Morphologial , physiological and phylogenetic characterization and functional verification of an anaerobic strain SN 22-2 with simultaneous capabilities of denitrification and sulfate reduction

Anaerobic bacterium (strain SN22-2) with simultaneous capabilities of denitrification and sulfate reduction was enriched and isolated in a specially designed medium by anaerobic Hungate technology. The characterisation of SN22-2 was conducted from the morphological, physiological and phylogenetic aspects. It was a rod-shaped, gram-negative, 0.2 to 0.7 μm in width and 3.0 to 5.5 μm in length. Most of the fatty acids of SN22-2 were distributed among C12:0–C19-CYC-9 and the main fatty acids consisted of C16:1-CIS-9-FAME, C16:0-FAME, C18:0-FAME and C18:0 DMA. Based on the morphological and physiological characteristics as well as the phylogenetic analysis, SN22-2 was identified as Bacillus coagulans since its 16S rDNA bore 99% resemblance to that of B. coagulans (Accession No. AB240205). The dissimilatory sulfite reductase (Dsr) and the nitrite reductase (nirS) genes were successfully amplified and cloned from strain SN22-2. The removal efficiencies of NO3 and SO4 2were up to 96.48 and 97.98% when the initial concentrations of NO3 and SO4 2were 4368.45 and 586.70 mg/L, respectively. High activities of nitrite reductase and sulfate reductase were detected in the culture medium of SN22-2. The analysis of gene sequences and the enzyme activities as well as the nitrate and sulfate removal efficiency data confirmed that strain SN22-2 had simultaneous denitrifying and sulfate reducing capabilities. The characterization of the present isolate had important ecological implications in treating organic wastewater containing high concentration of sulfate and nitrogen.


INTRODUCTION
The sulfate reducing bacteria (SRB) were characterized by their ability to reduce sulfate to sulfide with the simultaneous oxidation of the organic substrates (Jenneman et al., 1986).Sulfide production is a major concern to the petroleum industry since it is toxic and corrosive and causes plugging due to the formation of insoluble iron sulfides (Reinsel et al.,1996 ).Inhibition of SRB by nitrate (NO 3 -) injection had been widely investi-*Corresponding author.E-mail: weilihit@126.com.gated.Sulfide production was temporarily inhibited due to the preferential use of nitrate as an electron acceptor.The prolonged decrease of SRB as a result of prolonged exposure to an oxidzing envrionment was due to the buildup of N 2 O or NO or both by nitrate reduction.Similarly, other studies (Senez and Pichinoty,1958) also concluded that the production of intermediate metabolites (N 2 O and NO) by denitrification led to the raise of redox potential and thus long-term inhibition sulfate reduction was realized.
It was reported that the genera of Desulfovibrio, Desulfobulbus and Desulfomonas could utilize nitrate as electron acceptor and obtain energy for their growth.Successful dissimilatory nitrate reduction to ammonia was achieved by a strain of Desulfovibrio desulfuricans (a strict anaerobic SRB), which confirmed that dissimilatory nitrate reduction to ammonia was not confined to facultatively anaerobic bacteria (Keith and Herbert,1983).Marietou et al. (2005) found that the nitrate reduction by strain D. desulfuricans ATCC 27774 was catalyzed by a periplasmic nitrate reductase system including a unique tetraheme c-type cytochrome, NapM.Liu et al. (1994) detected the hexaheme nitrite reductase for the first time in nitrate-respiring D. desulfuricans ATCC 27774.It demonstrated that the nitrate reductase isolated from D. desulfuricans ATCC 27774 was a periplasmic enzyme and seemed to belong to a class of monomeric enzymes (Moura et al.,1997).K. Mori isolated a novel type of moderate thermophilic autotrophic SRB from a hot spring, which could grow with nitrate in place of sulfate (Mori et al., 2003).Medium applied for the isolation of SRB contained NaNO 3 as nitrogen source, which indicated that the isolated strain could utilize nitrate (Hong et al.,2010).
In an anaerobic baffled reactor (ABR) process operated in our lab for the inhibition of SRB with the dosage of nitrate, the presence of bacteria with simultaneous capabilities of denitrification and sulfate reduction was proposed.In the present study, a special medium for the isolation of strains with simultaneous denitrifying and sulfate reducing capablities was designed.An anaerobic strain with simultaneous denitrifying and sulfate-reducing capablities, named as SN22-2, was isolated.Morphological observation, physiological tests, fatty acids analysis, phylogenetic analysis of 16S rDNA, cloning and sequencing of Dsr and nirS genes and detection of enzyme activities were conducted on SN22-2 for the function verification of its simultaneous denitrifying and sulfate reducing capablities.The characterization of the present isolate had important ecological implications in treating organic wastewater containing high concentration sulfate and nitrogen generating from light chemical engineering industries, food processing and pharmaceutical factories.

