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Investigation of the denitrification activity of immobilized Pseudomonas butanovora cells in the presence of different organic substrates
Water Research 36 (2002) 1565–1571
Investigation of the denitrification activity of immobilized Pseudomonas butanovora cells in the presence of different organic substrates + Istva! n Kiss, Zolta! n Bihari, Be! la Polya! k Pe! ter Kesseru*, ! Bay Foundation for Applied Research, Institute for Biotechnology, Derkovits fasor 2, Szeged, H-6726, Hungary Zoltan Received 18 January 2001; received in revised form 2 July 2001; accepted 16 July 2001
Abstract An investigation was made of the effects of variation of the hydraulic retention time (HRT) and the concentrations of three different carbon sources (succinic acid, ethanol and acetic acid) on the denitrification activity of immobilized Pseudomonas butanovora cells. The highest denitrification activity was in all cases measured at a C : N ratio of 6 : 1 and a 3 1 relatively low HRT (2.5 h). The highest denitrification rates were 1.17 kg NO d for succinic acid, 1.63 kg 3 -N m 3 1 3 1 NO3 -N m d for ethanol and 1.53 kg NO3 -N m d for acetic acid. At the same C : N ratios, ethanol and acetic acid proved to be better substrates for the reduction of nitrate and nitrite. The determined optimum C : N ratios were 1.7870.31, 0.9570.17, 1.7670.42 for succinic acid, ethanol and acetic acid, respectively. The optimum C : N ratios for the different substrates did not change in response to an increased flow rate. At a C : N ratio of 3 : 1 and a HRT of 1.5 h, the immobilized cells did not retain their activity. Apart from the difference in the effectivity between the electron donors, the main influence on the denitrification rate was exerted by the flow rate The results of this study demonstrated that Pseudomonas butanovora can utilize all three of these carbon sources to achieve a high rate of denitrification. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Acetic acid; Cell immobilization; Denitrification; Ethanol; Pseudomonas butanovora; Succinic acid
1. Introduction As a consequence of the intensive industrialized agricultural production during the past 50 years, the residual ions of fertilizers and chemicals spread onto the land now pose ever-increasing problems both in the soil and in the groundwater, which is used as drinking water in certain areas. As in most European countries, nitrate contamination is a problem even in Hungary. In many areas, the nitrate concentration in the groundwater exceeds the limit of 50 mg l1 NO 3 set by the World Health Organization and the European Community.
*Corresponding author. Tel.: +36-62-432-252; fax: +36-62432-250. + E-mail address: [email protected] (P. Kesseru).
Although the primary toxicity is low, the consumption of drinking water contaminated with nitrate causes serious health risks because of the secondary and tertiary effects, i.e. the reduction to nitrite and the formation of nitrosamines [1]. Both chemical/physical and biological processes have been developed for the removal of nitrate from water. Although reverse osmosis, electrodialysis and ionexchange are very effective in removing nitrate from contaminated water, they also result in a concentrated contaminated waste brine, which must still be disposed of. In biological treatment procedures, the denitrifying bacteria make use of the chemically bound oxygen in nitrate as a terminal electron acceptor, and nitrogen is released as gaseous N2. Various biological denitrification technologies have been established in order to solve the problem of nitrate
0043-1354/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 3 - 1 3 5 4 ( 0 1 ) 0 0 3 6 4 - 5
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P. Kesseru+ et al. / Water Research 36 (2002) 1565–1571
in drinking water and groundwater. The existing technologies differ in the nature of the bacterium used for this purpose and in the technical setup of the equipment. Depending on the biochemical properties of the nitrateutilizing bacteria, they can be divided into two main groups: chemolithotrophic and heterotrophic bacteria. The chemolithotrophic cells are able to grow on the HCO 3 content of water and use H2 to transform nitrate into N2. They play an important role in bio-electrochemical systems developed for the elimination of nitrate [2,3]. In this approach, nitrate is reduced by bacteria immobilized on the surface of the cathode inside an electrochemical cell. Paracoccus denitrificans is the wellknown chemolithotrophic bacteria [4,5]. Most denitrifying bacteria, however, are heterotrophic. They require an organic carbon source for cell growth and nitrate reduction. Methanol, ethanol and acetic acid are commonly used as organic substrates to provide the reducing power for nitrate elimination [6–9]. The best-characterized heterotrophic bacteria are from the Pseudomonas genus. Following the discovery that Pseudomonas denitrificans is able to achieve complete denitrification, other species of Pseudomonas have also been reported to be capable of reduction of nitrate and nitrite [10–13]. The present investigation was undertaken in order to characterize the denitrification ability of Pseudomonas butanovora. A study was carried out for the effects of the variation of the hydraulic retention time (HRT) and the concentrations of succinic acid, ethanol and acetic acid as substrates on the operation of bioreactors containing immobilized Pseudomonas butanovora cells.
