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Denitrifying sulfide removal by Pseudomonas sp. C27 at excess carbon supply: Mechanisms
Bioresource Technology 180 (2015) 381–385
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Denitrifying sulfide removal by Pseudomonas sp. C27 at excess carbon supply: Mechanisms Hongliang Guo a, Chuan Chen a, Duu-Jong Lee a,b,c,⇑, Aijie Wang a, Nanqi Ren a a
State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan c Department of Chemical Engineering, National Taiwan University, Taipei 106, Taiwan b
h i g h l i g h t s Mixotrophic growth tests of C27 were conducted at different C/N ratios. At C/N = 1.63, C27 enhanced heterotrophic denitrification pathway. At C/N = 3.0, C27 accelerated metabolism via coupled-cycles pathway. With the coupled-cycles pathway, the accessible regime for C27 is enlarged. Revision of the coupled-cycles pathway was made.
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Article history: Received 7 December 2014 Received in revised form 4 January 2015 Accepted 9 January 2015 Available online 16 January 2015 Keywords: Sulfide Nitrate C/N ratio DSR Pseudomonas sp. C27
a b s t r a c t Pseudomonas sp. C27 can effectively conduct mixotrophic denitrifying sulfide removal (DSR) reactions using both organic matters and sulfide as electron donors. This study conducted DSR tests using C27 and quantitatively analyzed the protein abundances at C/N = 1.26, 1.63 and 3.0. At C/N = 1.26, C27 principally adopted autotrophic denitrification pathway in DSR reaction. As C/N ratio was increased to 1.63, C27 enhanced heterotrophic denitrification pathway for removing nitrous compounds. As the C/N ratio was further increased to 3.0, C27 accelerated metabolism via coupled-cycles pathway. The C/N ratio for coupled-cycles pathway was estimated ranging 2.0–2.3 in the studied medium. Optimal C/N ratio of traditional DSR processes ranged 1.05–1.26. With the coupled-cycles pathway, the accessible C/N/S regime for C27 on DSR reactions is enlarged. Minor revision of the coupled-cycles pathway considering production of ammonium step was made. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Nitrogen and sulfur wastes in industrial wastewaters need sufficient treatment before being disposed of (Show et al., 2013). Biological processes are preferred compared with the physiochemical treatments to remove nitrate, sulfide and organic matters from waters (Lee et al., 2013a,b). Recently, the researches related to denitrifying sulfide removal (DSR) reactions were conducted (Lee and Wong, 2014a,b). The DSR process is composed of two sequential steps: S2 + NO 3 ? S0 + NO 2 by autotrophic denitrifiers and Ac + NO2 ? CO2 + N2 by heterotrophic denitrifiers (Reyes-Avila et al., 2004; Chen et al., 2008a,b). To maintain balanced growth of the autotrophic ⇑ Corresponding author at: Department of Chemical Engineering, National Taiwan University, Taipei 106, Taiwan. E-mail address: [email protected] (D.-J. Lee). http://dx.doi.org/10.1016/j.biortech.2015.01.030 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
denitrifiers and heterotrophic denitrifiers are difficult in long-term DSR operations (Wang et al., 2010). The DSR process was proposed to be synergistically performed by autotrophic and heterotrophic denitrifiers at C/N of 1.05–1.26 (Chen et al., 2009; Show et al., 2013). A relatively narrow C/N window is available for satisfactory DSR performance with mixed cultures. The use of single facultative autotrophic bacterium (FAB) for mixotrophic denitrification, such as Thiobacillus delicates, Thiobacillus pantotropha, Pseudomonas stutzeri, and Paracoccus denitrificans, can lift the balanced-growth limitations by conventional DSR process (Robertson and Kuenen, 1983). Pseudomonas sp. C27 was isolated from DSR granules in the expanded granular sludge bed (EGSB) reactor that can effectively conduct mixotrophic growth using organic matters and sulfide as electron donors (Chen et al., 2013). The pathways of C27 in DSR reactors were studied (Guo et al., 2013, 2014a). Guo et al. (2014b) showed that C27 has functional
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enzymes for both autotrophic and heterotrophic denitrification, and has an enzyme system for DSR with coupled C + N + S cycles. Understandings on this newly proposed coupled-cycles pathway remains limited. This study performed DSR reactions with C27 at C/N > 1.26. The obtained experimental results and literature results provides information on how C27 utilized the three pathways in DSR medium with excess carbon supply.
