Antonie van Leeuwenhoek (2009) 96:7987

DOI 10.1007/s10482-009-9338-3

ORIGINAL PAPER

Coordinate induction of dissimilatory ammonification

and fermentative pathways in rhizobia
Wadysaw Polcyn Justyna Podeszwa

Received: 9 December 2008 / Accepted: 25 March 2009 / Published online: 7 April 2009
Springer Science+Business Media B.V. 2009

Abstract Dissimilatory ammonification was indicated as the common feature of ten rhizobial strains
representing six species and three genera. In the
absence of external electron acceptors, all investigated strains were capable of ethanolic fermentation.
However, induction of anaerobic nitrite reduction
was shown to be coupled with a shift of fermentation
towards acetate in all the strains tested. Three
metabolic groups could be distinguished with regard
to nitrite regulation of ethanolic fermentation. It was
shown for Bradyrhizobium sp. strain USDA 3045 that
nitrite is the signal for switching between fermentative pathways although both ammonia and acetate
excretion could not accelerate until nitrate had been
utilized first. In the absence of N oxyanions, ethanol
was indicated as the main product of mannitol
fermentation, five-fold more abundant than acetate.
An inverse composition was found in nitrite-amended
cultures, due to a four-fold increase in acetate
excretion whereas ethanol was kept at low level.
Nitrite-supported fermentation towards acetate has
not been previously reported for rhizobia. This
benefit of this pathway was a two-fold shorter
doubling time on 1% mannitol and 2.5 mM nitrite
compared to no-nitrite media variants but also

W. Polcyn (&)
J. Podeszwa

Institute of Experimental Biology, Faculty of Biology,
A. Mickiewicz University, Umultowska 89, 61-614
Poznan, Poland
e-mail: polcyn@rose.man.poznan.pl

enabled fermentation of the more reduced carbon

compound glycerol.
Keywords Bradyrhizobium sp.

Dissimilatory ammonification
Fermentation

Nitrite regulation
Rhizobia

Introduction
Our previous work showed that anaerobically grown
free-living cells of Bradyrhizobium sp. strain USDA
3045 had nitrate and nitrite reductase activities,
efficiently linked with energy conservation (Polcyn
and Lucinski 2003). Utilization of nitrate as a terminal
electron acceptor is preferential for bacteria since this
is the most efficient respiratory pathway for oxidation
of ubiquinol, generation of transmembrane proton
gradient and thereby ATP synthesis when oxygen is
absent (Berks et al. 1995; Stewart 1988; Unden and
Bongaerts 1997). Respiratory nitrate reduction is
driven by a membrane-bound, cytoplasmically-oriented enzyme encoded by the narGHI genes, which
has also been identified in Bradyrhizobium sp. strain
USDA 3045 (Polcyn 2008a).
As a result of respiratory nitrate reduction, toxic
nitrite accumulates rapidly in the cytoplasm. Two
alternative pathways of dissimilatory nitrite reduction
could be employed. One of them leads to emission of
nitric oxide and therefore is the hallmark of true

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Antonie van Leeuwenhoek (2009) 96:7987

denitrifiers. However, some bacteria could use cytoplasmic, NADH-dependent enzymes for anaerobic
nitrite reduction with subsequent ammonia excretion
to environment (Cole 1996; Mohan and Cole 2007;
Nakano and Zuber 1998; Zumft 1997). It is thought
that nitrite detoxification is the secondary benefit of
the pathway, since it could serve as electron sink
which allows a very efficient reoxidation of NADH
and thereby ATP generation during glycolysis (Tiedje
1988; Zumft 1997). Alternatively, NADH recycling
could be accomplished through fermentative pathways by conversion of pyruvate to more reduced endproducts such as ethanol, which leads to the oxidation
of two molecules of NADH. However, the capacity
for accepting respectively, two and six electrons per
nitrate or nitrite ion reduced through dissimilatory
ammonification renders most of the fermentation
pathway superfluous. As aresult, in ammonifying
bacteria the expression of alternative anaerobic
respiratory pathways and fermentation are subject to
hierarchical control. This ensures maximal ATP yield
since first nitrate and then nitrite is used preferentially
as an electron acceptor (Nakano and Zuber 1998;
Stewart 1988; Unden and Bongaerts 1997). Interestingly, denitrifying bacteria, in contrast to ammonifiers, are not able to carry out efficient fermentation.
This seems to be the basis of the domination of
dissimilatory ammonification over denitrifying activity in environments rich in carbon compounds but
poor in nitrate (Tiedje 1988).

