N ratio, endogenous and exogenous compounds

Available online at www.sciencedirect.com

Bioresource Technology 99 (2008) 1293–1299

Kinetics of denitrification using sulphur compounds: Effects of S/N ratio, endogenous and exogenous compounds J.L. Campos *, S. Carvalho, R. Portela, A. Mosquera-Corral, R. Me´ndez Department of Chemical Engineering, Institute of Technological Research, School of Engineering, University of Santiago de Compostela, Rua Lope Go´mez de Marzoa, E-15782 Santiago de Compostela, Spain Received 23 August 2004; received in revised form 19 December 2006; accepted 9 February 2007 Available online 30 March 2007

Abstract The influence of different sulphur to nitrogen (S/N) ratios on the specific autotrophic denitrification activity was studied in batch experiments using thiosulphate and nitrate as substrates. Transitory accumulations of nitrite were observed for assays with S/N ratios of 3.70 and 6.67 g/g, probably due to the higher specific reduction rate of nitrate compared to that of nitrite. Nitrite was the main end product when S/N ratios of 1.16 and 2.44 g/g were tested. 2  2 The effects of endogenous ðNO 3 ; NO2 ; S2 O3 and SO4 Þ and exogenous compounds (acetate and NaCl) on the specific denitrifying activity of the sludge were tested. Nitrite and sulphate did exert clear inhibitory effects over the process while thiosulphate, acetate and NaCl did not have strong effects at the concentrations tested. Similar experiments also showed that sulphur was not a suitable electron donor for these microorganisms, but sulphide was used successfully. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Autotrophic denitrification; Inhibition; Kinetics; Nitrate; Thiosulphate

1. Introduction Denitrification with sulphur compounds as electron donors is an alternative to heterotrophic denitrification for wastewaters with high nitrate concentration and low organic matter content. This process is carried out by autotrophic denitrifiers such as Thiobacillus denitrificans and Thiomicrospira denitrificans. The energy required by these microorganisms is derived from oxidation–reduction reactions with elements such as hydrogen or various reduced2 2 sulphur compounds ðH2 S; S; S2 O2 3 ; S4 O6 ; and SO3 Þ acting as the electron donors. Autotrophic denitrifiers utilize inorganic carbon compounds e.g. ðCO2 ; HCO2 3 Þ as their carbon source for growth. Therefore, compared with conventional heterotrophic denitrification, autotrophic

*

Corresponding author. Tel.: +34 981 563100x16777; fax: +34 981 528050. E-mail address: [email protected] (J.L. Campos). 0960-8524/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.02.007

denitrification has two clear advantages: (1) no need for an external organic carbon source, e.g. methanol or ethanol, which decreases the cost of the process; and (2) less biomass production, which minimizes the handling of sludge (Claus and Kutzner, 1985; Zhang and Lampe, 1999). The common values of maximum growth rates for autotrophic denitrifying microorganisms, 0.11–0.20 h1 (Claus and Kutzner, 1985; Oh et al., 2000), are similar to the heterotrophic ones (0.062–0.108 h1 (Wiesmann, 1994)), while the biomass yields are lower for the autotrophic denitrifying microorganisms (0.40–0.57 g VSS/g NO 3 –N (Claus and Kutzner, 1985; Oh et al., 2000)) compared to 0.8–1.2 g VSS/g NO x –N (Wiesmann, 1994) for the denitrifying heterotrophic ones. The presence of the sulphur compounds used and nitrate in stoichiometric amounts is of prime importance for biomass growth and operation of the autotrophic denitrifying system. The stoichiometries of several denitrification reactions using sulphur compounds as energy sources are given in the following Eqs. (1)–(3):

1294

J.L. Campos et al. / Bioresource Technology 99 (2008) 1293–1299

Table 1 Autotrophic denitrification using reduced-S compounds as electron donors Authors Krishnakumar and Manilal (1999) Gommers and Kuenen (1988a) Kleerebezem and Me´ndez (2002) Claus and Kutzner (1985) Trouve et al. (1998) Bisogni and Driscoll (1977) Justin and Kelly (1978) Furumai et al. (1996) Kimura et al. (2002) Soares (2002)

Biomass Enriched sludge Enriched sludge Enriched sludge Enriched sludge Enriched sludge Enriched sludge Pure culture Enriched sludge Enriched sludge Enriched sludge