(M), deamidated (NQ) were the potential variable modifications, and carbamidomethyl (C), iTRAQ8plex (N-term), iTRAQ8plex (K) were fixed modifications. Other details for data Analysis and function method description were available in Guo et al. (2014a). 3. Results and discussion 3.1. The DSR performance of C27 at different C/N ratio
2. Methods 2.1. Bacterial strain and tests The Pseudomonas sp. C27 is a Gram-negative bacterium of rodshaped with GenBank under accession number GQ241351 (Chen et al., 2013). Three parallel growth tests were conducted at C/N = 1.26, 1.63 and 3.0 mol/mol media. The C27 strain was cultivated anaerobically in 250-ml culture bottles containing 200 ml liquid medium at 30 °C and pH 8. The composition of liquid medium was (per liter): 0.76 g of KNO3, 0.64, 0.83 or 1.52 g of NaAC3H2O for C/N = 1.26, 1.63 or 3.0, 1.0 g of NH4Cl, 0.5 g of NaHCO3, 1.8 g of KH2PO4, 3.0 g of Na2HPO412H2O, 0.5 g of MgSO47H2O, 0.39 g of Na2S (equivalent to 160 mg l1 S2) and 0.1 g trace elements. The concentration of the S, N and C species in liquid media were sampled and measured. Measure protocols of these compounds were available in Chen et al. (2010). 2.2. Protein preparation and measurements After 18 h cultivation, the liquid medium in bottle was collected and the cells were harvested at 10,000g for 10 min at 4 °C. The collected sediment was washed by PBS buffer at pH 7.4 (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) for three times, and then was ground into powder in liquid nitrogen, extracted with lysis buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 40 mM Tris–HCl, 1 mM PMSF, 2 mM EDTA, 10 mM DTT, pH 8.5). Protein mix was obtained according to Guo et al. (2014a), which were quantified using Bradford assay, and then were kept at 80 °C before further analysis. Trypsin Gold (Promega, Madison, WI, USA) with protein:trypsin = 30:1 was used to digest the obtained protein powders at 37 °C for 16 h. After digestion, the peptides were dried by vacuum centrifugation and were reconstituted in 0.5 M TEAB and processed according to the manual for 8-plex iTRAQ reagent (Applied Biosystems). The labeled peptide mixtures were then dried again by vacuum centrifugation for strong cation exchange (SCX) chromatography analysis. The collected samples were re-suspended in buffer A (5% ACN, 0.1% FA) and centrifuged at 20,000g for 10 min. 10 ll supernatant was loaded on an LC-20AD nanoHPLC (Shimadzu, Kyoto, Japan) equipped with a 2 cm C18 trap column. Then, the peptides were eluted onto a 10 cm pack-in-house analytical C18 column. The samples were loaded at 8 ll min1 for 4 min, then the 35 min gradient was run at 300 ll min1 by 2–35% buffer B (95% ACN, 0.1% FA), followed by 5 min linear gradient to 60%, then, followed by 2 min linear gradient to 80%, and maintained at 80% B for 4 min, and finally returned to 5% in 1 min. 2.3. Data analysis and function method description The data were processed using Proteome Discoverer software (Version 1.2.0.208) (Thermo Fisher, Waltham, MA, USA). The proteins were identified using Mascot search engine ver. 2.3.02 (Matrix Science, London, UK) against database containing Pseudomonas sequences. Gln ? pyro-Glu (N-term Q), oxidation
Rates of sulfide removal by C27 followed C/N = 1.26 > 3.0 > 1.63. For example, in the first 2 h of reaction, 49.8 and 36.2 mg l1 sulfide was reduced in C/N = 1.26 and 3.0 media, respectively; only 21.4 mg l1 sulfide was reduced in the C/N = 1.63 medium (Fig. S1). It took 12 h in the C/N = 1.26 and 3.0 media to reduce over 90% sulfide; while it needed 24 h for C27 to remove 90% sulfide in the C/N = 1.63 medium. In the first 8 h testing, the rates of nitrate reduction of C27 at C/N = 1.26 were similar to C/N = 3.0, and were only slightly higher than the C/N = 1.63 medium (Fig. S2). 99% of NO 3 -N was removed in 16 h at all three C/N ratios, with nitrite being accumulated in the media. The NO 2 -N concentrations at 16 h were 49.4, 37.1 and 52.6 mg l1 in the C/N = 1.26, 1.63 and 3.0 media, respectively. The accumulated NO 2 -N was further reduced in the C/N = 1.63 and 3.0 media, but was not successfully reduced in the C/N = 1.26 medium. At the end of the 32 h test, 106.0, 139.2 and 134.7 mg l1 Ac-C were consumed in the C/N = 1.26, 1.63 and 3.0 media, respectively (Fig. S3). Restated, the Ac-C consumption rates followed C/N = 1.63 > 3.0 > 1.26. 3.2. Abundances of proteins related to DSR for C27 at different C/N ratio The coupled pathway for C27 was presented based on that in Guo et al. (2014a) in Fig. 1. Nos. 1–26 were the identified proteins in Guo et al. (2014a) and Nos. 27 and 28 were incorporated into the new pathway since these two proteins were corresponding to ammonium metabolism. Further discussions were made on the expressions of these 28 proteins. Most of the identified proteins were differently expressed at different C/N ratio, including 18 proteins relating to carbon metabolic pathway, 1 protein relating to sulfur metabolic pathway and 2 proteins relating to nitrogen metabolic pathway (Fig. 1). Three comparable groups were set, including C/N = 1.63 vs 1.26, 3.0 vs 1.26 and 3.0 vs 1.63. The abundance changes of 21 proteins were higher than 1.20-folds in at least one comparable group (Table 1). Other proteins displayed less than 1.2-folds change in abundance in all three comparable groups. In the 18 proteins relating to carbon metabolic pathway, using C/N = 1.26 as the basis, 4 proteins were up-regulated and 14 proteins were down-regulated in the C/N = 1.63 sample. When using C/N = 1.26 as control, increase C/N ratio to 3.0 up-regulated 12 proteins and down-regulated 6 proteins. Using C/N = 1.63 as control, 14 proteins were up-regulated and 4 proteins were downregulated in the C/N = 3.0 sample. In sum, the C-relating proteins were down-regulated at C/N ratio from 1.26 to 1.63, and then up-regulated at C/N ratio from 1.63 to 3.0. The cytochrome c551 was related to sulfide oxidation (Then and Trüper, 1983). Compared with the C/N = 1.26 sample, cytochrome c551 was down-regulated 1.92 and 1.33-folds in the C/N = 1.63 and C/N = 3.0 sample, respectively. Comparing the C/N = 1.63 sample, c551 was up-regulated by 1.44-folds in the C/N = 3.0 sample. Restated, the expression of S-relating protein followed C/N = 1.26 > 3.0 > 1.63 (Table 1). The nitrite reductase and denitrification regulatory protein nirQ were relating to nitrite reduction reaction (Arai et al., 1995). The
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Fig. 1. The pathways of acetate cycle and sulfide conversion for C27. Nos. 1–26 are proteins in Table 1 of Guo et al. (2014a). Nos. 27 and 28 are newly identified enzymes. Left arrows are changes in protein abundance from C/N = 1.26–1.63, and the latter, 1.26–3.0.