Recently, Bradyrhizobium sp. strain USDA 3045

was shown to be capable of dissimilatory ammonification (Polcyn and Lucinski 2003) which was a
novel finding since nitrate respiring rhizobia are
perceived as denitrifiers (Bedmar et al. 2005; Delgado et al. 2007; OHara and Daniel 1985). In the
present work we designed experiments to indicate
whether both dissimilatory ammonification and fermentative pathways would be simultaneously active
in Bradyrhizobium sp. and nine other rhizobial strains
representing six species and three genera.

Materials and methods

Culture conditions
The origin and source of used rhizobial strains was
listed in Table 1. Strains were maintained on agar
slants containing yeast extract-mannitol medium
(YM) (Somasegaran and Hoben 1994) supplemented
with calcium glycerophosphate (0.1 g L-1) and
casein hydrolysate (1 g L-1). Aerobic cultures were
grown to an OD580 of *1, divided into aliquots,
centrifuged and frozen in liquid nitrogen with 20%
(v/v) glycerol. Cells were stored at -20C until used.
Stored cultures were then transferred to 70 mL
batches of the fresh medium to reach an initial
OD580 of *0.7. Anaerobic conditions were achieved
in sealed flasks after a short time through the

Table 1 Strains used in this study

Acronym

Strain

Host plant

Origin

Source

Bradyrhizobium sp. USDA 3045

Lupinus luteus

USA

USDA

JZ 3.1.3

Bradyrhizobium sp. JZ 3.1.3

Lupinus luteus

Poland

ZBB AU

JZ 4.2.1
JZ 5.2.1

Bradyrhizobium sp. JZ 4.2.1

Bradyrhizobium sp. JZ 5.2.1

Lupinus luteus
Lupinus luteus

Poland
Poland

ZBB AU
ZBB AU

Mam

Mesorhizobium amorphae ICMP 15022

Amorpha fruticosa

China

ICMP

Mlo

Mesorhizobium loti USDA 1129

Lotus corniculatus

USA

USDA

Ret

Rhizobium etli USDA 9032

Phaseolus vulgaris

USA

USDA

RvL

Rhizobium leguminosarum bv. viciae: L

Lathyrus sativus

Poland

ZMR IUNG

RvS

Rhizobium leguminosarum bv. viciae: ST

Lens culinaris

Poland

ZMR IUNG

Sme

Sinorhizobium meliloti USDA 1021

Medicago sativa

USA

USDA

ICMP International Collection of Microorganisms from Plants, Landcare Research, Auckland, New Zealand; USDA United States
Department of Agriculture, Beltsville; ZBB AU Department of Biochemistry and Biotechnology, August Cieszkowski Agricultural
University, Poznan, Poland; ZMR IUNG Department of Agricultural Microbiology, Institute of Soil Science and Plant Cultivation,
Puawy, Poland