Experiment type Batch Continuous Continuous Batch Batch Continuous Continuous Continuous Continuous Continuous

  þ 0:844 S2 O2 3 þ NO3 þ 0:347 CO2 þ 0:086 HCO3 þ 0:086 NH4 þ 0:434 H2 O ! þ ð1Þ 1:689 SO2 4 þ 0:500 N2 þ 0:086 C5 H7 O2 N þ 0:697 H  þ 0:421 H2 S þ 0:421 HS þ NO þ 0:346 CO þ 0:086 HCO þ 0:086 NH ! 2 3 3 4 þ 0:842 SO2 4 þ 0:500 N2 þ 0:086 C5 H7 O2 N þ 0:434 H2 O þ 0:262 H þ 1:10 So þ NO þ 0:76 H O þ 0:40 CO þ 0:08 NH ! 2 2 4 3 þ 1:10 SO2 4 þ 0:50 N2 þ 0:08 C5 H7 O2 N þ 1:28 H

ð2Þ ð3Þ

Results found in the literature show the importance of the type of sulphur source on the intermediate and final products of autotrophic denitrification obtained (Table 1). Therefore, it is of interest to know the reactions involved in the process as a preliminary step prior to the design of lab- or pilot-scale units. Furthermore, batch experiments are reported as being very useful to describe the effects of short-term changes in the operation of autotrophic denitrifying reactor systems (Gommers and Kuenen, 1988a). Autotrophic denitrification has been successfully applied as a post-treatment to achieve low concentrations of nitrogen in small-scale wastewater treatment facilities (Kuai and Verstraete, 1999) and to remove nitrate from aquaria (Vidal et al., 2002), from groundwater (Moon et al., 2004, 2006) and from drinking water (Soares, 2002). However, there is a lack of information about its application to the treatment of industrial wastewaters. Since it is common that these streams contain compounds that may inhibit biological processes, research studies to test their possible toxicity effects are necessary to know the feasibility of this treatment. The objectives of this work were to study in batch assays: (1) the effects of the S/N ratio used on the type of products obtained; (2) the inhibitory effects of both endogenous (nitrate, nitrite, thiosulphate and sulphate) and exogenous compounds (acetate and NaCl); and (3) the feasibility of using sulphur or sulphide as electron donors. 2. Methods

Sulphur source 2

S S2 S2 S2 O2 3 S2 O2 3 S2 O2 3 S2 O2 3 S2 O2 3 S° S°

Nitrogen source

Intermediate products formed

NO 3 NO 3 NO 3 NO 3 NO 3 NO 3 NO 3 NO 3 NO 3 NO 3

S°, NO 2 S°, NO 2 S°  NO2 NO 2 NO 2 NO 2 NO 2 NO 2 NO 2

was maintained in closed vials with a total volume of 1 l and 800 ml of liquid phase. Temperature was maintained at 28–30 °C and mixing achieved in a shaker at 150 rpm. Vials were periodically fed in batch mode with nitrate and thiosulphate to maintain biomass activity. Required amounts of sludge were collected from the vials to carry out the batch experiments. 2.2. Batch assays to estimate the effects of the S/N ratio A stirred tank reactor with a useful volume of 3 l containing autotrophic biomass and equipped with a water jacket connected to a thermostatic bath was operated in batch mode. The anoxic conditions required to use nitrate as electron acceptor were achieved through continuous gassing with argon, while complete mixing was achieved by means of mechanical stirring at 150 rpm. In these experiments, the effects of different initial S/N ratios and temperature were tested. To test the effects of the initial S/N ratio the reactor was filled with a medium containing 1.5 g NaHCO3/l, 1.5 g Na2HPO4/l, 0.3 g KH2PO4/l, 0.5 g MgSO4 Æ 7H2O/l, 0.1 g NH4Cl/l, trace elements, 2.96 g Na2S2O3/l and variable concentrations of NaNO3 (1.21– 5.46 g NaNO3/l). The effects of temperature were tested by changing the temperature of the water in the thermostatic jacket at 25, 30 and 35 °C. The pH value was maintained at 8 for all experiments through the addition of 1 M NaOH. The maximum specific denitrification rates were estimated from the maximum slopes of the curves obtained describing the nitrate and nitrite (intermediate of the reaction) concentration decreases in the liquid phase during the experiment and related to the biomass concentration in the reactor. 2.3. Batch experiments to estimate the effects of endogenous and exogenous compounds

