FEMS Microbiology Letters Advance Access published June 8, 2016

Nitrate reduction in sulfate reducing bacteria

Angeliki Marietou

Center for Geomicrobiology, Department of Bioscience, Aarhus University, Ny Munkegade 114, DK-8000 Aarhus C, Denmark

Sulfate is the most abundant and thermodynamically stable form of sulfur in our planet with a great diversity of sulfate reducing
bacteria (SRBs) isolated from a wide range of environments. Since the isolation of the first sulfate reducing bacterium at the end of
the 19th century, there has been an increasing understanding of their ecology, physiology, and their role in carbon mineralization.
SRBs gain their cell energy by coupling the oxidation of organic substrates to the reduction of sulfate to sulfide via a stepwise eight-
electron transfer process. Several SRBs are able to use alternative terminal electron acceptors to sulfate such as sulfonates, nitrate,
nitrite, iron, and manganese. Contrary to iron and manganese reduction there are very few studies addressing the ecological and
physiological significance of nitrate reduction in SRBs. The use of nitrate as an alternative terminal acceptor could confer a significant
advantage to nitrate-respiring SRBs under sulfate-limited or oxygen-stress conditions.

Desulfovibrio desulfuricans 27774 is an example of a SRB that can use nitrate reduction for growth in the absence of sulfate
(Marietou et al. 2009). Nitrate is reduced to ammonia via a two step process; first nitrate is reduced to nitrite by the periplasmic
nitrate reductase NapA and then nitrite is reduced to ammonia by the membrane anchored nitrite reductase complex NrfHA
(Marietou et al. 2009). Interestingly, a recent SRB isolate from acidic river sediments, Desulfosporosinus acididurans, has the ability
to reduce nitrate but lacks a putative napA gene (Sánchez-Andrea et al. 2015). In silico analysis of the D. acididurans genome
showed the presence of a putative narG gene (Table 1). NarG is the catalytic subunit of a respiratory membrane-bound nitrate
reductase with the active site localized on the cytoplasmic face of the membrane (Kristjansson and Hollocher, 1979). Further in silico
analysis showed that additional SRBs contain genes in their genomes that encode putative NarG proteins (Table 1). Studies in
Escherichia coli that has both the periplasmic (NapA) and respiratory (NarG) nitrate reductase systems suggest that the Nap system
is active at low nitrate concentrations while the NarG system is utilized at high nitrate concentrations (Potter et al. 1999).

Even though there are limited studies that examine nitrate reduction in SRBs the ability to reduce nitrate is broadly distributed in
three phyla of SRBs (Table 1). Isolates capable of nitrate reduction have been recovered from environments as diverse as the
mammalian oral cavity and gut and marine or freshwater ecosystems (Table 1). Desulfovibrio species isolated from the human oral
cavity, which are exposed to high concentrations of nitrate, contain in their genomes putative respiratory nitrate reductase genes in
addition to putative periplasmic nitrate reductase genes (Table 1). The human oral cavity and the intestinal tract are examples of
habitats with low sulfate concentration (μM) and high nitrate concentrations (mM) while the aquatic environments are characterized
by low nitrate concentrations (μM) and varying sulfate concentrations; from milimolar (marine) to micromolar (freshwater) (Lundberg
et al. 2004). SRB isolates with genes encoding either NapA or NarG were recovered from a wide range of environments with variable
nitrate and sulfate concentrations (Table 1). Therefore, the ability to respire nitrate might not only be linked to habitat adaptation in
the presence of high nitrate concentrations but might confer additional advantages in nitrate-limited environments.

Earlier culture-dependent approaches to isolate and characterize SRBs capable of nitrate reduction might have been compromised
by false negative results due to the use of conventional sulfate-based SRB media. Previous studies have demonstrated that sulfide
concentrations above 700 μM have a strong inhibitory effect on nitrate reduction, while the presence of sulfate in the culture medium
repressed the transcription of genes involved in nitrate reduction (Dalsgaard and Bak, 1994; Marietou et al. 2009). Sulfide inhibition
probably explains the unsuccessful attempts to grow SRBs with nitrate as the sole terminal electron acceptor using conventional,
sulfide reduced, SRB media (Rabus et al. 2006). But even when sulfide is omitted from the medium the use of 10% inoculum of
sulfate-grown cells could transfer sulfide to the fresh medium at concentrations high enough to slow significantly or inhibit nitrate
reduction in SRBs. In addition, the prolonged lag phase (of up to 80 hours) observed for the sulfate-adapted D. desulfuricans when
transferred to a nitrate based medium might could further contribute to the reporting of false negative growth for other SRBs
(Marietou et al. 2009).

Oxygen on the other hand does not have such deleterious effects on the nitrate reductase activity of SRBs. D. desulfuricans 27774
was able to grow at the presence of 18% oxygen by reducing nitrate in the absence of sulfate (Lobo et al, 2007). Even though SRBs
are described as obligate anaerobic microbes their presence and activity has been recorded in oxic environments (Hastings and
Emerson, 1988; Abdollahi and Wimpenny, 1990; Bryukhanov et al. 2011). Some SRBs were able to tolerate exposure and respire
oxygen for the generation of ATP including several Desulfovibrio species (Table 2). A number of strategies have been documented
in SRBs in order to survive exposure to oxygen and its reactive species but there is evidence to support that such strategies are used
as maintenance mechanisms and do not support growth at oxic conditions (Dolla et al. 2006). Oxygen defense strategies include
behavioral changes such as aggregation and aerotaxis, and enzymatic systems for the reduction and removal of oxygen (Dolla et al.
2006).

