Download Nitrogen fixation by reductively dechlorinating bacteria

Environmental Microbiology (2007) 9(4), 1078–1083
doi:10.1111/j.1462-2920.2006.01199.x
Brief report
Nitrogen fixation by reductively dechlorinating bacteria
Xiongfei Ju, Liping Zhao and Baolin Sun*
Hefei National Laboratory for Physical Sciences at
Microscale and School of Life Sciences, University of
Science and Technology of China, Hefei, Anhui 230027,
China.
Summary
Significant advances in the ecology, physiology and
genetics of reductively dechlorinating bacteria have
revealed their important environmental roles in bioremediation and in the global chlorine cycle. N2 fixation
has been widely observed in symbiotic, associative
and free-living bacteria. Here we show physiological
and molecular evidence that reductively dechlorinating bacteria are capable of fixing atmospheric
nitrogen. Furthermore, N2 fixation in some of these
dechlorinating bacteria stimulated reductive dechlorination, which should help predict and regulate the
environmental function of dechlorinating bacteria in
in situ bioremediation of chlorinated pollutants.
These results imply that N2 fixation in dechlorinating
bacteria interacts with other biogeochemical cycles
to control the nitrogen status of the anaerobic
ecosystem.
Introduction
A wide range of man-made and naturally produced
chlorinated hydrocarbons has been released into the
environment. These compounds include many of the most
toxic and environmentally persistent pollutants. Their wide
distribution causes a significant risk to public health and
the environment. Fortunately, reductively dechlorinating
bacteria are capable of transforming chlorinated hydrocarbons through reductive dechlorination in anaerobic
environments such as anoxic soils, groundwaters and
sediments (Mohn and Tiedje, 1992; Fetzner, 1998), in an
energy-generating process termed halorespiration. Bacterial culture, carbon isotope fractionation and molecular
biology studies have showed that halorespiring anaerReceived 20 July, 2006; accepted 30 October, 2006. *For
correspondence. E-mail [email protected]; Tel. (+86) 551 360 6748;
Fax (+86) 551 360 7438.
obes are widely distributed in nature, implying their important environmental roles in bioremediation and in the
global chlorine cycle (Drenzek et al., 2001; Sherwood
Lollar et al., 2001; Smidt and de Vos, 2004; Watts et al.,
2005). Some reductively dechlorinating bacteria also
conduct alternative physiological activities such as nitrate
and sulfate reduction, depending on environmental
conditions. In addition, reductive dechlorination may be
compatible with other important physiological features
such as nitrogen fixation through long-term evolutionary
adaptation.
N2 fixation is an energy-consuming process and has
been widely observed in symbiotic, associative and freeliving bacteria. This bacterial physiological feature plays a
tremendous role in ecological sustenance, world agriculture and global nitrogen cycle. Nitrogen availability can
limit microbial growth and affect ecosystem activity (Dixon
and Kahn, 2004). Although N2 fixation is widely distributed
among Bacteria and Archaea (Raymond et al., 2004), this
significant physiological feature has not been revealed
in reductively dechlorinating bacteria. Results from
sequencing of three whole genomes of the halorespiring
bacteria Desulfitobacterium hafniense and Dehalococcoides indicated that nitrogen fixation-related genes are
found in D. hafniense strain Y51 (Nonaka et al., 2006) and
Dehalococcoides strain 195 (Seshadri et al., 2005), but
are missing in Dehalococcoides strain CBDB1 (Kube
et al., 2005). In this research we provide experimental
evidence that reductively dechlorinating bacteria are
capable of fixing atmospheric nitrogen.
Results and discussion
nifH cloning and phylogenetic analysis
We detected N2 fixation capability in halorespiring anaerobes Desulfomonile tiedjei (Shelton and Tiedje, 1984),
Sulphurospirillum multivorans (Scholz-Muramatsu et al.,
1995), Desulfovibrio dechloracetivorans (Sun et al., 2000)
and Desulfitobacterium dehalogenans (Utkin et al., 1994),
which were isolated from distinct habitats and represent
Gram-negative and Gram-positive bacteria. Partial nifH
fragments (approximate 390 bp) were amplified using
degenerate primers (Mehta et al., 2003) corresponding to
the conserved regions of nifH. All these dechlorinating
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd
N2 fixation by reductively dechlorinating bacteria 1079
Fig. 1. Unrooted Neighbour-Joining phylogenetic tree of deduced NifH sequences of diazotrophic and reductively dechlorinating bacteria.