2.1. Biomass Autotrophic denitrifying biomass was pre-enriched from granular anaerobic sludge collected from a UASB reactor treating fish canning wastewater by Kleerebezem and Me´ndez (2002), using nitrate and hydrogen sulphide. The sludge

In this case vials of 120 ml, with a liquid volume of 100 ml and 20 ml of gas phase, were used. The basal medium consisted of a buffer solution with 1.43 g KH2PO4/l and 7.47 g K2HPO4/l, the initial concentrations of thiosulphate and nitrate being 300 mg S2 O2 3 –S=l and 100 mg

J.L. Campos et al. / Bioresource Technology 99 (2008) 1293–1299

NO 3 –N=l, respectively (when thiosulphate or nitrate effects were studied these initial concentrations were changed). Different amounts of concentrated solutions were added in order to obtain the desired concentrations of the compounds tested (Table 2). When the effects of organic matter were tested a vial containing neither thiosulphate nor acetate was used as a control to show endogenous denitrifying activity and another vial was fed only with acetate as electron donor to study heterotrophic denitrification. The pH value was adjusted to 7.5. To obtain anoxic conditions, the vials were sealed and flushed with N2 gas for 5 min and placed in a shaker (150 rpm) at 30 °C. Aliquots of sludge were weighed exactly and added in order to have a biomass concentration of 1 g VSS/l in the vials. Biogas production was monitored during the experiments by means of a pressure transducer (Buys et al., 2000). Biogas was analysed at the end of the tests, the N2 percentage being always higher than 99%. The maximum specific denitrifying activity was calculated from the maximum slope of the curve representing the cumulative nitrogen production over time and related to the biomass concentration in the vials. The percentage of activity was calculated as the ratio of the maximum specific activity at each concentration of the compound tested to the maximum specific activity without the addition of this compound. For the assays where the effect of nitrate and thiosulphate were tested, 100% of activity was considered to occur at concentrations of 100 NO 3 –N=l and 300 mg S2 O2 –S=l, respectively. 3

1295

2.4. Analytical methods Biomass concentration was determined in terms of volatile suspended solids (VSS) as proposed in Standard Methods (APHA, 1985). Nitrite, nitrate and sulphate ions were analysed by capillary electrophoresis using a Waters Quanta 4000 system with sodium sulphate as the electrolyte for the nitrogen compounds and chromate as the electrolyte for the sulphate analysis (Vilas-Cruz et al., 1994). Sulphide concentration was determined by ion-selective electrode (Orion, model 9616), while thiosulphate was estimated spectrophotometrically according to the standard methods (APHA, 1985). Biogas composition (N2, CO2, CH4 and N2O) was analysed by gas chromatography (Hewlett–Packard 5890 series II) using He as carrier gas and a thermal conductivity detector (Ferna´ndez et al., 1995). 3. Results and discussion 3.1. Effects of the S/N ratio Several activity tests using thiosulphate and nitrate at different initial S/N ratios (1.16, 2.44, 3.70 and 6.67 g/g) were carried out at pH 8 and a temperature of 30 °C. The stoichiometric value of the S/N ratio corresponding to autotrophic denitrification using thiosulphate as electron donor was calculated as 3.84 g/g from Eq. (1), which is quite similar to the experimental value of 3.72 g

Table 2 Batch assays to estimate effects of endogenous and exogenous compounds and electron donor Series

Compound

Endogenous compounds A-I Nitrate A-II

Nitrite

A-III

Thiosulphate

A-IV

Sulphate

Exogenous compounds B-I Acetate

B-II

NaCl

Electron donor sources C-I Sulphur

C-II

Sulphide

Initial concentrations S2 O2 3 ðmg S=lÞ NO 3 ðmg N=lÞ S2 O2 3 ðmg S=lÞ NO 3 ðmg N=lÞ NO 2 ðmg N=lÞ S2 O2 3 ðmg S=lÞ NO 3 ðmg N=lÞ S2 O2 3 ðmg S=lÞ NO 3 ðmg N=lÞ SO2 4 ðmg S=lÞ