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consumption of residual oxygen by the inoculated

bacteria. Anaerobic batch cultures were carried out at
30C in YM broth, amended with 15 mM NH4Cl, to
minimize consumption of dissimilatory excreted
ammonium. Ammonium addition had no influence
on the rate of nitrite or nitrate reduction (data not
shown). The medium contained mannitol at 0.1 or 1%
concentration, substituted with 0.2% glycerol in some
variants, and nitrate or nitrite at various concentrations. Under these conditions, the cultures entered
early-stationary phase after 6 h of anaerobic growth.
Analytical methods
Nitrite was determined according to Senn et al.
(1976). Nitrate and ammonium ions were measured
using Spectroquant test kits (Merck KGaA, Darmstadt, Germany). The initial ammonium concentration
(15 mM) of the media was subtracted from the
measured values. Ethanol and acetate excretion were
monitored with enzymatic test systems (Boehringer,
Mannheim, Germany). Analytical methods were
adapted to microtiter plates and high reproducibility
of calibration curves within the concentration ranges
designed for experiments were obtained.

Results
For Bradyrhizobium sp. strain USDA 3045 the most
effective ammonification (100% conversion) was
achieved with 1.5 mM nitrite (Fig. 1a) whereas
2.5 mM nitrite was reduced to ammonium with at
least 80% efficiency (Fig. 1b). When mannitol was
substituted with glycerol, the stoichiometry of nitrite
to ammonium reduction was found to be unchanged
despite the significantly slower rate of this process
(Fig. 1c). Dissimilatory ammonification of higher
initial nitrite concentrations was impaired in strain
USDA 3045. Only 15% of 6 mM nitrite had been
converted to ammonium within the 6-hour experimental time frame, resulting in 5 mM of unreduced
nitrite still remaining in the medium (Fig. 1d).
Figure 2 shows that in the absence of N oxyanions
strain USDA 3045 made efficient use of fermentative
pathways with ethanol as the main product. Fermentation of 0.1% (5.5 mM) mannitol resulted in the
production of 6.28 (0.57) mM of ethanol and 1.17
(0.53) mM of acetate. A ten-fold higher supply of

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mannitol did not result in a significant increase in the

level of fermentation products, indicating the limits
of fermentative metabolism of this carbon source by
rhizobia.
In the presence of nitrite, the fermentation products showed an inverse composition of up to four-fold
increase of acetate excretion, with the level as high as
4.88 (0.23) mM for 6 mM nitrite (Fig. 1eh). The
level of ethanol was below 1 mM until the nitrite
concentration was higher than 0.36 (0.15) mM
(Fig. 1a). However, as soon as the nitrite concentration dropped below this threshold value a backward
shift of the fermentative pathway towards ethanol
was observed. This effect occurred after 3 h of
growth on 1.5 mM nitrite (compare Fig. 1a, e) and
after 4.5 h of culture amended with 2.5 mM nitrite
(compare Fig. 1b, f). When nitrite consumption was
slower, e.g., on glycerol (Fig. 1c) or on 6 mM nitrite
(Fig. 1d), ethanol excretion did not increase within
the time frame of the experiments (Fig. 1g, h).
Dissimilatory metabolism of rhizobia grown on
3 mM nitrate exhibited two successive stages of
nitrate reduction followed by nitrite ammonification
(Fig. 3a). Initially, nitrate reduction resulted in nitrite
excretion whereas only 20% of NNO3- was converted to ammonium. Ammonification of accumulated external nitrite proceeded in the period
following, beginning from the time when no more
than 0.5 mM nitrate remained in the medium
(Fig. 3a). During the nitrate reduction stage, fermentation apparently could not proceed and only low
amounts of acetate and ethanol were detected
(Fig. 3b). However, once nitrate became exhausted
(after ca. 3 h of growth), the level of acetate excretion
started to increase [up to 3.97 (0.58) mM], simultaneously with an acceleration of nitrite reduction,
whereas the low ethanol concentration remained
unchanged.
Table 2 summarizes the effect of alternative carbon
sources (two mannitol concentrations and glycerol) on
the growth yield of Bradyrhizobium sp. strain USDA
3045 and the efficiency of the ammonifying and
fermentative pathways. For soil bacterial communities a maximal induction of dissimilatory ammonification is expected in conditions characterized by a
high ratio of available carbon to nitrate (Tiedje 1988).
However, little difference was found between 0.1 and
1% mannitol with respect to the levels of ammonia or
fermentation products excretion. Nevertheless, the