Polymerase chain reaction products (approximate 390 bp) were cloned with TOPO TA Cloning Kit (Invitrogen) and 10 colonies were randomly
selected for sequencing. The obtained nifH partial sequences have been submitted to GenBank databases under Accession number
DQ337203 for nifH-1 of D. dechloracetivorans (ATCC700912); DQ337204 for nifH-2 of D. dechloracetivorans; DQ337205 for nifH of D. tiedjei
(ATCC49306); DQ337206 for nifH of S. multivorans (DSMZ12446) and DQ337207 for nifH of D. dehalogenans (DSMZ9161). The previously
determined nifH gene sequences used for comparisons in this study were selected from other representative diazotrophs that fell into four
clusters and retrieved from the GenBank database at the National Center for Biotechnology Information. Deduced NifH amino acid sequences
were aligned using ClustalW version 1.81, and Unrooted Neighbour-Joining phylogenetic tree was created with the program MEGA version 3.1.
Analysis was bootstrapped and bootstrap values greater than 60 are indicated at respective nodes (1000 replications). The scale indicates the
number of amino acid substitutions per site.
bacteria
possessed
nifH
homologues.
In
D. dechloracetivorans, two divergent nifH homologues
(nifH-1 and nifH-2) were found. nifH-2 grouped within
cluster I of the NifH phylogenetic tree (Fig. 1), which
includes a-, b- and g-proteobacterial nitrogenases; nifH-1
fell into cluster III, which includes diverse anaerobic bacteria like sulfate reducers and reductive dechlorinators.
Single nifH homologues were found in three other bacteria and grouped into different clusters: S. multivorans into
cluster I, D. tiedjei into cluster III and D. dehalogenans
into cluster IV respectively. The distribution of nifH homologues in these dechlorinating bacteria coincides with
conventional opinion that nifH was phylogenetically
diverse in nature (Ueda et al., 1995; Zehr et al., 1998;
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1078–1083
1080 X. Ju, L. Zhao and B. Sun
Fig. 2. Growth optical density (OD600) or reductive dechlorinating product concentration and rate of acetylene reduction to ethylene.
A. Growth of D. dechloracetivorans under sulfate reduction conditions (32 mM sulfate + 10 mM lactate).
B. Reductive dechlorination of 2-chlorophenol (2-CP) to phenol with pyruvate (10 mM) as electron donor by D. dechloracetivorans.
C. Fumarate respiration by S. multivorans (10 mM fumarate + 10 mM pyruvate).
D. Reductive dechlorination of tetrachloroethene (PCE) to DCE with pyruvate (10 mM) as electron donor by S. multivorans. Addition of 5.6 mM
NH4Cl into culture resulted in complete suppression of acetylene reduction (data not shown). The error bars indicate the standard errors of the
measurements. Symbols: 䉭, nitrogenase activity determined by the acetylene reduction with N2 as the sole nitrogen source; 䉬, addition of
ammonia as nitrogen source; 䉲, bacterial growth with N2 as the sole nitrogen source; 䉱, addition of Ar instead of N2 as negative control.
2003; Mehta et al., 2003), suggesting that lateral transfer
of nifH genes may have occurred in diverse
environments. Previous studies have shown that some
symbiotic and free-living bacteria harbour multiple nifH
homologues (Lilburn et al., 2001; Choo et al., 2003), but
how these nifH homologues respond to environmental
cues and are expressed remains unknown. The divergence of nifH genes in these bacteria may proffer them
ecological advantages in natural environments.
N2-dependent growth and acetylene reduction activity
The nitrogenase activity in D. dechloracetivorans and
S. multivorans was demonstrated by their N2-dependent
growth and their NH4+-repressible acetylene reduction
activity (Fig. 2). Dechlorination products such as phenol
and benzoate were analysed by high-performance liquid
chromatography (Agilent 1100 Series) with reverse C18
column (Phenomenex). Dichloroethene (DCE) was monitored by gas chromatography (Agilent 6890 N) equipped
with a flame ionization detector with a DB-1 column
(30 m ¥ 250 mm ¥ 1 mm). For acetylene reduction assay,
6 ml of acetylene was injected into headspace of each
bottle when bacterial growth entered exponential phase.