300 100 300 100 0 300 100 300 100 0

300 250 300 0 50 500 100 300 100 500

300 500 300 0 100 1000 100 300 100 1000

300 1000 300 0 250 2500 100 300 100 2500

300 2000 300 0 500 5000 100 300 100 5000

S2 O2 3 ðmg S=lÞ NO 3 ðmg N=lÞ Acetate (mg C/l) S2 O2 3 ðmg S=lÞ NO 3 ðmg N=lÞ NaCl (mg/l)

300 100 0 300 100 0

300 100 250 300 100 500

300 100 1000 300 100 1000

300 100 2500 300 100 5000

300 100 5000 300 100 10000

S2 O2 3 ðmg S=lÞ NO 3 ðmg N=lÞ S0 (mg S/l) S2 O2 3 ðmg S=lÞ NO 3 ðmg N=lÞ S2 (mg S/l)

300 100 0 300 100 0

0 100 100 0 100 50

0 100 300 0 100 100

0 100 500

0 100 1000

Sources: summary of conditions studied.

300 0 1000

0 100 0

0 100 250

1296

J.L. Campos et al. / Bioresource Technology 99 (2008) 1293–1299

mg S/l

a

1500

250

1200

200

900

150

600

100

300

50

0

mg N/l

 S2 O2 3 –S=g NO3 –N found by Oh et al. (2000). Nitrite was measured as an intermediate of the autotrophic denitrifying reaction, being partially or totally reduced into N2 gas as end product. When the activity assays were carried out under nitrate limiting or stoichiometric conditions (S/N ratio of 6.67 and 3.70 g/g), a maximum concentration of nitrite was achieved immediately after complete depletion of nitrate in the media (Fig. 1a). In these cases, nitrite formed was also fully reduced to nitrogen gas during the batch experiment. When the S/N ratio was lower than that required by stoichiometry, thiosulphate was the limiting compound (1.16 and 2.44 g/g) and nitrate was never depleted at the end of the experiment (Fig. 1b). Nitrite was also present at the end of the test due to the incomplete denitrification. The profiles of nitrate and nitrite concentrations indicate that the specific utilization rates of nitrate and nitrite were affected neither by substrate concentrations (zero-order reactions) nor by the initial S/N ratio, their values being 1.30 ± 0.36 g NO and 0.49 ± 0.03 g 3 –N=ðg VSS dÞ NO 2 –N=ðg VSS dÞ, respectively. Therefore, nitrite accumulation might be explained by the higher value of the specific utilization rate of nitrate compared to that of nitrite. Taking into account that the overall rate in the serial reactions corresponds to the rate of the limiting stage (nitrite reduction), the latter controls the consumption rate of nitrogenous compounds (nitrate plus nitrite) (Ble´con et al., 1983).

3.2. Effects of endogenous compounds

0 0

5

10

15

20

25

30

35

Time (h)

b 2000

1200

1500

900

1000

600

500

300

0

mg N/l

mg S/l

Sulphur balances based on measurements of sulphate and thiosulphate indicated a constant sulphur concentration over time, meaning that no elemental sulphur or other sulphur compounds (not measured) were produced in the system. There are different speculations about the factors that influence accumulation of nitrite in the autotrophic denitrification process, such as a delay in the induction of NO 2 reducing enzymes in the presence of nitrate, different saturation rates and affinities of electron acceptors, the type of bacteria, etc. (Krishnakumar and Manilal, 1999). Nitrite has been found to accumulate under very different conditions according to results from literature. YamamotoIkemoto et al. (2000) observed that nitrite was only detected when the S/N ratio was lower than 4.35 g/g, while Krishnakumar and Manilal (1999) and Justin and Kelly (1978) found accumulations under nitrate limiting conditions using sulphide and thiosulphate as sulphur sources, respectively. Previous workers have reported that elemental sulphur was formed when sulphide was used as electron donor (Bisogni and Driscoll, 1977; Kleerebezem and Me´ndez, 2002; Gommers and Kuenen, 1988b). However, when thiosulphate was used as electron donor no formation of elemental sulphur is referenced (Table 1), which agrees with the results obtained in the present study. Activity tests were carried out at temperatures of 25 and 35 °C (initial S/N ratio of 6.67 g/g and pH value of 8) in order to research the effects of the temperature on specific utilization rates of both nitrate and nitrite (Table 3). An increase in both nitrate and nitrite specific consumption activities was observed with the increase in temperature. However, the temperature did not affect the ratio between the specific utilization rates of nitrate and nitrite, its value being around 2.5, which is rather lower than that of 14.7 found by Furumai et al. (1996).