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Antonie van Leeuwenhoek (2009) 96:7987

Fig. 1 Dissimilatory nitrite

reduction (ad) and
fermentation products
formation (eh) by
Bradyrhizobium sp. USDA
3045, grown with 0.1%
mannitol and 1.5 mM nitrite
(a and e); 2.5 mM nitrite
(b and f); 6 mM nitrite (d
and h) or with 0.2%
glycerol and 2.5 mM nitrite
(c and g). The nitrite (m)
reduction and ammonium
(e), ethanol (j) and
acetate (d) excretion were
determined in the growth
media. The values are
means and standard
deviations of measurements
performed in duplicate and
determined in four to eight
independent experiments
for each culture condition.
Microbial growth was
measured as OD580 (X).
Standard deviations of
growth rates are not shown,
however, less than 20%
variation was found

condition promoting most growth of rhizobia was a

1% mannitol-rich culture with 2.5 mM nitrite. In
contrast, the highest doubling time was found for
0.1% mannitol and 6 mM nitrite, in spite of the high
level of acetate-excreting fermentation. In addition,
nitrate and nitrite dissimilation also allowed growth
on glycerol, a carbon source more reduced than
mannitol. Substitution of mannitol with glycerol did
not change the levels of ammonia and excretion of
fermentation products. However, growth of cultures
on nitrite was significantly slower.
The model of dissimilatory metabolism established for strain USDA 3045 was then compared with
several other rhizobial strains (Table 3). In addition

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to fermentative growth, all of investigated strains

were able to grow by nitrate and nitrite dissimilation.
The strains investigated strains differed from each
other in the rate of nitrate reduction, although 2 mM
nitrite could be entirely consumed until 4.5 hgrowth
(data not shown). The end product of anaerobic nitrite
reduction was ammonium, with 75% average efficiency. However, Mesorhizobium amorphae and
Rhizobium etli ammonified 100% of the available
nitrite, whereas Mesorhizobium loti converted no
more than 55% of the available nitrite to ammonium.
In the case of nitrate-fed cultures, rhizobia did not
excrete ammonium until the nitrate disappeared from
the medium, similarly to strains USDA 3045. An

Antonie van Leeuwenhoek (2009) 96:7987

83

Fig. 2 Fermentation products formation by Bradyrhizobium

sp. USDA 3045 grown without N oxyanions addition with
a 0.1% mannitol [ethanol (j) and acetate (d)] or b 1%
mannitol [ethanol (h) and acetate (s)]. The values are means
and standard deviations of measurements performed in
duplicate and determined in three independent experiments.
Microbial growth was measured as OD580 (X). Standard
deviations of growth rates are not shown, however, less than
20% variation was found

Fig. 3 a Dissimilatory nitrate reduction and b fermentation

products formation by Bradyrhizobium sp. USDA 3045 grown
with 0.1% mannitol and 3 mM nitrate. The nitrate (4) or
nitrite (m) reduction and ammonium (e), ethanol (j) or
acetate (d) excretion were determined in the growth media.
The values are means and standard deviations of measurements
performed in duplicate and determined in four independent
experiments. Microbial growth was measured as OD580 (X).
Standard deviations of growth rates are not shown, however,
less than 20% variation was found

exception was Bradyrhizobium sp. JZ 3.1.3 strain, for

which ammonification proceeded simultaneously
with nitrate reduction (data not shown).
The strains investigated strains could be divided
into three metabolic groups with respect to the course
of ethanolic fermentation (Table 2). In the absence of
N oxyanions group I, consisting of bradyrhizobial
strains and Sinorhizobium meliloti, resembled the
metabolism of Bradyrhizobium sp. strain USDA 3045
and excreted 6.6 to 7 mM ethanol. This was at a 1.5fold higher level than strains in group II, which
consisted of rhizobial strains and Mesorhizobium
amorphae. An exceptionally low level of ethanolic
fermentation in no-nitrite cultures was found in
Mesorhizobium loti (group III), resulting in no more
than 1.57 (0.12) mM ethanol excreted.
After nitrite or nitrate addition, ethanol excretion
was initially at very low level (ca. 0.4 mM) in all tested
strains (Table 2). After nitrite depletion, the ethanol