Strain source
Sample for strain isolation was the activated sludge collected from a denitrification-based SRB inhibition bioreactor.The reactor was fed with a synthetic wastewater, which contained 600 mg/L SO4 2-, 600 mg/L NO3 -, 2350 mg/L COD (pH, 8.0).

Medium and isolation
Techniques of Hungate, the most probable number (MPN) and the roll tube were applied for the isolation of the bacterial strain.A special medium was designed for the isolation of strains with simultaneous denitrifying and sulfate-reducing capabilities.The liquid medium was composed of 1750 mL distilled water and the following salts: Na2SO4, 4 g; KNO3, 2.0 g; NaNO3, 2.0 g; MgSO4•7H2O 1g; K2HPO4 0.5 g; KNaC4H4O6•4H2O 5 g; KH2PO4 1.0 g; CaCl2•2H2O 0.2 g (final pH, 7.5).Resazurin (0.2%, wt/vol) was added as redox indicator to the medium.Then, the medium was boiled for complete dissolution and 0.5 g L-cysteine was added.After that, high purity nitrogen was introduced to drive away oxygen for 30 min.The medium was autoclaved for 20 min at 121°C.The sterilized medium was cooled and 0.1 mL 3% FeSO4•(NH4)2SO4 was added.Powdered agar (1.6%, wt/vol) was added when solid medium was prepared before sterilization.

Characterization of the isolated strain
Gram-stained strains were observed by electron microscope (CX31, Olympus, Japan).Morphological observations of the cells were also carried out through atomic force microscope (AFM) (Di BioScope, Veeco, USA).The physiochemical tests of the isolate were conducted according to the guidance of "Bergey's Manual of Determinative Bacteriology".The microbial fatty acids of the isolated strain were identified using the Sherlock MIS (MIDI Sherlock, MIDI, USA) equipped with the gas chromatograph (6890N, Agilent, USA).

Identification of the isolate by 16S rDNA-based phylogenetic analysis
The DNA extraction of the isolate was conducted with a bacterial genomic DNA extraction kit (TaKaRa, Dalian, China).The 16S rDNA genes were amplified by using universal primers (TaKaRa, Dalian, China).The 50 µL PCR mixture contained 2 µL template DNA, 0.5 µL rTaq polymerase (TaKaRa, Dalian, China, 5 U/µL), 5 µL 10×PCR buffer, 4 µL of dNTPs (2.5 mM of each dNTP ), 1 µL of each primer (50 mM) and 37.5 µL deionized water.The thermal cycling included: 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 30s, annealing at 58 °C for 45 s, extension at 72°C for 90 s, and a final extension at 72°C for 10 min.PCR product was subjected to agarose gel electrophoresis.Target fragment was purified using an agarose gel DNA purification kit (TaKaRa, Dalian, China) and ligated to a TA type vector pGEM-T (Promega, Madison, WI, USA).Ligated product was transformed to an E. coli chemical competent cell TOP10 (Tiangen Biotech, Beijing, China).Transformed E. coli was selected on Lysogeny Broth (LB) solid culture medium with Amp (50 µg/mL) and X-gal (200 µg/mL).The transformed E. coli was analyzed using the universal primers T7 and SP6 and sequenced by the Shanghai Sangon Biological Engineering Co., Ltd, China.The sequenced 16S rDNA were compared with a nonredundancy nucleotides database by using the basic local alignment search tool (BLAST).Multiple sequence alignment was conducted using BioEdit Version 5.06, and a phylogenetic tree was constructed by MEGA Software Version 4.1 with a neighbor-joining method.