0 0

10

20

30

Time (h)

Fig. 1. Effects of the S/N ratio: typical profile of a test carried out with: (a) S/N of 6.67 and (b) S/N of 1.16. Thiosulphate ( ), nitrate (m), sulphate (s) and nitrite (D).



3.2.1. Nitrogen compounds An initial thiosulphate concentration of 300 mg S2 O2 3 –S=l was used to determine the effects of nitrate concentration on the autotrophic denitrifying activity (series A-I, Fig. 2). Thiosulphate was the limiting substrate when nitrate concentrations were higher than 100 mg NO 3 –N=l. Gas production was lower than that expected from stoichiometry for concentrations higher than 100 mg NO 3 –N=l.

Table 3 Effects of temperature on the specific nitrate and nitrite utilization rates Temperature (°C)

qNO3 (g N/g VSS d)

qNO2 (g N/g VSS d)

qNO3 /qNO2

25 30 35

1.24 ± 0.18 1.30 ± 0.36 2.00 ± 0.13

0.52 ± 0.10 0.49 ± 0.03 0.80 ± 0.15

2.4 2.6 2.5

J.L. Campos et al. / Bioresource Technology 99 (2008) 1293–1299 8

5

A-I

A-II 6 mg N2-N

mg N2-N

4 3 2

4 2

1 0

0

0

100

200

300

400

500

0

100

Time (min)

200 300 400 Time (min)

500

600

6

6

A-IV

A-III

5

5

4

mg N2-N

mg N2-N

1297

3 2

4 3 2 1

1

0

0 0

100 200 300 400 500 600

0

100

200

300

400

500

Time (min)

Time (min)



Fig. 2. Effects of endogenous compounds. Nitrate (A-I): 100 mg N/l (s), 250 mg N/l ( ), 500 mg N/l (D), 1000 mg N/l (m) and 2000 mg N/l (); nitrite (A-II): 0 mg N/l (s), 50 mg N/l ( ), 100 mg N/l (D), 250 mg N/l (m), 500 mg N/l () and 1000 mg N/l (r); thiosulphate (A-III): 300 mg S/l (s), 500 mg S/l (d), 1000 mg S/l (D), 2500 mg S/l (m) and 5000 mg S/l (); sulphate (A-IV): 0 mg S/l (s), 500 mg S/l (d), 1000 mg S/l (D), 2500 mg S/l (m) and 5000 mg S/l ().



This low gas production can be attributed to the specific consumption rate of nitrate being higher than that of nitrite, causing a large part of the electron donor to be used to reduce nitrate to nitrite without gas production. This fact was confirmed by the presence of nitrite, around 100 mg NO 2 –N=l, in the liquid phase at the end of the experiments. High nitrate concentrations around 670 mg NO 3 –N=l have been found to inhibit the process (Oh et al., 2000; Claus and Kutzner, 1985), but in this work, a clear detrimental effect of nitrate on the overall denitrification rate was not detected. The effect of nitrite was studied at an initial thiosulphate concentration of 300 mg S2 O2 3 –S=l. Inhibitory effects were only observed at concentrations of 500 and 1000 mg NO 2 –N=l, which caused inhibition percentages of 38 and 67%, respectively (series A-II, Fig. 2). Detrimental effects were detected at lower concentrations of 30 and 60 mg NO 2 –N=l by Krishnakumar and Manilal (1999) and Claus and Kutzner (1985), respectively. Since nitrite accumulation depends on the S/N ratio used and the initial nitrate concentration the control of both parameters is very important to avoid the inhibitory effects of this compound. 3.2.2. Sulphur compounds Thiosulphate had an inhibitory effect of around 20% at concentrations of 2.5 and 5 g S2 O2 3 –S=l (39 and 78 mM) (series A-III, Fig. 2). This value is quite similar to that observed with NaCl; therefore, this inhibition may have been due to a saline effect similar to that observed by Claus and Kutzner (1985).