concentration remained unchanged within the time

frame of experiment for strains in groups II and III.
However, for strains in group I the ethanol concentration rapidly increased to 5 (0.5) mM (data not
shown). Nevertheless, all these groups reproduced the
pattern of induction of the acetate-producing pathway
found in strain USDA 3045. When nitrite had been
present in the cultures, the amount of excreted acetate
was 3 fold higher than in control no-nitrite variants
(Table 2). In addition, strains in group I were able to
reuse at least half of the excreted acetate as a carbon
source (data not shown). Similar features were exhibited by strain USDA 3045 (Fig. 3b).

Discussion
In the present work the operation of fermentative
pathways in relation to the course of dissimilatory

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Antonie van Leeuwenhoek (2009) 96:7987

Table 2 Effect of various culture conditions on efficiency of ammonifying and fermentative pathways and on the growth yield of
Bradyrhizobium sp. USDA 3045
Culture conditionsa

Doubling time

Ammonium produced (mM)

Ethanol produced (mM)b

Acetate produced (mM)b

0.1 M/0 N

4.19

6.28 (0.57)

1.17 (0.53)

1.0 M/0 N

6.12

6.70 (0.7)

1.50 (0.49)

0.1 M/1.5 N

4.41

1.49 (0.27)

1.42 (0.39)

3.71 (1.36)

0.1 M/2.5 N

4.19

2.22 (0.29)

1.00 (0.08)

3.99 (0.3)

1.0 M/2.5 N

2.59

2.13 (0.23)

1.05 (0.11)

4.23 (0.24)

0.1 M/6.0 N

8.04

0.87 (0.15)

0.86 (0.15)

4.52 (0.54)

1.0 M/6.0 N

5.67

1.08 (0.28)

0.83 (0.15)

4.88 (0.23)

0.2G/2.5 N

7.36

1.40 (0.3)

0.89 (0.05)

4.18 (1.21)

0.1 M/3 N

6.25

1.18 (0.2)

1.11 (0.13)

3.97 (0.58)

0.2G/3 N

5.98

1.55 (0.2)

0.77 (0.16)

3.88 (1.06)

Nitrite dissimilation

Nitrate dissimilation

The values are means and standard deviations of measurements performed in duplicate and determined in four to eight independent
experiments for each culture condition. Standard deviations of growth rates are not shown, however, less than 20% variation was
found
a
N-initial nitrite or nitrate concentration (mM), M-percent mannitol concentration (0.1% = 5.5 mM), G-percent glycerol
concentration (0.2% = 21.7 mM)
b

The level of ethanol or acetate excretion measured when ammonium production reached maximal level but nitrite remained at no
less than 0.4 mM concentration. Below this threshold value (see Fig. 1a, e) nitrite regulation was abolished and the backward shift of
fermentative pathways towards ethanol was observed

Table 3 Effect of ammonification of 2 mM nitrate or nitrite on fermentative pathways in various rhizobial strains
Ammonium produced (mM)

Ethanol produced (mM)a

NO2-

NO3-

0NOx

NO2-

NO3-

0NOx

NO2-

NO3-

JZ 3.1.3

1.3 (0.5)

1.9 (0.1)

7.1 (0.3)

1.4 (0.1)

0.3 (0.0)

0.4 (0.2)

2.6 (0.4)

2.7 (0.2)

JZ 4.2.1

1.3 (0.5)

1.5 (0.2)

6.6 (0.3)