The cloning and sequencing of nirS gene
The nirS gene of the isolate was PCR amplified from its DNA extract by using the primer pair 832F (5'-TCACACCCCGAGCCGCGCGT-3') and 1606R (5'-AGKCGTTGAACTT KCCGGTCGG-3') (Kutyavin et al., 2000), and otherwise the same PCR reaction mixture and thermal cycling procedure as described above.The cloning and sequencing of the nirS gene were identical to that of the 16S rDNA.

Analytical methods
The concentrations of NO3 -, NO2 -and SO4 -were detected by ion chromatography (ICS-3000, Dionex, USA).The activity of nitrite reductase was determined according to the method of Cole et al. (1974).The determination of sulfite reductase followed the method described by Ostrowski et al. (1989).

Morphological and physiochemical characterization of the isolated strain SN22-2
An anaerobic strain, named as SN22-2, was isolated for its simultaneous denitrification and sulfate-reducing capabilities.Figure 1 shows that strain SN22-2 was long rod-shaped, 0.2 to 0.7 µm in width and 3.0 to 5.5 µm in length.It was Gram-positive with polar flagella.Its colony on the agar plate was white and round in moderate size with convex surface.The results of physiochemical tests are listed in Table 1.It shows that strain SN22-2 was mesophilic and grew at temperatures ranging from 20 to 40°C with an optimum growth temperature of 36°C.Optimal growth occurred at pH 7.5.Growth was observed on the following substrates: glucose, citrate and grease.Strain SN22-2 was not able to grow using fructose, lactose, sucrose, ethanol, starch, urea and gelatin as electron donors.The nitrate reduction and H 2 S production tests confirmed that strain SN22-2 had denitrification and sulfate reduction capabilities.Besides, the analysis of fatty acids analysis indicated that most of the fatty acids were distributed around C 12:0 -C 19-CYC-9 .The main fatty acids consisted of C 16:1-CIS-9-FAME , C 16:0-FAME , C 18:0-FAME and C 18:0 DMA , which accounted for 62.76% of the total fatty acids with 6.35, 21.42, 28.95 and 6.04%, respectively.

Phylogenetic analysis based on the 16S rDNA sequences
A 1468 bp sequence of 16S rDNA gene from SN22-2 was obtained.The nucleotide sequences of 16S rDNA of SN22-2 had been deposited in the GenBank database under accession numbers DQ450463.Representative strains that had high homology with SN22-2 were selected and the phylogenetic tree was constructed.As shown in Figure 2, the average genetic distances of the 16 strains was 0.043.The resemblance between strain SN22-2 and B. coagulans (Accession No. AB240205) was 99%.Consequently, based on the results of morphological observation, physiological tests, fatty acids and 16S rDNA analysis, strain SN22-2 was identified as B. coagulans and named as B. coagulans SN22-2.

Analysis of nirS sequence
Nitrite reductase reduces nitrite to NO and is encoded by two structurally different genes: nirK, encoding a coppercontaining enzyme, and nirS encoding cytochrome cd1 (Garbeva et al., 2007).These genes are functionally and physiologically equivalent and appear to be mutually exclusive.Figure 3 shows an 893 bp fragment of nirS gene obtained and the gene sequences were deposited in the GenBank database under accession number    DQ450463.A 97% resemblance was obtained when the sequenced nirS gene was compared with reference sequence of gene under accession number of AF549053, which were the conserved sequences of nir gene.

Analysis of Dsr sequence
For SRB, the Dsr gene, which encodes the dissimilatory sulfite reductase , was a key enzyme in sulfate reduction that catalyzes the reduction of sulfite to sulfide and hence is required by all sulfate reducers.As shown in Figure 4, a 243 bp fragment of Dsr gene was obtained.A 98% similarity was obtained when the sequenced Dsr gene was compared with the sequence of α-subunit Dsr gene of D. desulfuricans (AF273034), which were the conserved sequences of nir gene.