The effect of sulphate was studied at concentrations between 0 and 5000 mg SO2 4 –S=l (0–156 mM). Inhibition appeared at a concentration of 500 mg SO2 4 –S=l. The autotrophic denitrifying activity was inhibited to 85% of that in the control test at a concentration of 5000 mg SO2 4 –S=l (series A-IV, Fig. 2). Oh et al. (2000) reported that sulphate inhibition began at concentrations above 2000 mg SO2 4 –S=l using a mixed culture, while Claus and Kutzner (1985) found that this compound started to inhibit at 1600 mg SO2 4 –S=l and caused total inhibition at 6400 mg SO2 –S=l when working with a pure 4 culture of T. denitrificans. The sulphate seems to exert an inhibitory effect beyond the salinity effect attributed to other compounds such as thiosulphate and sodium chloride. 3.3. Effects of exogenous compounds 3.3.1. Organic matter The effects of the organic matter were studied by adding acetate at concentrations from 0 to 5000 mg NaAc–C/l to 2 vials containing 100 mg NO 3 –N=l and 300 mg S2 O3 –S=l (series B-I, Fig. 3). At the concentrations used the presence of organic matter did not have significant effects on the autotrophic denitrifying activity and the measured heterotrophic denitrifying activity was negligible. This fact could be attributed to the high selection for autotrophic denitrifiers in the sludge used. In batch experiments working with mixed cultures containing heterotrophic denitrifying bacteria no significant effects of the organic matter at concentra-

1298

J.L. Campos et al. / Bioresource Technology 99 (2008) 1293–1299 8

4

B-II

B-I 6 mg N2-N

mg N2-N

3 2 1

4 2

0

0 0

100

200 300 Time (min)

400

500

0

100

200 300 Time (min)

400

500

Fig. 3. Effects of exogenous compounds. Acetate (B-I): 0 mg C/l (s), 250 mg C/l (d), 1000 mg C/l (D), 2500 mg C/l (m), 5000 mg C/l (), 0 mg C/l and 0 mg thiosulphate/l (r) and 250 mg C/l and 0 mg thiosulphate/l (h); NaCl (B-II): 0 mg NaCl/l (s), 500 mg NaCl/l (d), 1000 mg NaCl/l (D), 5000 mg NaCl/l (m) and 10000 mg NaCl/l ().

tions as high as 2 g glucose–C/l (Oh et al., 2000) or up to 400 g acetate–C/l in a sulphur packed bed reactor (Kim and Son, 2000), were observed. Furthermore, the addition of small amounts of organic compounds within an autotrophic environment during batch assays was beneficial in helping the process to maintain alkalinity (Oh et al., 2002). In contrast to this, Krishnakumar and Manilal (1999), working with a pure culture of T. denitrificans, found total inhibition of the process at concentrations of 150 mg NaAc–C/l in their tests. 3.3.2. Salts An activity decrease of only 10% with respect to the control was found at 10 g NaCl/l (172 mM) (series B-II, Fig. 3). These results agree with those of Claus and Kutzner (1985), who observed no effects at concentrations lower than 30 g NaCl/l in batch tests. On the other hand, Gu et al. (2004) successfully operated an autotrophic denitrifying reactor fed with a medium containing 33 g NaCl/l, which could confirm the resistance of microorganisms to high salinity conditions. 3.4. Influence of electron donor source 3.4.1. Sulphur Results indicated that this compound was not a good electron donor for the sludge used (series C-I, Fig. 4). Trouve et al. (1998) found that elemental sulphur was

not a suitable energy source for the nine strains of T. denitrificans they assayed. These authors classified the efficiency of the sulphur source in the following order 0 S2 O2 3 > FeS > FeS2 > S . This result disagrees with the findings of Kim and Bae (2000) and Soares (2002), who used sulphur packed filters to carry out autotrophic denitrification for wastewater and drinking water, respectively. On the other hand, nitrate loading rates treated using thio3 sulphate (6.3 kg NO (Yamamoto-Ikemoto 3 –N=m dÞ 3 et al., 2000) or sulphide ð5 kg NO –N=m dÞ (Gommers 3 and Kuenen, 1988a) as energy source were higher than those achieved using elemental sulphur ð0:20 kg N–NO 3= m3 dÞ (Soares, 2002), which again confirms this fact. Yamamoto-Ikemoto et al. (2000) also observed that specific sulphur denitrification activities using thiosulphate were 2–5 fold higher than those using sulphur granules. The different results obtained by these authors can be attributed to the different sources of the inocula tested and also to acclimation to the sulphur sources which differed from those tested. 3.4.2. Sulphide Sulphide seems to be a suitable electron donor for microorganisms adapted to thiosulphate because biogas production, without lag phase, was observed during experiments carried out in the presence of 50 and 100 mg S2–S/l (series C-II, Fig. 4). The profile of gas production using sulphide was different to that corresponding to thiosulphate,