1.5 (0.4)

0.3 (0.0)

0.3 (0.0)

2.6 (0.7)

2.3 (0.2)

JZ 5.2.1

1.7 (0.1)

1.3 (0.2)

6.9 (0.3)

1.7 (0.3)

0.3 (0.0)

0.5 (0.2)

3.1 (0.6)

3.1 (0.3)

Sme

1.3 (0.0)

1.0 (0.4)

6.6 (0.2)

1.7 (0.7)

0.4 (0.1)

0.6 (0.3)

3.2 (0.8)

3.1 (0.1)

RvS

1.3 (0.5)

1.5 (0.3)

5.5 (0.3)

0.4 (0.0)

0.4 (0.0)

0.6 (0.1)

2.8 (0.2)

1.9 (0.1)

RvL

1.6 (0.4)

1.6 (0.5)

4.5 (0.6)

0.4 (0.0)

0.4 (0.0)

0.9 (0.3)

3.4 (0.3)

2.9 (0.5)

Ret

2.0 (0.1)

2.0 (0.1)

4.4 (0.3)

0.4 (0.0)

0.4 (0.0)

1.0 (0.0)

3.1 (0.4)

2.6 (0.3)

Mam

2.0 (0.1)

2.0 (0.1)

4.5 (0.2)

0.4 (0.0)

0.4 (0.0)

1.2 (0.2)

2.7 (0.1)

2.9 (0.3)

1.1 (0.1)

1.2 (0.1)

1.6 (0.1)

0.4 (0.0)

0.4 (0.0)

1.7 (0.4)

4.4 (0.4)

4.4 (0.1)

Rhizobial strain

Acetate produced (mM)a

Group Ib

Group II

Group III
Mlo

The values are means and standard deviations of measurements performed in duplicate and determined in two to four independent
experiments for each strain and culture condition
a
The maximal level of ethanol or acetate excretion in the presence of nitrate or nitrite (see comments to Table 1). About 0.1%
(5.5 mM) mannitol was used as the carbon source for fermentation
b

Three metabolic groups distinguished with regard to the course of ethanolic fermentation in the absence or in the presence of nitrite

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nitrate or nitrite ammonification was studied. We

documented for Bradyrhizobium sp. strain USDA
3045 that the almost exclusive product of anaerobic
nitrite reduction was ammonium, although such a
yield from this pathway was restricted up to 2.5 mM
initial nitrite concentrations. In the absence of
external electron acceptors, the strain investigated
was able to carry out fermentative growth on
mannitol, with ethanol as the main product (five-fold
abundant than acetate). Mannitol fermentation was
differentially regulated in the presence of nitrite since
a shift of the fermentative pathways towards acetate
was observed. Higher initial nitrite concentrations
had a very similar effect on fermentation despite the
low efficiency of ammonia production, with more
than 80% of 6 mM nitrite remaining unreduced. This
suggests that it is not the intensity of ammonification
but the reception of the nitrite signal that is responsible for switching between fermentative pathways.
Bacterial nitrite ammonification could be driven
by periplasmic NrfA and/or cytoplasmic NirBD
(NasDE) reductases (Cole 1996; Mohan and Cole
2007; Simon 2002). However, inspection of a database compiled from complete rhizobial genomes
(Oct. 3, 2008, http://bacteria.kazusa.or.jp/rhizobase)
showed only the latter enzyme to be present. Cytoplasmic nitrite reductases have not been studied in
rhizobia and may primarily have an assimilatory
function. However, it has been suggested for other
bacteria that, under anaerobiosis, cytoplasmic nitrite
reductase could supply NAD? to allow the proper
functioning of glycolysis (Tiedje 1988). This pathway may be considered fermentative nitrite ammonification and the well-documented examples are
Bacillus subtilis (Nakano and Zuber 1998) and
Escherichia coli (Stewart 1988).
In this study, nitrate-amended cultures showed
tendencies similar to the nitrite containing variants.
However, both ammonia and acetate excretion did
not accelerate until nitrate had been utilized first. This
contrasts with the elevated acetate production at the
initial stages of cultures grown on nitrite. Such
regulatory effects support a hypothesis that fermentation towards acetate is coupled with nitrite ammonification but not with nitrate respiration. Inhibition
by nitrate of both nitrite reduction and fermentative
pathways is known from enteric bacteria and is
explained by competition from nitrate reductase for
electron donors (Stewart 1988).