Ethylene was analysed by gas chromatography (Agilent
6890 N) equipped with a HP-PLOT/AL203 ‘S’ Deactivated
column (50 m ¥ 530 mm ¥ 15 mm). Ethylene production
rate was determined from the slope of the linear regression of time versus ethylene concentration using software
Origin version 7.5.
N2 fixation in D. tiedjei was evidenced by bacterial
growth and reductive dechlorination when N2 was the sole
nitrogen source, although acetylene reduction activity was
not observed in this organism. No growth or reductive
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1078–1083
N2 fixation by reductively dechlorinating bacteria 1081
Fig. 3. Reductive dechlorination and growth under various culture conditions.
A. Reductive dechlorination of 2-CP to phenol with acetate (2.5 mM) as electron donor by D. dechloracetivorans.
B. Reductive dechlorination of 3-chlorobenzoate (3-CB) to benzoate with lactate (10 mM) as electron donor by D. tiedjei. The error bars
indicate the standard errors of the measurements. Symbols: 䉬, addition of ammonia as nitrogen source; 䉲, N2 as the sole nitrogen source; 䉱,
addition of Ar instead of N2 as negative control.
dechlorination was observed in D. dehalogenans under
any growth conditions when ammonia was omitted, indicating that nifH is not functional in N2 fixation in the
bacterium. This result is consistent with the previous
observation that diverse nifH homologues in cluster IV are
not involved in N2 fixation (Raymond et al., 2004; Mehta
et al., 2005).
It is interesting to note that N2 fixation gave rise
to
higher
reductive
dechlorination
rates
in
D. dechloracetivorans and D. tiedjei, which were sustained over repeated feedings of chlorinated compounds
as terminal electron acceptors. When N2 was provided as
the sole nitrogen source and pyruvate was used as
electron donor in D. dechloracetivorans, dechlorination
rate (4.24 mmol l-1 h-1) was the same as with ammonia
(Fig. 2B). On the other hand, when N2 was provided as
the sole nitrogen source and acetate was used as
electron
donor,
reductive
dechlorination
rate
(3.94 mmol l-1 h-1) was higher than that (3.26 mmol l-1 h-1)
of ammonia (Fig. 3A); under both growth conditions,
biomass production was about 3 g of cells (dry weight) per
mol of 2-chlorophenol dechlorinated. This suggests that a
more efficient halorespiration occurred when acetate
served as electron donor, considering that N2 fixation is an
energy-consuming process. Similar dechlorination pattern
was observed in D. tiedjei (Fig. 3B) and this phenomenon
was only observed under dechlorination conditions. Comparing with acetate, pyruvate oxidation produces reducing
equivalents with much more negative redox potential,
which are preferred by both reductive dechlorination and
N2 fixation. The competition for some components of the
electron transport chain may affect dechlorination activity.
Other possible explanations for this phenomenon include
that acetate oxidation provides a more efficient and balanced carbon and nitrogen flux, and/or ammonia assimilation affects reductive dechlorination. Although we do not
have convincing data to explain this phenomenon, it has
significant implications in in situ bioremediation. In some
cases, nitrogen input is necessary in bioremediation of
haloorganics-contaminated sites, where nitrogen source
is limited. Nitrogen input may lead to the dominance of
bacterial populations other than dechlorinators, thereby
decreasing their environmental function. In addition,
excess nitrogen input may cause nitrate contamination in
these sites. This finding not only may provide clues of how
reductively dechlorinating bacteria take advantage of their
diverse physiological features in natural environment, but
also help us optimize nitrogen input and regulate the
environmental function of dechlorinating bacteria in contaminated sites.
Reverse transcription polymerase chain reaction
(RT-PCR)
Reverse transcription polymerase chain reaction was
executed to further detect the capability of N2 fixation in
dechlorinating bacteria. Anticipated RT-PCR products
were amplified from D. tiedjei and S. multivorans cultures
(Fig. 4C and D), further demonstrating the nifH expression and N2 fixation in these bacteria. As predicted, no
RT-PCR product was observed from D. dehalogenans
cultures
(data
not
shown).