8

8

C-II

C-I 6 mg N2-N

mg N2-N

6 4 2

4 2

0

0 0

100

200

300

Time (min)

400

500

0

100

200

300

400

500

Time (min)

Fig. 4. Effects of the electron donor sources: Sulphur (C-I): 0 mg S/l and 300 mg thiosulphate/l (s), 100 mg S/l (d), 300 mg S/l (D), 500 mg S/l (m) and 1000 mg S/l (); sulphide (C-II): 0 mg S/l and 300 mg thiosulphate/l (s), 50 mg S/l (d) and 100 mg S/l (D).

J.L. Campos et al. / Bioresource Technology 99 (2008) 1293–1299

which could indicate another reaction mechanism. Sulphide inhibition has been reported at concentrations higher than 800 mg S2–S/l by Krishnakumar and Manilal (1999) and Xiushan et al. (1993). However, in this work, the effect of sulphide concentrations higher than 100 mg S2–S/l was not tested because the buffer medium was not able to maintain an adequate pH value. 4. Conclusions Batch tests indicated that the specific utilization rate of nitrate was around 2.5 fold higher than that of nitrite and, therefore, nitrite accumulation was observed. As high concentrations of this compound inhibit autotrophic denitrification, it is necessary to control both S/N ratio and initial nitrate concentration fed to the system to maintain its efficiency. No important effects of the concentrations of thiosulphate, acetate and NaCl tested on autotrophic denitrifying activity were observed, but both sulphate and nitrite seemed to inhibit the process strongly. Sulphide can be used as electron donor, but the tests showed that these microorganisms might need an acclimation period to use elemental sulphur as energy source. Acknowledgements This work was funded by the Spanish CICYT through the BIOGRAMEN project (Ref: CTQ2005-04935/PPQ) and the Xunta de Galicia which funded this research through the project (PGIDIT03PXIC 20906 PN). References APHA–AWWA–WPCF, 1985. Standard Methods for Examination of Water and Wastewater, sixteenth ed. Washington DC, USA. Bisogni, J.J., Driscoll, C.T., 1977. Denitrification using thiosulphate and sulphide. J. Environ. Eng. Div., Am. Soc. Civil Eng. 103, 31–36. Ble´con, G., Gillet, M., Martı´n, G., Philipot, J.M., 1983. Autotrophic denitrification process by Thiobacillus denitrificans on sulphur-Mae¨rl. Revue Franc¸aise des Sciencies de l’Eau 2, 267–279. Buys, B.R., Mosquera-Corral, A., Sa´nchez, M., Me´ndez, R., 2000. Development and application of a denitrification test based on gas production. Water Sci. Technol. 41 (12), 113–120. Claus, G., Kutzner, H.J., 1985. Physiology and kinetics of autotrophic denitrification by Thiobacillus denitrificans. Appl. Microbiol. Biotechnol. 22, 283–288. Ferna´ndez, J.M., Me´ndez-Pampı´n, R.J., Lema, J.M., 1995. Anaerobic treatment of eucalyptus fiberboard manufacturing wastewater by a hybrid USBF lab-scale reactor. Environ. Technol. 15, 677–684. Furumai, H., Tagui, H., Fujita, K., 1996. Effects of pH and alkalinity on sulphur-denitrification in a biological granular filter. Water Sci. Technol. 34 (1–2), 355–362. Gommers, P.J.F., Kuenen, J.G., 1988a. Simultaneous sulphide and acetate oxidation in a denitrifying fluidized bied reactor-I. Start-up and reactor performance. Water Res. 9, 1075–1083. Gommers, P.J.F., Kuenen, J.G., 1988b. Simultaneous sulphide and acetate oxidation in a denitrifying fluidized bed reactor-II. Measurements of activities and conversion. Water Res. 9, 1085–1092.