abundances of these two proteins were not changed much at different C/N ratios (Table 1). 3.3. Denitrification of C27 at different C/N ratios When C/N ratio was increased from 1.26 to 1.63, and then to 3.0, the cytochrome c551 (No. 22 in Table 1) that catalyzes sulfide oxidization and most of proteins relating to coupled-cycles pathway were first down-regulated (from 1.26 to 1.63), and then upregulated (from 1.63 to 3.0). This occurrence is attributable to the regulation of C27 between the autotrophic denitrification, heterotrophic denitrification, and the coupled-cycles pathway upon different C/N ratios. In C/N = 1.26 medium, the C27 rapidly consumed sulfide, suggesting that it inclined to autotrophic denitrification pathway. We noted that S + N reaction was enhanced with related proteins being up-regulated, and C + N reaction was weaken with related proteins being down-regulated and nitrite being accumulated. Guo et al. (2014a) tested performance of C27 in the C/N = 0.75 medium. Comparing their data and the 1.26 medium test conducted herein, C27 has higher sulfide reduction and nitrite accumulation rates in the 0.75 medium, which suggests that C27 is even more inclined to autotrophic denitrification in 0.75 than in 1.26. In C/N = 1.63 medium, C27 was inclined to heterotrophic denitrification pathway, with high rates of acetate consumption and nitrate removal, and low rate of sulfide oxidization. At C/N = 3.0, removals of sulfide and acetate were increased compared with those at C/N = 1.63 (Figs. S1 and S3). Also, most of proteins relating to carbon metabolism of C27 were upregulated compared with those at C/N = 1.63 (Fig. 1). This occurrence suggested sufficient carbon supply enhances coupled-cycles pathway. 3.4. Revised coupled-cycles pathway of C27 The couple-cycles pathway needs ammonium as substrate. Guo et al. (2014a) showed that under insufficient carbon source
condition (C/N = 0.75) the D-3-phosphoglycerate dehydrogenase was up-regulated, which is attributable to increased demand of NH3. Pyruvate can be produced from phosphoenolpyruvate. L-Serine, an intermediate of the coupled-cycles pathway, can be converted to pyruvate and ammonia by threonine dehydratase (No. 27 in Table 1) catalyzing (Husain et al., 2010). In addition, produced L-alanine can be converted to pyruvate by glutamate-pyruvate aminotransferase (No. 28 in Table 1) catalyzing (Peña-Soler et al., 2014). Based on the present experimental observations, two reversible reactions were incorporated into Fig. 1: L-Serine
M pyruvate + ammonia + 2-oxoglutarate M pyruvate + L-glutamate
L-Alanine
Reaction of NH3 with 2-oxoglutarate forms L-glutamate cycle (Guo et al., 2014a). In the present study, reaction of L-alanine with 2-oxoglutarate also formed the same L-glutamate cycle. L-Alanine was reacted with 2-oxoglutarate to produce L-glutamate. The produced L-glutamate can react with 3-phosphonooxypyruvate to produce O-phospho-L-serine and 2-oxoglutarate. The so-yielded 2-oxoglutarate can react again with L-alanine to produce L-glutamate. The revised scheme uses NH3 as a substrate recoverable at the end of reaction, which correlates with most studies noting minimal net changes in NH3 concentrations in the DSR tests.
3.5. Implications to DSR practice The enzymatic reactions relating to the coupled-cycles pathway are listed in Fig. 1. Based on the stoichiometry, consumption of one mole of phosphoenolpyruvate can lead to formation of one mole of 3-phosphonooxypyruvate. Since phosphoenolpyruvate can be consumed by other reactions such as reaction 13, hence, we cannot confirm the fixed ratio of C/N/S in the coupled-cycles pathway using reaction scheme presented in Fig. 1. An estimation of the contributions of each pathway can be made. The sequential reaction scheme proposed by Reyes-Avila et al. (2004) is as follows.
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Table 1 Proteins related to denitrifying sulfide removal for the strains C27. No. Accession
1 2 3 4 5 6 7
gi|104782577 gi|472327168 gi|333901560 gi|146282233 gi|339486839 gi|146282649 gi|104782661
8 gi|397687799 9 gi|397687793 10 gi|28871476 11 gi|70733947 12 gi|431929058
13 gi|146308281 14 gi|472324702 15 gi|512688177
16 gi|146280793 17 gi|512619571 18 19 20 21 22 23
gi|346642691 gi|28868628 gi|28871054 gi|104780229 gi|431926008 gi|152987611
24 gi|397685765 25 gi|146283850 26 gi|116051410 27 gi|392985190 28 gi|146307606
Description
Species
Acetyl-CoA synthetase Acetate kinase A Phosphate acetyltransferase Type II citrate synthase Aconitate hydratase Isocitrate dehydrogenase 2-Oxoglutarate dehydrogenase E1 component Succinyl-CoA synthetase subunit alpha Succinate dehydrogenase flavoprotein subunit Fumarate hydratase, class I NADP-dependent malic enzyme ATP-dependent phosphoenolpyruvate carboxykinase Pyruvate kinase Enolase 2,3-Bisphosphoglycerateindependent phosphoglycerate mutase
Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas
D-3-Phosphoglycerate dehydrogenase Phosphoserine aminotransferase Phosphoserine phosphatase Serine O-acetyltransferase Cysteine synthase A Cysteine desulfurase Cytochrome c551 Nitrate reductase subunit alpha Nitrite reductase Denitrification regulatory protein nirQ Nitrous-oxide reductase Threonine dehydratase Glutamate-pyruvate aminotransferase
entomophila L48 denitrificans ATCC 13867 fulva 12-X stutzeri A1501 putida S16 stutzeri A1501 entomophila L48
Score Mass (kDa)
Cov Unique Change folds at different C/N ratio (%) spectrum (C/N = 1.63) vs (C/N = 3.0) vs (C/N = 3.0) vs (C/N = 1.26) (C/N = 1.26) (C/N = 1.63)
21 144 539 1518 1361 1909 1268
2.6 4.5 3.6 15.4 14 20.8 5.5
64.5 45.5 83.3 55.0 106.6 54.6 119.0