85

Using NADH primarily for anaerobic nitrate

reduction is advantageous for bacteria since it enables
respiratory ATP production. However, after nitrate
depletion, ammonifying nitrite reduction may be used
to accept electrons and thereby aid survival when
nitrate respiration is limited. In recent work on strain
USDA 3045 (Polcyn 2008b), proteolytic degradation
of membrane-bound nitrate reductase was shown as
the result of nitrate depletion. The data from the
present paper support the hypothesis that such a downregulation of respiratory nitrate reductase might be
followed by the switch from respiratory metabolism to
fermentative pathways coupled with ammonifying
nitrite reduction.
The present study demonstrated that fermentative
reduction to ethanol ceased after induction of nitrite
ammonification and a less reduced compound (acetate) was formed instead. Such a mechanism for
coordinating dissimilatory pathways has not been
reported so far in rhizobia. In enteric bacteria,
fermentation towards acetate is energetically the
most favorable due to additional ATP synthesis
driven by Acetyl CoA kinase, which is not possible
through alternative synthesis of the reduced compounds such as ethanol (Stewart 1988). Since
mannitol-rich rhizobial cultures had two-fold shorter
doubling times on 2.5 mM nitrite compared to
control no-nitrite variants (Table 2), we suggest that
rhizobia also benefit from nitrite-supported acetate
formation (Fig. 4). In E. coli, proceeding with this
pathway depends on the reduced state of the available
carbon source or the presence of an external electron
acceptor (Dharmadi et al. 2006). As we demonstrate,
disposing of reducing equivalents through nitrite
ammonification was efficient enough to make rhizobia competent to ferment glycerol (a highly reduced
carbon substrate) to acetate. Nevertheless, strain
USDA 3045 strain was unable to ferment glycerol
in the absence of external electron acceptors (Polcyn
and Lucinski 2003), as has been reported for E. coli
(Dharmadi et al. 2006).
Subsequent experiments showed that dissimilatory
metabolism of several other rhizobial species resembled, with minor modifications, the model determined
for Bradyrhizobium sp. USDA 3045. Induction of
ammonifying nitrite reduction coordinated with a shift
of the fermentative pathways towards acetate was an
attribute of all the strains tested, which represent six
rhizobial species and three genera. These results

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Antonie van Leeuwenhoek (2009) 96:7987

2 P06R 077 27 from the Polish State Committee for Scientific
Research.

References

Fig. 4 Proposed model of fermentation processes in relation

to dissimilatory nitrite ammonification in rhizobia during
anaerobic growth on mannitol. Note that the presence of
additional fermentative pathways is also possible

indicate that dissimilatory ammonification and nitritesupported fermentation towards acetate may be catabolic pathways that are widely distributed among
rhizobial species, which has not been reported previously. Nevertheless, three metabolic groups could be
distinguished with regard to the level of ethanol
excretion in the absence or in the presence of N
oxyanions. For strains in group I, ethanolic fermentation started dynamically as soon as nitrite had been
consumed whereas in the two latter groups this
remained unchanged. This suggests that the inhibitory
effect of N oxyanions on the synthesis or activity of
the enzymes of the ethanol-excretion pathway differs
depending on the rhizobial species.
Acknowledgments We are grateful to Prof. Wanda Maek,
Prof. Cezary Madrzak and Dr. Joanna Kroliczak for providing
the strains used in this study. This work was supported by grant

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