1299

Gu, J.D., Qiu, W., Koenig, A., Fan, Y., 2004. Removal of high NO 3 concentrations in saline water through autotrophic denitrification by the bacterium Thiobacillus denitrificans strain MP. Water Sci. Technol. 49 (5–6), 105–112. Justin, P., Kelly, D.P., 1978. Growth kinetics of Thiobacillus denitrificans in anaerobic and aerobic chemostat culture. J. Gen. Microbiol. 107, 123–130. Kim, E.W., Bae, J.H., 2000. Alkalinity requirements and the possibility of simultaneous heterotrophic denitrification during sulphur-utilizing autotrophic denitrification. Water Sci. Technol. 42 (3–4), 233–238. Kim, I.S., Son, J.H., 2000. Impact of COD/N/S ratio on denitrification by the mixed cultures of sulphate reducing bacteria and sulphur denitrifying bacteria. Water Sci. Technol. 42 (3–4), 69–76. Kimura, K., Nakamura, M., Watanabe, Y., 2002. Nitrate removal by a combination of elemental sulphur-based denitrification and membrane filtration. Water Res. 36 (7), 1758–1766. Kleerebezem, R., Me´ndez, R., 2002. Autotrophic denitrification for combined hydrogen sulphide removal from biogas and post-denitrification. Water Sci. Technol. 45 (10), 349–356. Krishnakumar, B., Manilal, V.B., 1999. Bacterial oxidation of sulphide under denitrifying conditions. Biotechnol. Lett. 21, 437–440. Kuai, L., Verstraete, W., 1999. Autotrophic denitrification with elemental sulphur in small-scale wastewater treatment facilities. Environ. Technol. 20, 201–209. Moon, H.S., Ahn, K.H., Lee, S., Nam, K., Kim, J.Y., 2004. Use of autotrophic sulfur-oxidizers to remove nitrate from bank filtrate in permeable reactive barrier system. Environ. Pollut. 129, 499–507. Moon, H.S., Chang, S.W., Nam, K., Choe, J., Kim, J.Y., 2006. Effect of reactive media composition and co-contaminants on sulphur-based autotrophic denitrification. Environ. Pollut. 144, 802–807. Oh, S.E., Kim, K.S., Choi, H.C., Cho, J., Kim, I.S., 2000. Kinetics and physiological characteristics of autotrophic denitrification by denitrifying sulphur bacteria. Water Sci. Technol. 42 (3–4), 59–68. Oh, S.E., Bum, M.S., Yoo, Y.B., Zubair, A., Kim, I.S., 2002. Nitrate removal by simultaneous sulphur utilizing autotrophic and heterotrophic denitrification under different organics and alkalinity conditions: batch experiments. Water Sci. Technol. 47 (1), 237–244. Soares, M.I.M., 2002. Denitrification of groundwater with elemental sulphur. Water Res. 36 (5), 1392–1395. Trouve, C., Chazal, P.M., Gueroux, B., Sauvaitre, N., 1998. Denitrification by new strains of Thiobacillus denitrificans under non-standard conditions. Effect of temperature, pH and sulphur source. Environ. Technol. 19, 601–610. Vidal, S., Rocha, C., Galvao, H., 2002. A comparison of organic and inorganic carbon controls over biological denitrification in aquaria. Chemosphere 48, 445–451. Vilas-Cruz, M., Go´mez, G., Me´ndez, R., Lema, J.M., 1994. Simultaneous  determination of NO 2 and NO3 in wastewater by capillary electrophoresis. In: Proc. of the III International Symposium of Analytical Methodology for the Environment, vol. II, Ref. P1, pp 50, Barcelona, Spain, 23–24 March (in Spanish). Wiesmann, U., 1994. Biological nitrogen removal from wastewater. In: Fletcher, A. (Ed.), Advances in Biochemical Engineering Biotechnology, vol. 51. Springer-Verlag, Berlin, Germany, pp. 113–154. Xiushan, Y., Garuti, G., Tilche, A., 1993. Denitrification with Thiobacillus denitrificans in the ANANOX process. Biotechnol. Lett. 15 (5), 531– 536. Yamamoto-Ikemoto, R., Komori, T., Nomura, M., Matsukami, T., 2000. Nitrogen removal from hydrophonic culture wastewater by autotrophic denitrification using thiosulphate. Water Sci. Technol. 42 (3– 4), 369–376. Zhang, T.C., Lampe, D.G., 1999. Sulfur:limestone autotrophic denitrification processes for treatment of nitrate-contaminated water: batch experiments. Water Res. 33 (3), 599–608.