2 2 13 35 8 15 16
– 1.27 1/1.22 1/1.20 1.31 1/1.28 1/1.25
– 1.43 1.10 1/1.28 1.07 1/1.27 1.20
– 1.13 1.24 1/1.05 1/1.20 1/1.01 1.39
Pseudomonas stutzeri DSM 10701
544 36.3
27.3 24
1/1.20
1.14
1.37
Pseudomonas stutzeri DSM 10701
1581 72.0
10.2 21
1/1.22
1/1.03
1.14
977 62.7 932 52.9
14.6 15 21.8 9
1/1.23 1/1.14
1.19 1.11
1.36 1.26
1694 64.3
10.3 45
1.15
1/1.16
1/1.30
Pseudomonas mendocina ymp Pseudomonas denitrificans ATCC 13867 Pseudomonas putida NBRC 14164
474 59.6 668 54.2 111 62.4
11.6 11 5.4 17 3.5 8
1/1.37 – –
1/1.27 – –
1.08 – –
Pseudomonas stutzeri A1501
576 60.0
7.1
3
1/3.23
1/2.04
1.58
Pseudomonas resinovorans NBRC 106553
339 46.7
7.5
1
–
–
–
5.9 7 13.6 12 11.1 12 14.6 4 15.4 1 12.5 15
1.12 1/1.20 1/1.01 1/1.16 1/1.92 –
1.37 1.07 1.20 1.07 1/1.33 –
1.23 1.29 1.22 1.30 1.44 –
1945 76494 1119 28380
12.2 9 21.7 11
1.24 1.16
1.44 1.45
1.12 1.26
183 83381 378 60717 54 52810
4.1 27 11.1 1 5.1 4
– – 1/1.15
– – 1.38
– – 1.59
Pseudomonas syringae DC3000 Pseudomonas protegens Pf-5 Pseudomonas stutzeri RCH2
Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas
protegens Pf-5 syringae DC3000 syringae DC3000 entomophila L48 stutzeri RCH2 aeruginosa PA7
231 343 744 965 26 2023
Pseudomonas stutzeri DSM 10701 Pseudomonas stutzeri A1501 Pseudomonas aeruginosa UCBPP-PA14 Pseudomonas aeruginosa DK2 Pseudomonas mendocina ymp
S2 þ NO3 þ H2 O ! S0 þ NO2 þ 2OH
ð1Þ
3CH3 COO þ 8NO2 þ H2 O ! 4N2 þ 6CO2 þ 11OH
ð2Þ
If both reactions (1) and (2) occur in the DSR reactor, C/N = 1.33. The acetate to pyruvate process is energy consuming; therefore, sulfide oxidization to elemental sulfur should occur first to drive the coupled-cycle pathway. Hence, we consider the two reactions (1) and (2) take place as the autotrophic and heterotrophic denitrification pathways by C27 with moles of a and b. Then at C/N = 1.26, 1 the maximum NO , giving 2 -N accumulation was 49.4 mg l 2 a = 3.53 mM; that is, 3.53 mM S was reacted with 3.53 mM NO 3 -N by autotrophic denitrification pathway. NO2 -N accumulation was dropped to 40.0 mg l1 (2.86 mM) at 32 h. Therefore, 3.53–2.86 = 0.67 mM NO 2 -N was reduced by heterotrophic denitrification, giving b = 0.084. A total of 156.7 mg l1 (4.90 mM) S2, 1 107.9 (7.7 mM) NO (8.83 mM) Ac-C were 3 -N, and 106.0 mg l consumed in the 32 h test, giving 1.37 mM S2, 4.17 mM N and 8.33 mM Ac-C was consumed in the coupled-cycles pathway. The C/N/S for coupled-cycles pathway at C/N = 1.26 is 6/3/1. Using similar calculations, at C/N = 1.63, the C/N/S is 4/2/1 for coupled-cycles pathway. At C/N = 3.0, the corresponding C/N/S for coupled-cycles pathway is estimated to be 8/3.5/1. Traditional DSR processes with mixed cultures adopting sequential autotrophic and heterotrophic denitrifications have optimal C/N ratio range
50.4 31.6 40.0 51581 14265 159386
1.05–1.26 (Chen et al., 2009; Show et al., 2013). As noted above, at fixed dosed sulfide, the C/N consumption rates by the coupledcycles pathway were estimated ranging 2.0–2.3. This estimation should be considered preliminary since fixed denitrification reactions were assumed in calculations. However, the estimation still indicates that the coupled-cycles pathway presents a scheme C27 can conduct DSR reactions beyond C/N = 2. The products pyruvate and alanine can be used by cells for metabolisms, which link the coupled-cycles pathways to other biological pathways in cells. This occurrence can make C27 to work under C/N > 2. The finding has implication to DSR practice for handling C + N + S wastewaters at wider C/N window compared with the traditional DSR processes.
4. Conclusions This study performed DSR reactions with C27 at C/N > 1.26. At C/N = 1.26, C27 principally adopted autotrophic denitrification pathway in DSR reaction. As C/N ratio was increased from 1.26 to 1.63, the removal rates of sulfide and nitrate were decreased; while as C/N ratio was further increased to 3.0, the rates of sulfide and nitrate removal were increased. With excess carbon supply, C27 first enhanced heterotrophic denitrification pathway. As the carbon supply was in large surplus, C27 accelerated coupled-cycles pathway for DSR reaction. Implication of incorporation of the
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newly proposed coupled-cycles pathway to DSR practice was discussed. Acknowledgements This work is partially supported by Ministry of Science and Technology, Taiwan projects. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.01. 030. References Arai, H., Igarashi, Y., Kodama, T., 1995. Expression of the nir and nor genes for denitrification of Pseudomonas aeruginosa requires a novel CRP/FNR-related transcriptional regulator, DNR, in addition to ANR. FEBS Lett. 371, 73–76. Chen, C., Wang, A.J., Ren, N.Q., Kan, H., Lee, D.J., 2008a. Biological breakdown of denitrifying sulfide removal process in high-rate expanded granular bed reactor. Appl. Microbiol. Biotechnol. 81, 765–770. Chen, C., Ren, N.Q., Wang, A.J., Yu, Z.G., Lee, D.J., 2008b. Simultaneous biological removal of sulfur, nitrogen and carbon using EGSB reactor. Appl. Microbiol. Biotechnol. 78, 1057–1063. Chen, C., Wang, A.J., Ren, N.Q., Lee, D.J., Lai, J.Y., 2009. High-rate denitrifying sulfide removal process in expanded granular sludge bed reactor. Bioresour. Technol. 100, 2316–2319. Chen, C., Ren, N.Q., Wang, A.J., Liu, L.H., Lee, D.J., 2010. Enhanced performance of denitrifying sulfide removal process under micro-aerobic condition. J. Hazard. Mater. 179, 1147–1151. Chen, C., Ho, K.L., Liu, F.C., Ho, M.N., Wang, A.J., Ren, N.Q., 2013. Autotrophic and heterotrophic denitrification by a newly isolated strain Pseudomonas sp. C27. Bioresour. Technol. 145, 351–356.