Could there be a link between the ability of certain D. desulfuricans strains to reduce nitrate and their improved tolerance for oxygen
and survival in oxic environments? The nitrate reducing D. desulfuricans strains 27774 and Essex exhibited higher tolerance to
oxygen compared to the non-nitrate reducing strains (Table 2). Similarly, SRBs isolated from oxic sediments showed higher oxygen
tolerance and oxygen respiration capacity compared to isolates from anoxic sediments (Sass et al. 1997). Freshwater SRB isolates
also appear to have higher oxygen reduction potential compared to marine strains, suggesting a possible adaptation to sulfate-limited
environments (Dolla et al. 2006). The mammalian oral cavity and gut are examples of microaerophilic habitats with low sulfate
concentration (μM) and high nitrate concentrations (mM) that could potentially select for SRBs with increased oxygen tolerance and
ability to use nitrate as a terminal electron acceptor (Lundberg et al. 2004). Several Desulfovibrio species originally isolated from
such environments (mouth and rumen) have the ability to reduce nitrate or the genes for nitrate reduction, including D. desulfuricans
27774 (Table 1). However, even in environments where nitrate concentrations are not high, the ability to reduce nitrate could still be
advantageous in the presence of oxygen concentrations inhibitory for sulfate reduction. Dalsgaard and Bak (1994) reported that the
uptake affinity for nitrate was significantly higher (0,05 μM) compared to sulfate uptake affinity (5 μM) in a D. desulfuricans strain
isolated from a rice field. This allowed effective utilization of nitrate at micromolar concentrations.

SRBs have been found in oxic layers of marine and freshwater sediments, in a brackish water column, and recovered from
oxic/anoxic interfaces such as cyanobacterial mats and biofilms (Frund and Cohen, 1992; Niel et al. 1996, Sass et al. 1997). Their
survival in sediments is dependent on their ability to compete for the available organic material, which in turn is controlled by the
availability of a suitable terminal electron acceptor. Under oxic conditions, when sulfate reduction is inhibited and sulfate is no longer
a suitable oxidant, the ability to use nitrate as terminal electron acceptor would allow SRBs to remain active during prolonged oxygen
exposure and compete with the rest of the community for access to the available organic material. Even though nitrate
concentrations are low at such environments (μM), the free energy associated with the reduction of nitrate to ammonia is higher
resulting to improved growth yield compared to sulfate reduction (Marietou et al. 2009). Sass et al. (1997) carried a detailed study of
the oxic/anoxic interface of an oligotrophic lake which revealed that the number of SRB was similar in the two zones, while they
recovered 13 Desulfovibrio-like SRB strains from the oxic zone, five of which were able to reduce nitrate.

Many aspects of the SRB physiology remain unclear since there are very few studies addressing questions such as oxygen
respiration, nitrate reduction, and the distribution and activity of SRB in oxic environments. Further studies combining improved
culturing approaches, physiological experiments, and in situ measurements would allow us to determine the importance of nitrate
reduction in the ecophysiology of SRBs and the evolution of such a diverse group of microbes.

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Table 1 In silico survey of putative nitrate reductase proteins in selected sulfate reducing bacteria.
− #
Phylum Organism NO3 Growth NapA* NarG Isolation Site Ref
% Identity
Proteobacteria Desulfobulbus japonicus ND 46 - estuarine sediment Suzuki et al. 2007
Proteobacteria Desulfobulbus mediterraneus - 42 - marine sediment Sass et al. 2002
Proteobacteria Desulfobulbus propionicus + 46 - freshwater sediment Widdel and Pfenning, 1982
Proteobacteria Desulforhopalus singaporensis + 45 - sulfidic sediment Lie et al. 1999
Proteobacteria Desulfovibrio cuneatus - 78 - lake sediment Sass et al, 1998
Proteobacteria Desulfovibrio desulfuricans Essex + 90 - tar and sand mix Seitz and Cypionka, 1986
Proteobacteria Desulfovibrio desulfuricans 27774 + 100 - sheep rumen Marietou et al. 2009
Proteobacteria Desulfovibrio profundus + ND ND Marine sediment Bale et al, 1997
Proteobacteria Desulfovibrio sp. 3_1_syn3 ND 89 49 oral cavity NCBI
Proteobacteria Desulfovibrio sp. 6_1_46AFAA ND 89 49 oral cavity NCBI
Proteobacteria Desulfovibrio termitidis + 80 - termite hindgut Trinkerl et al. 1990
Proteobacteria Desulfovibrio zosterae DSM 11974 - 31 - rhizosphere Nielsen et a l. 1999
Proteobacteria Desulfobacterium autotrophicum ND - 52 marine sediment Brysch et al. 1987
Nitrospirae Thermodesulfovibrio islandicus + 31 - microbial mat Sonne-Hansen and Ahring, 1999
Firmicute Desulfosporosinus acididurans + - 49 acidic river sediment Sanchez-Andrea et al. 2015
+, positive; - negative/absent; ND, not determined

* sequences compared to D. desulfuricans 27774 NapA sequence (CAI72603)

# sequences compared to Paracoccus denitrificans NarG sequence (ABL72300)

Table 2 Ability to grow in the presence of oxygen and/or nitrate as terminal electron acceptor for selected Desulfovibrio species.

Desulfovibrio species Nitrate Reduction % O2 Isolation Site Reference

napA Growth Growth
D. desulfuricans 27774 + + 18 sheep rumen Lobo et al. 2007
D. desulfuricans NCIB 8301 ND ND 0.4 clay soil Abdollahi and Wimpenny, 1990
D. desulfuricans Essex + + 1.5 waterlogged clay Marschall et al. 1993
D. desulfuricans CSN ND + 1.5 freshwater Marschall et al. 1993
D. oxyclinae - - 5 cyanobacterial mat Krekeler et al. 1997
D. cuneatus + - 20 littoral sediment Sass et al. 1998
ND, not determined