the fraction of N taken
up via the direct route in a microcosm study with amendment of
wheat residues to be 55 and 62% for N-rich and N-poor residues,
respectively. The increased importance of the direct route in the soil
amended with N-poor residues was likely due to the lower mineral
N availability in this treatment (Geisseler et al., 2009). This finding
is supported by the fact that the uptake of organic N molecules is
subject to N regulation. The pathway of N acquisition, however, is
not only dependent on N availability, but also on that of C and
energy. In the presence of readily available C, organic N molecules
are primarily used as N sources, while in the absence of other C
sources, organic N molecules are used for both C and N favoring the
direct route (Gibbs and Barraclough, 1998). This hypothesis is
supported by studies reported earlier; the expression of the genes
encoding for amino acid oxidases have been found to be induced by
amino acids in the presence of a C source (Vallon et al., 1993; Davis
et al., 2005), suggesting that under C limiting conditions, the direct
uptake of the amino acids may be favored.
5.1. Conceptual model for the nitrogen uptake pathway
How is the relative importance of the MIT and direct route
affected by the composition of residues and soil properties? The
literature indicates that N regulation is the dominant mechanism
governing the enzyme systems involved in the utilization of N
containing compounds. Therefore, when the availability of NHþ
4 is
high, enzyme systems for the utilization of alternative N sources are
repressed. Under these conditions, the direct route may be insignificant (Fig. 2). However, the synthesis of extracellular N mineralizing enzymes is also repressed by high NHþ
4 levels reducing the
supply of NHþ
4 (Palenik and Morel, 1990; Vallon et al., 1993). But
enzymes already excreted are no longer under the control of
microorganisms and may continue to contribute to the availability
þ
of NHþ
4 . As long as their activity is high enough to maintain the NH4
concentration at an elevated level, enzymes systems for the uptake
of other N sources are not de-repressed and the MIT route remains
the dominant N uptake pathway. In contrast, when the NHþ
4
availability is low, enzyme systems for the uptake of organic N
molecules are de-repressed and the direct route becomes dominant. In aerated soils, the NHþ
4 concentration is generally low due to
plant uptake and nitrification, which favors the direct route.
When the ratio between available C to N is high, N becomes
limiting relative to C. Under these conditions, net N immobilization
occurs, which results in a depletion of the mineral soil N pool. This
in turn should result in the de-repression of enzyme systems used
for the acquisition of alternative N sources. The direct route should
Fig. 2. Conceptual model of the factors affecting the relative importance of the MIT
and direct route of N uptake by soil microorganisms.
D. Geisseler et al. / Soil Biology & Biochemistry 42 (2010) 2058e2067
therefore be favored over the MIT route, as long as N is limiting
relative to C (Fig. 2). When C becomes limiting relative to N, N
containing organic molecules may be used as C sources. As long as
the NHþ
4 concentration in soil solution is low, the direct route
remains the dominant N uptake pathway. However, when microorganisms use large amounts of N containing molecules as C
sources, excess N is released as NH3, which results in a shift from
the direct to the MIT route. Therefore, the relative importance of the
two pathways depends on the relative availability of C and N as well
as on the concentration of NHþ
4 in soil solution. Carbon and N
availability most likely have the strongest effect on the N uptake
route between a C to N ratio of the available substrates of 20 and 40.
While a C to N ratio greater than about 40 results in net N immobilization, a C to N ratio below 20 normally leads to net N mineralization (Stevenson, 1986; Whitmore, 1996; Cabrera et al., 2005).
However, other properties of residues, such as lignin and tannin
content, may affect N mineralization rates from litter (Fox et al.,
1990; Valenzuela-Solano and Crohn, 2006).
When most of the available C is in the form of molecules that
also contain N, such as amino acids and amino sugars, the direct
route may even be important when NHþ
4 is available at high
concentrations because microorganisms have to meet their C
demand by taking up organic N molecules. In contrast, when
carbohydrates or other readily available C sources are available, the
C of organic N molecules is not needed and NHþ
4 is again preferred
as N source over organic N molecules, favoring their extracellular
deamination. This may explain why extracellular amino acid
oxidase has been found to be induced by amino acids in the presence of a C source (Vallon et al., 1993; Davis et al., 2005). In plant
residues, a large proportion of the C is in the form of carbohydrates,
which reduces the need to take up N containing molecules as C
sources. The MIT route may therefore be dominant when plant
residues are the main source of organic material, as long as the C to
N ratio of the residues is not too high to result in a depletion of the
NHþ
4 pool. In contrast, a large proportion of the C in microbial
residues is in the form of amino sugars and amino acids, which also
contain N. Therefore, when a large proportion of the available
organic material is of microbial origin, the direct route may be more
important. However, the narrow C to N ratio of the organic molecules will result in the release of NH3. If alternative C sources
become available, the microorganisms may switch to the MIT route.
Therefore, we hypothesize that the relative importance of the
direct and MIT route during the decomposition of residues is
determined by three factors: (i) the form of N available, (ii) the
source of C, and (iii) the availability of N relative to C (Fig. 2). In our
conceptual model, the relative amount of N acquired by the MIT and
the direct route is not static, but dynamically changes over time in
response to changing availabilities of different C and N sources.
Carbon and N availability in turn is affected by environmental
factors such as temperature, soil moisture or aeration. In addition,
the heterogeneity of soil makes it likely that both pathways may be
dominant at the same time in different microsites.
6. Conclusions
Nitrogen turnover in soil, including N mineralization and N
uptake by soil microorganisms have been investigated in a large
number of studies. The enzyme systems responsible for these
processes and their regulation, however, have found less attention.
Our review of the enzyme systems involved in the utilization of N
by microorganisms and their regulation shows that in soils, being
an environment with many different N sources and often limited
supply of N, the conditions favoring the direct as well as the MIT
route may be met.
2065
The pathway of N uptake not only affects the competition
between plants and microorganisms, but also the interpretation of
gross N mineralization rates as measured by isotope dilution
methods. If the MIT route is dominant, all the N passes through the
NHþ
4 pool. Therefore, gross N mineralization represents the total
amount of bioavailable N. If in contrast the direct route is dominant,
gross N mineralization determined by isotope pool dilution only
measures the surplus N released from cells.
Based on the literature, the factors most likely affecting the
relative importance of the two pathways during the decomposition
of residues are the form of N available, the source of C, and the
availability of N relative to C. However, most of our knowledge
about the enzyme systems involved in the microbial N utilization
comes from studies conducted with cultured microorganisms. As
the species used as well as the experimental conditions may not be
representative for soil, it is important to determine the relative
importance of the direct and MIT route as well as the factors
affecting the enzyme systems required for these two pathways in
soil directly. This knowledge should result in a better understanding of the soil N cycle in general and the competition between
soil microorganisms and plants for N in particular.
Acknowledgements
We would like to thank two anonymous reviewers for their
valuable comments on the manuscript.
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