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Short Communication
Denitrifying sulfide removal by Pseudomonas sp. C27 at excess carbon supply: Mechanisms Hongliang Guo a, Chuan Chen a, Duu-Jong Lee a,b,c,⇑, Aijie Wang a, Nanqi Ren a a
State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 106, Taiwan c Department of Chemical Engineering, National Taiwan University, Taipei 106, Taiwan b
h i g h l i g h t s Mixotrophic growth tests of C27 were conducted at different C/N ratios. At C/N = 1.63, C27 enhanced heterotrophic denitrification pathway. At C/N = 3.0, C27 accelerated metabolism via coupled-cycles pathway. With the coupled-cycles pathway, the accessible regime for C27 is enlarged. Revision of the coupled-cycles pathway was made.
a r t i c l e
i n f o
Article history: Received 7 December 2014 Received in revised form 4 January 2015 Accepted 9 January 2015 Available online 16 January 2015 Keywords: Sulfide Nitrate C/N ratio DSR Pseudomonas sp. C27
a b s t r a c t Pseudomonas sp. C27 can effectively conduct mixotrophic denitrifying sulfide removal (DSR) reactions using both organic matters and sulfide as electron donors. This study conducted DSR tests using C27 and quantitatively analyzed the protein abundances at C/N = 1.26, 1.63 and 3.0. At C/N = 1.26, C27 principally adopted autotrophic denitrification pathway in DSR reaction. As C/N ratio was increased to 1.63, C27 enhanced heterotrophic denitrification pathway for removing nitrous compounds. As the C/N ratio was further increased to 3.0, C27 accelerated metabolism via coupled-cycles pathway. The C/N ratio for coupled-cycles pathway was estimated ranging 2.0–2.3 in the studied medium. Optimal C/N ratio of traditional DSR processes ranged 1.05–1.26. With the coupled-cycles pathway, the accessible C/N/S regime for C27 on DSR reactions is enlarged. Minor revision of the coupled-cycles pathway considering production of ammonium step was made. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Nitrogen and sulfur wastes in industrial wastewaters need sufficient treatment before being disposed of (Show et al., 2013). Biological processes are preferred compared with the physiochemical treatments to remove nitrate, sulfide and organic matters from waters (Lee et al., 2013a,b). Recently, the researches related to denitrifying sulfide removal (DSR) reactions were conducted (Lee and Wong, 2014a,b). The DSR process is composed of two sequential steps: S2 + NO 3 ? S0 + NO 2 by autotrophic denitrifiers and Ac + NO2 ? CO2 + N2 by heterotrophic denitrifiers (Reyes-Avila et al., 2004; Chen et al., 2008a,b). To maintain balanced growth of the autotrophic ⇑ Corresponding author at: Department of Chemical Engineering, National Taiwan University, Taipei 106, Taiwan. E-mail address: [email protected] (D.-J. Lee). http://dx.doi.org/10.1016/j.biortech.2015.01.030 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
denitrifiers and heterotrophic denitrifiers are difficult in long-term DSR operations (Wang et al., 2010). The DSR process was proposed to be synergistically performed by autotrophic and heterotrophic denitrifiers at C/N of 1.05–1.26 (Chen et al., 2009; Show et al., 2013). A relatively narrow C/N window is available for satisfactory DSR performance with mixed cultures. The use of single facultative autotrophic bacterium (FAB) for mixotrophic denitrification, such as Thiobacillus delicates, Thiobacillus pantotropha, Pseudomonas stutzeri, and Paracoccus denitrificans, can lift the balanced-growth limitations by conventional DSR process (Robertson and Kuenen, 1983). Pseudomonas sp. C27 was isolated from DSR granules in the expanded granular sludge bed (EGSB) reactor that can effectively conduct mixotrophic growth using organic matters and sulfide as electron donors (Chen et al., 2013). The pathways of C27 in DSR reactors were studied (Guo et al., 2013, 2014a). Guo et al. (2014b) showed that C27 has functional
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enzymes for both autotrophic and heterotrophic denitrification, and has an enzyme system for DSR with coupled C + N + S cycles. Understandings on this newly proposed coupled-cycles pathway remains limited. This study performed DSR reactions with C27 at C/N > 1.26. The obtained experimental results and literature results provides information on how C27 utilized the three pathways in DSR medium with excess carbon supply.
(M), deamidated (NQ) were the potential variable modifications, and carbamidomethyl (C), iTRAQ8plex (N-term), iTRAQ8plex (K) were fixed modifications. Other details for data Analysis and function method description were available in Guo et al. (2014a). 3. Results and discussion 3.1. The DSR performance of C27 at different C/N ratio
2. Methods 2.1. Bacterial strain and tests The Pseudomonas sp. C27 is a Gram-negative bacterium of rodshaped with GenBank under accession number GQ241351 (Chen et al., 2013). Three parallel growth tests were conducted at C/N = 1.26, 1.63 and 3.0 mol/mol media. The C27 strain was cultivated anaerobically in 250-ml culture bottles containing 200 ml liquid medium at 30 °C and pH 8. The composition of liquid medium was (per liter): 0.76 g of KNO3, 0.64, 0.83 or 1.52 g of NaAC3H2O for C/N = 1.26, 1.63 or 3.0, 1.0 g of NH4Cl, 0.5 g of NaHCO3, 1.8 g of KH2PO4, 3.0 g of Na2HPO412H2O, 0.5 g of MgSO47H2O, 0.39 g of Na2S (equivalent to 160 mg l1 S2) and 0.1 g trace elements. The concentration of the S, N and C species in liquid media were sampled and measured. Measure protocols of these compounds were available in Chen et al. (2010). 