The denitrification and sulfate-reduction capabilities of strain SN22-2
The bacterial solution of SN22-2 was inoculated in the specially designed medium with a 5% volume ratio to the medium.The concentrations of SO 4 2-, NO 3 -and NO 2 were detected at daily intervals for the assessment of its denitrification and sulfate-reduction capabilities.As shown in Figure 5, the concentration of NO 3 -declined from 4368.45 mg/L in the beginning to 88.12 mg/L at the end.The removal efficiency of NO 3 -was 82.75% on the 4 th day and the highest was 97.98%.The greatest concentration of NO 2 -was detected on the 4 th day (1493.60mg/L), which was produced as the intermediate product when the concentration of NO 3 -dropped dramatically.Along with the cultivation of strain SN22-2, the NO 2 -concentration was gradually declined without  accumulation.Thus, strain SN22-2 had great denitrification capability.Figure 6 shows that the concentration of SO 4 2-dropped from 586.71 to 20.63 mg/L during the denitrification process of strain SN22-2.The removal efficiency of SO 4 2-on the 4 th day was up to 90.84% and the highest was 96.48%.It demonstrated that strain SN22-2 was efficient for sulfate reduction.Consequently, SN22-2 was a strain with simultaneous capabilities of denitrification and sulfate reduction.

The detection of enzyme activities
Nitrite reductases are a group of enzymes that catalyze the reduction of nitrite.Dissimilatory sulfite reductase was a key enzyme in sulfate reduction that catalyzes the reduction of sulfite to sulfide. Figure 7 shows that the activity of the nitrite reductase enhanced from 0.22 U/mL on the 1 st day to 0.543 U/mL on the 6 th day.For the sulfite reductase, its activity increased from 0.38 U/mL on the 1 st day to 0.69 U/mL on the 7 th day.It gave further evidence that SN22-2 was a strain with denitrification and sulfate reduction capabilities.
Evidence had been presented that SRB could utilize various types of refractory organics (such as polycyclic aromatic hydrocarbons and n-alkenes) as carbon sources (Tsai et al., 2009).The use of SRB for metal and radioactive waste remediation was also widely studied (Zhengji, 2010).However, the application of SRB in the treatment of wastewater containing high concentration of sulfate and nitrogen was rare.Generally, the transformations of sulfate and nitrogen were performed in individual reactors or with different technology (Wang et al., 2009).The isolated SN22-2 with simultaneous capabilities of denitrification and sulfate-reduction had great potential in the treatment of wastewaters produced from light chemical engineering industries, food processing and pharmaceutical factories.These wastewaters were rich in sulfate and nitrogen and the isolated strain could be applied without the consideration of interspecific competition.Further studies are necessary to explore the feasibility and the characteristics of the isolate in practical wastewater treatment.

Conclusions
An anaerobic strain, named as SN22-2 with simultaneous denitrifying and sulfate-reducing ablities was isolated.It was a rod-shaped, Gram-negative, 0.20 to 0.70 µm in width and 3.0 to 5.5 µm in length.Most of the fatty acids of SN22-2 were distributed among C 12:0 -C 19-CYC-9 and the main fatty acids consisted of C 16:1-CIS-9-FAME , C 16:0-FAME , C 18:0-FAME and C 18:0 DMA .Strain SN22-2 was identified as Bacillus coagulans as its 16S rDNA bored 99% resemblance to that of Bacillus coagulans (Accession No. AB240205).The dissimilatory sulfite reductase (Dsr) and the nitrite reductase (nirS) genes were successfully amplified and cloned from strain S22-2.The removal efficiencies of NO 3 -and SO 4 2-were up to 96.48 and 97.98% when the initial concentrations of NO

Figure 2 .
Figure2.Phylogenetic tree based on the 16S rDNA gene sequences of strain SN22-2 and the selected species from GenBank Database using the neighbour-joining method.

Figure 3 .
Figure 3. Sequences of the nirS gene amplified by primer pair 832 F and 1606R.

Figure 4 .
Figure 4. Sequences of the Dir gene amplified by primer pair DSR1F and DSR5R.

Figure 5 .
Figure 5.The daily variations of NO3 -and NO2 -concentrations and the removal efficiency of NO3 -.

Figure 6 .
Figure 6.The daily variations of SO4 2-concentrations and the removal efficiency of SO4 2-.

Table 1 .
The results of the physiochemical tests.
4368.45 and 586.70 mg/L.High activities of nitrite reductase and sulfate reductase were detected in the culture medium of SN22-2.In conclusion, strain SN22-2 had simultaneous denitrifying and sulfate reducing capablities.The characterization of the present isolate had important ecological implications in treating organic wastewater containing high concentraions of sulfate and nitrogen.
were Time (days)