Interestingly,
in
D. dechloracetivorans an expression discrepancy of
nifH-1 and nifH-2 was observed (Fig. 4A and B). Only
nifH-1 was expressed under sulfate reduction conditions;
both nifH-1 and nifH-2 were expressed under pyruvate
© 2006 The Authors
Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1078–1083
1082 X. Ju, L. Zhao and B. Sun
Fig. 4. Expression of nifH genes in reductively dechlorinating bacteria. RNA was extracted using SV total RNA Isolation System (Promega)
and DNA was removed using DNase I (RNase-free, TaKaRa) treatment and phenol : chloroform extraction (Burgmann et al., 2003). Reverse
transcription PCR primers were designed based on our nifH sequences and reaction procedure was performed using ImProm-II™ Reverse
Transcription System (Promega) with 4 mM MgCl2.
A. Reverse transcription PCR products (280 bp) by D. dechloracetivorans nifH-1 primers which were forward
5′-CGAGATGGGCCGCAAGGTCAT-3′ and reverse 5′-GTCCAGACCCTCGGACTCCTC-3′.
B. Reverse transcription PCR products (299 bp) by D. dechloracetivorans nifH-2 primers which were forward
5′-CCTGGCATCACTGGGGAAAAAAATC-3′ and reverse 5′-CATAAAAAACGAAATCGAGATCGGGAG-3′.
C. Reverse transcription PCR products (296 bp) by strain D. tiedjei nifH primers which were forward 5′-CACACAAAACACTGTGGCAG-3′ and
reverse 5′-CACTCTCGTCGTATGCTCCAAG-3′.
D. Reverse transcription PCR products (207 bp) by S. multivorans nifH primers which were forward 5′-CTAGCTTCAGAGGCAGGTACAG-3′
and reverse 5′-GTCATAAGCACCCTCTTCCTCA-3′. The reaction conditions were 30 cycles of denaturation at 94°C for 30 s, annealing at
65°C for 30 s and extension at 72°C for 30 s. Symbols: sulfate + N2, sulfate reduction with N2 as the sole nitrogen source; sulfate + NH4+,
addition of NH4+ under sulfate reduction conditions; sulfate + Ar, addition Ar instead of N2 under sulfate reduction conditions; pyruvate + N2,
pyruvate fermentation with N2 as the sole nitrogen source; pyruvate + NH4+, addition of NH4+ under pyruvate fermentation conditions;
2-CP + N2, 2-CP dechlorination with N2 as the sole nitrogen source; 2-CP + NH4+, addition of NH4+ under 2-CP dechlorination conditions;
3-CB + N2, 3-CB dechlorination with N2 as the sole nitrogen source; 3-CB + NH4+, addition of NH4+ under 3-CB dechlorination conditions;
3-CB + Ar, addition of Ar instead of N2 under 3-CB dechlorination conditions; fumarate + N2, fumarate reduction with N2 as the sole nitrogen
source; fumarate + NH4+, addition of NH4+ under fumarate reduction conditions; fumarate + Ar, addition of Ar instead of N2 under fumarate
reduction conditions; PCE + N2, PCE dechlorination with N2 as the sole nitrogen source; PCE + NH4+, addition of NH4+ under PCE
dechlorination conditions; PCE + Ar, addition of Ar instead of N2 under PCE dechlorination conditions.
fermentation and reductive dechlorination conditions.
Further elucidation of the nifH expression pattern in this
organism should aid in our understanding of genetic regulation of N2 fixation in reductively dechlorinating bacteria.
The diversity and global distribution of reductively
dechlorinating bacteria have been revealed in recent
years, suggesting the significant role they play in the
biogeochemical transformations of elements and ecosystem sustenance (Smidt and de Vos, 2004). This
study reveals a heretofore unrecognized role for reductively dechlorinating bacteria in the anoxic ecosystem
and in the global nitrogen cycle. Our results may also
lead to a better understanding of the dynamics, the specific activities and the conditions required for environmental function of dechlorinating populations in in situ
bioremediation.
Acknowledgements
We thank Zhang J. and Wang J. for technical assistance. This
work was supported by the One Hundred Talent Project of
Chinese Academy of Sciences.
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Journal compilation © 2006 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 9, 1078–1083