2.2. Protein preparation and measurements After 18 h cultivation, the liquid medium in bottle was collected and the cells were harvested at 10,000g for 10 min at 4 °C. The collected sediment was washed by PBS buffer at pH 7.4 (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) for three times, and then was ground into powder in liquid nitrogen, extracted with lysis buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 40 mM Tris–HCl, 1 mM PMSF, 2 mM EDTA, 10 mM DTT, pH 8.5). Protein mix was obtained according to Guo et al. (2014a), which were quantified using Bradford assay, and then were kept at 80 °C before further analysis. Trypsin Gold (Promega, Madison, WI, USA) with protein:trypsin = 30:1 was used to digest the obtained protein powders at 37 °C for 16 h. After digestion, the peptides were dried by vacuum centrifugation and were reconstituted in 0.5 M TEAB and processed according to the manual for 8-plex iTRAQ reagent (Applied Biosystems). The labeled peptide mixtures were then dried again by vacuum centrifugation for strong cation exchange (SCX) chromatography analysis. The collected samples were re-suspended in buffer A (5% ACN, 0.1% FA) and centrifuged at 20,000g for 10 min. 10 ll supernatant was loaded on an LC-20AD nanoHPLC (Shimadzu, Kyoto, Japan) equipped with a 2 cm C18 trap column. Then, the peptides were eluted onto a 10 cm pack-in-house analytical C18 column. The samples were loaded at 8 ll min1 for 4 min, then the 35 min gradient was run at 300 ll min1 by 2–35% buffer B (95% ACN, 0.1% FA), followed by 5 min linear gradient to 60%, then, followed by 2 min linear gradient to 80%, and maintained at 80% B for 4 min, and finally returned to 5% in 1 min. 2.3. Data analysis and function method description The data were processed using Proteome Discoverer software (Version 1.2.0.208) (Thermo Fisher, Waltham, MA, USA). The proteins were identified using Mascot search engine ver. 2.3.02 (Matrix Science, London, UK) against database containing Pseudomonas sequences. Gln ? pyro-Glu (N-term Q), oxidation
Rates of sulfide removal by C27 followed C/N = 1.26 > 3.0 > 1.63. For example, in the first 2 h of reaction, 49.8 and 36.2 mg l1 sulfide was reduced in C/N = 1.26 and 3.0 media, respectively; only 21.4 mg l1 sulfide was reduced in the C/N = 1.63 medium (Fig. S1). It took 12 h in the C/N = 1.26 and 3.0 media to reduce over 90% sulfide; while it needed 24 h for C27 to remove 90% sulfide in the C/N = 1.63 medium. In the first 8 h testing, the rates of nitrate reduction of C27 at C/N = 1.26 were similar to C/N = 3.0, and were only slightly higher than the C/N = 1.63 medium (Fig. S2). 99% of NO 3 -N was removed in 16 h at all three C/N ratios, with nitrite being accumulated in the media. The NO 2 -N concentrations at 16 h were 49.4, 37.1 and 52.6 mg l1 in the C/N = 1.26, 1.63 and 3.0 media, respectively. The accumulated NO 2 -N was further reduced in the C/N = 1.63 and 3.0 media, but was not successfully reduced in the C/N = 1.26 medium. At the end of the 32 h test, 106.0, 139.2 and 134.7 mg l1 Ac-C were consumed in the C/N = 1.26, 1.63 and 3.0 media, respectively (Fig. S3). Restated, the Ac-C consumption rates followed C/N = 1.63 > 3.0 > 1.26. 3.2. Abundances of proteins related to DSR for C27 at different C/N ratio The coupled pathway for C27 was presented based on that in Guo et al. (2014a) in Fig. 1. Nos. 1–26 were the identified proteins in Guo et al. (2014a) and Nos. 27 and 28 were incorporated into the new pathway since these two proteins were corresponding to ammonium metabolism. Further discussions were made on the expressions of these 28 proteins. Most of the identified proteins were differently expressed at different C/N ratio, including 18 proteins relating to carbon metabolic pathway, 1 protein relating to sulfur metabolic pathway and 2 proteins relating to nitrogen metabolic pathway (Fig. 1). Three comparable groups were set, including C/N = 1.63 vs 1.26, 3.0 vs 1.26 and 3.0 vs 1.63. The abundance changes of 21 proteins were higher than 1.20-folds in at least one comparable group (Table 1). Other proteins displayed less than 1.2-folds change in abundance in all three comparable groups. In the 18 proteins relating to carbon metabolic pathway, using C/N = 1.26 as the basis, 4 proteins were up-regulated and 14 proteins were down-regulated in the C/N = 1.63 sample. When using C/N = 1.26 as control, increase C/N ratio to 3.0 up-regulated 12 proteins and down-regulated 6 proteins. Using C/N = 1.63 as control, 14 proteins were up-regulated and 4 proteins were downregulated in the C/N = 3.0 sample. In sum, the C-relating proteins were down-regulated at C/N ratio from 1.26 to 1.63, and then up-regulated at C/N ratio from 1.63 to 3.0. The cytochrome c551 was related to sulfide oxidation (Then and Trüper, 1983). Compared with the C/N = 1.26 sample, cytochrome c551 was down-regulated 1.92 and 1.33-folds in the C/N = 1.63 and C/N = 3.0 sample, respectively. Comparing the C/N = 1.63 sample, c551 was up-regulated by 1.44-folds in the C/N = 3.0 sample. Restated, the expression of S-relating protein followed C/N = 1.26 > 3.0 > 1.63 (Table 1). The nitrite reductase and denitrification regulatory protein nirQ were relating to nitrite reduction reaction (Arai et al., 1995). The
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Fig. 1. The pathways of acetate cycle and sulfide conversion for C27. Nos. 1–26 are proteins in Table 1 of Guo et al. (2014a). Nos. 27 and 28 are newly identified enzymes. Left arrows are changes in protein abundance from C/N = 1.26–1.63, and the latter, 1.26–3.0.
abundances of these two proteins were not changed much at different C/N ratios (Table 1). 3.3. Denitrification of C27 at different C/N ratios When C/N ratio was increased from 1.26 to 1.63, and then to 3.0, the cytochrome c551 (No. 22 in Table 1) that catalyzes sulfide oxidization and most of proteins relating to coupled-cycles pathway were first down-regulated (from 1.26 to 1.63), and then upregulated (from 1.63 to 3.0). This occurrence is attributable to the regulation of C27 between the autotrophic denitrification, heterotrophic denitrification, and the coupled-cycles pathway upon different C/N ratios. In C/N = 1.26 medium, the C27 rapidly consumed sulfide, suggesting that it inclined to autotrophic denitrification pathway. We noted that S + N reaction was enhanced with related proteins being up-regulated, and C + N reaction was weaken with related proteins being down-regulated and nitrite being accumulated. Guo et al. (2014a) tested performance of C27 in the C/N = 0.75 medium. Comparing their data and the 1.26 medium test conducted herein, C27 has higher sulfide reduction and nitrite accumulation rates in the 0.75 medium, which suggests that C27 is even more inclined to autotrophic denitrification in 0.75 than in 1.26. In C/N = 1.63 medium, C27 was inclined to heterotrophic denitrification pathway, with high rates of acetate consumption and nitrate removal, and low rate of sulfide oxidization. At C/N = 3.0, removals of sulfide and acetate were increased compared with those at C/N = 1.63 (Figs. S1 and S3). Also, most of proteins relating to carbon metabolism of C27 were upregulated compared with those at C/N = 1.63 (Fig. 1). This occurrence suggested sufficient carbon supply enhances coupled-cycles pathway. 3.4. Revised coupled-cycles pathway of C27 The couple-cycles pathway needs ammonium as substrate. Guo et al. (2014a) showed that under insufficient carbon source
condition (C/N = 0.75) the D-3-phosphoglycerate dehydrogenase was up-regulated, which is attributable to increased demand of NH3. Pyruvate can be produced from phosphoenolpyruvate. L-Serine, an intermediate of the coupled-cycles pathway, can be converted to pyruvate and ammonia by threonine dehydratase (No. 27 in Table 1) catalyzing (Husain et al., 2010). In addition, produced L-alanine can be converted to pyruvate by glutamate-pyruvate aminotransferase (No. 28 in Table 1) catalyzing (Peña-Soler et al., 2014). Based on the present experimental observations, two reversible reactions were incorporated into Fig. 1: L-Serine
M pyruvate + ammonia + 2-oxoglutarate M pyruvate + L-glutamate
L-Alanine
Reaction of NH3 with 2-oxoglutarate forms L-glutamate cycle (Guo et al., 2014a). In the present study, reaction of L-alanine with 2-oxoglutarate also formed the same L-glutamate cycle. L-Alanine was reacted with 2-oxoglutarate to produce L-glutamate. The produced L-glutamate can react with 3-phosphonooxypyruvate to produce O-phospho-L-serine and 2-oxoglutarate. The so-yielded 2-oxoglutarate can react again with L-alanine to produce L-glutamate. The revised scheme uses NH3 as a substrate recoverable at the end of reaction, which correlates with most studies noting minimal net changes in NH3 concentrations in the DSR tests.
3.5. Implications to DSR practice The enzymatic reactions relating to the coupled-cycles pathway are listed in Fig. 1. Based on the stoichiometry, consumption of one mole of phosphoenolpyruvate can lead to formation of one mole of 3-phosphonooxypyruvate. Since phosphoenolpyruvate can be consumed by other reactions such as reaction 13, hence, we cannot confirm the fixed ratio of C/N/S in the coupled-cycles pathway using reaction scheme presented in Fig. 1. An estimation of the contributions of each pathway can be made. The sequential reaction scheme proposed by Reyes-Avila et al. (2004) is as follows.
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Table 1 Proteins related to denitrifying sulfide removal for the strains C27. No. Accession
1 2 3 4 5 6 7
gi|104782577 gi|472327168 gi|333901560 gi|146282233 gi|339486839 gi|146282649 gi|104782661
8 gi|397687799 9 gi|397687793 10 gi|28871476 11 gi|70733947 12 gi|431929058
13 gi|146308281 14 gi|472324702 15 gi|512688177
16 gi|146280793 17 gi|512619571 18 19 20 21 22 23
gi|346642691 gi|28868628 gi|28871054 gi|104780229 gi|431926008 gi|152987611
24 gi|397685765 25 gi|146283850 26 gi|116051410 27 gi|392985190 28 gi|146307606
Description
Species
Acetyl-CoA synthetase Acetate kinase A Phosphate acetyltransferase Type II citrate synthase Aconitate hydratase Isocitrate dehydrogenase 2-Oxoglutarate dehydrogenase E1 component Succinyl-CoA synthetase subunit alpha Succinate dehydrogenase flavoprotein subunit Fumarate hydratase, class I NADP-dependent malic enzyme ATP-dependent phosphoenolpyruvate carboxykinase Pyruvate kinase Enolase 2,3-Bisphosphoglycerateindependent phosphoglycerate mutase
Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas
D-3-Phosphoglycerate dehydrogenase Phosphoserine aminotransferase Phosphoserine phosphatase Serine O-acetyltransferase Cysteine synthase A Cysteine desulfurase Cytochrome c551 Nitrate reductase subunit alpha Nitrite reductase Denitrification regulatory protein nirQ Nitrous-oxide reductase Threonine dehydratase Glutamate-pyruvate aminotransferase
entomophila L48 denitrificans ATCC 13867 fulva 12-X stutzeri A1501 putida S16 stutzeri A1501 entomophila L48
Score Mass (kDa)
Cov Unique Change folds at different C/N ratio (%) spectrum (C/N = 1.63) vs (C/N = 3.0) vs (C/N = 3.0) vs (C/N = 1.26) (C/N = 1.26) (C/N = 1.63)
21 144 539 1518 1361 1909 1268
2.6 4.5 3.6 15.4 14 20.8 5.5
64.5 45.5 83.3 55.0 106.6 54.6 119.0
2 2 13 35 8 15 16
– 1.27 1/1.22 1/1.20 1.31 1/1.28 1/1.25
– 1.43 1.10 1/1.28 1.07 1/1.27 1.20
– 1.13 1.24 1/1.05 1/1.20 1/1.01 1.39
Pseudomonas stutzeri DSM 10701
544 36.3
27.3 24
1/1.20
1.14
1.37
Pseudomonas stutzeri DSM 10701
1581 72.0
10.2 21
1/1.22
1/1.03
1.14
977 62.7 932 52.9
14.6 15 21.8 9
1/1.23 1/1.14
1.19 1.11
1.36 1.26
1694 64.3
10.3 45
1.15
1/1.16
1/1.30
Pseudomonas mendocina ymp Pseudomonas denitrificans ATCC 13867 Pseudomonas putida NBRC 14164
474 59.6 668 54.2 111 62.4
11.6 11 5.4 17 3.5 8
1/1.37 – –
1/1.27 – –
1.08 – –
Pseudomonas stutzeri A1501
576 60.0
7.1
3
1/3.23
1/2.04
1.58
Pseudomonas resinovorans NBRC 106553
339 46.7
7.5
1
–
–
–
5.9 7 13.6 12 11.1 12 14.6 4 15.4 1 12.5 15
1.12 1/1.20 1/1.01 1/1.16 1/1.92 –
1.37 1.07 1.20 1.07 1/1.33 –
1.23 1.29 1.22 1.30 1.44 –
1945 76494 1119 28380
12.2 9 21.7 11
1.24 1.16
1.44 1.45
1.12 1.26
183 83381 378 60717 54 52810
4.1 27 11.1 1 5.1 4
– – 1/1.15
– – 1.38
– – 1.59
Pseudomonas syringae DC3000 Pseudomonas protegens Pf-5 Pseudomonas stutzeri RCH2
Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas
protegens Pf-5 syringae DC3000 syringae DC3000 entomophila L48 stutzeri RCH2 aeruginosa PA7
231 343 744 965 26 2023
Pseudomonas stutzeri DSM 10701 Pseudomonas stutzeri A1501 Pseudomonas aeruginosa UCBPP-PA14 Pseudomonas aeruginosa DK2 Pseudomonas mendocina ymp
S2 þ NO3 þ H2 O ! S0 þ NO2 þ 2OH
ð1Þ
3CH3 COO þ 8NO2 þ H2 O ! 4N2 þ 6CO2 þ 11OH
ð2Þ
If both reactions (1) and (2) occur in the DSR reactor, C/N = 1.33. The acetate to pyruvate process is energy consuming; therefore, sulfide oxidization to elemental sulfur should occur first to drive the coupled-cycle pathway. Hence, we consider the two reactions (1) and (2) take place as the autotrophic and heterotrophic denitrification pathways by C27 with moles of a and b. Then at C/N = 1.26, 1 the maximum NO , giving 2 -N accumulation was 49.4 mg l 2 a = 3.53 mM; that is, 3.53 mM S was reacted with 3.53 mM NO 3 -N by autotrophic denitrification pathway. NO2 -N accumulation was dropped to 40.0 mg l1 (2.86 mM) at 32 h. Therefore, 3.53–2.86 = 0.67 mM NO 2 -N was reduced by heterotrophic denitrification, giving b = 0.084. A total of 156.7 mg l1 (4.90 mM) S2, 1 107.9 (7.7 mM) NO (8.83 mM) Ac-C were 3 -N, and 106.0 mg l consumed in the 32 h test, giving 1.37 mM S2, 4.17 mM N and 8.33 mM Ac-C was consumed in the coupled-cycles pathway. The C/N/S for coupled-cycles pathway at C/N = 1.26 is 6/3/1. Using similar calculations, at C/N = 1.63, the C/N/S is 4/2/1 for coupled-cycles pathway. At C/N = 3.0, the corresponding C/N/S for coupled-cycles pathway is estimated to be 8/3.5/1. Traditional DSR processes with mixed cultures adopting sequential autotrophic and heterotrophic denitrifications have optimal C/N ratio range
50.4 31.6 40.0 51581 14265 159386
1.05–1.26 (Chen et al., 2009; Show et al., 2013). As noted above, at fixed dosed sulfide, the C/N consumption rates by the coupledcycles pathway were estimated ranging 2.0–2.3. This estimation should be considered preliminary since fixed denitrification reactions were assumed in calculations. However, the estimation still indicates that the coupled-cycles pathway presents a scheme C27 can conduct DSR reactions beyond C/N = 2. The products pyruvate and alanine can be used by cells for metabolisms, which link the coupled-cycles pathways to other biological pathways in cells. This occurrence can make C27 to work under C/N > 2. The finding has implication to DSR practice for handling C + N + S wastewaters at wider C/N window compared with the traditional DSR processes.
4. Conclusions This study performed DSR reactions with C27 at C/N > 1.26. At C/N = 1.26, C27 principally adopted autotrophic denitrification pathway in DSR reaction. As C/N ratio was increased from 1.26 to 1.63, the removal rates of sulfide and nitrate were decreased; while as C/N ratio was further increased to 3.0, the rates of sulfide and nitrate removal were increased. With excess carbon supply, C27 first enhanced heterotrophic denitrification pathway. As the carbon supply was in large surplus, C27 accelerated coupled-cycles pathway for DSR reaction. Implication of incorporation of the
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newly proposed coupled-cycles pathway to DSR practice was discussed. Acknowledgements This work is partially supported by Ministry of Science and Technology, Taiwan projects. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.01. 030. References Arai, H., Igarashi, Y., Kodama, T., 1995. Expression of the nir and nor genes for denitrification of Pseudomonas aeruginosa requires a novel CRP/FNR-related transcriptional regulator, DNR, in addition to ANR. FEBS Lett. 371, 73–76. Chen, C., Wang, A.J., Ren, N.Q., Kan, H., Lee, D.J., 2008a. Biological breakdown of denitrifying sulfide removal process in high-rate expanded granular bed reactor. Appl. Microbiol. Biotechnol. 81, 765–770. Chen, C., Ren, N.Q., Wang, A.J., Yu, Z.G., Lee, D.J., 2008b. Simultaneous biological removal of sulfur, nitrogen and carbon using EGSB reactor. Appl. Microbiol. Biotechnol. 78, 1057–1063. Chen, C., Wang, A.J., Ren, N.Q., Lee, D.J., Lai, J.Y., 2009. High-rate denitrifying sulfide removal process in expanded granular sludge bed reactor. Bioresour. Technol. 100, 2316–2319. Chen, C., Ren, N.Q., Wang, A.J., Liu, L.H., Lee, D.J., 2010. Enhanced performance of denitrifying sulfide removal process under micro-aerobic condition. J. Hazard. Mater. 179, 1147–1151. Chen, C., Ho, K.L., Liu, F.C., Ho, M.N., Wang, A.J., Ren, N.Q., 2013. Autotrophic and heterotrophic denitrification by a newly isolated strain Pseudomonas sp. C27. Bioresour. Technol. 145, 351–356.
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