2. Materials and methods 2.1. Culturing conditions The Pseudomonas butanovora strain was isolated from the soil of highly contaminated land formerly used for military purposes. The isolate was maintained on DSM 1 agar plates (5 g peptone, 3 g meat extract and 15 g agar in 1000 ml distilled water). The bacterium was enriched in shaken cultures in DSM 1 broth, pH=7.0 (200 rpm, at 371C). The bacterial biomass for cell immobilization was fermented in the 80 l New Brunswick Mobile Pilot Plant fermentation system. The bacterium was identified by the staff of the National Collection of Agricultural and Industrial Microorganisms, University of Horticulture and Food Industry, Budapest, Hungary. The identification was based on the results of the API-20 NE test series. 2.2. Cell immobilization The pure grown culture was collected by centrifugation (Sorvall RC 3B, 2600 g, 30 min, 101C). The pellet
was resuspended in composite gel solution [14] at a final cell concentration of 5% (w/w). The composite solution was prepared by mixing 1.6% Na-alginate (Sigma) in 200 mg l1 Na2-EDTA solution and 1.6% Gelrite (Sigma) in 200 mg l1 Na2-EDTA solution in a volumetric ratio of 1 : 1. The two solutions were sterilized separately and mixed when hot. After the composite solution had cooled down, the collected bacterial cells were added. The mixed gel solution was added in drops to 1% CaCl2 solution. The average diameter of the resulting beads was 2 mm. The beads were stirred at 251C for 40 min and were then incubated at 41C for 12 h. 2.3. Analysis Nitrate and nitrite analyses were performed in accordance with Hungarian Standard No. 448/12-82 [15]. For nitrate concentration determination, the water sample and fresh 5 g l1 Na-salicylate solution were mixed in a volumetric ratio of 5 : 1. The samples were evaporated to dryness and dehydrated at 2001C for 30 min. Concentrated H2SO4 was then added in a volume equal to that of the initial Na-salicylate solution. After standing for 10 min at room temperature, the acidic solution was neutralized by the addition of 10 N NaOH solution and the optical density (OD) was measured at 410 nm on a Unicam Helios a spectrophotometer. Nitrite ion concentration was determined via reaction with sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride and the OD was measured at 540 nm. The measurements of succinic acid, ethanol and acetic acid were carried out on a Gynkotek HPLC instrument, with a SARASEP CAR-H column. Detection: with a refractive index detector and UV detector. Eluent: 0.01 N H2SO4, flow rate: 0.9 ml/min. Column temperature: 501C. Data evaluation: with CHROMELEON software. 2.4. Bioreactor operation The composite beads were placed in three 700 ml columns (46 cm long by 5 cm diameter). The volume of the beads was 510 cm3 in the case of succinate, and 300 cm3 for the ethanol and acetic acid bioreactors. The beads in the reactors were supported on the surface of hollow disks. Artificial nitrate-contaminated water was prepared by adding KNO3 to the ion-exchanged water 1 to attain a final NO 3 -N concentration of 45 mg l . The vertical flow in the columns was laminar flow. The pH of the water entering the bioreactors was adjusted to 7.0 and this water in all cases contained 0.05% CaCl2 in order to preserve the stability of the composite beads. The average effluent pH values were 7.4870.13, 7.6870.22 and 7.6470.14 for ethanol, acetic acid and succinic acid, respectively. The bioreactors were operated continuously for 36 days at 251C in three periods of
P. Kesseru+ et al. / Water Research 36 (2002) 1565–1571
12 days, in which the C : N ratio was initially 9 : 1, then 6 : 1 and finally 3 : 1. The initial HRT was decreased throughout the operation time so as to reach the maximum denitrification rates for the immobilized cells, and to investigate the effects of variation of the HRT and the concentration of the organic substrate in the medium on the activities of the bioreactors. 2.5. Calculation of HRT and denitrification rate In the determination of the average values of the HRT, the total volume of the bioreactors (700 ml) was taken into consideration. Denitrification rates, in 3 1 eliminated NO d , were calculated as 3 -N kg m ðinput NO 3 -N conc:2output NO3 -N conc:Þ flowrate : volume of the beads
The beads serve as the biologically active volume of the given bioreactor. T and F probes were used for the statistical analysis of the data.
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the activities of the bioreactors originated only from the degrees of effectiveness of the individual substrates. The denitrification rates increased in all cases, as may be seen in Fig. 1(a–c). The ethanol and the acetic acid bioreactors had attained higher denitrification rates than that for the succinic acid bioreactor by the end of this period. The final denitrification rate for the succinic acid bioreactor was similar to half those for the other two substrates (Table 1). The slopes of the straight lines fitted to the experimentally measured points differed significantly for the succinic acid and ethanol bioreactors (P=2 ¼ 0:0279) and for the succinic acid and acetic acid bioreactors (P=2 ¼ 0:0428). There was not a significant difference between the slopes of the straight lines fitted to the points for the ethanol and acetic acid bioreactors (Table 1). The substrate degradation activity, the amount of substrate-C eliminated, in mg l1, and the determined optimum C : N ratio for the different bioreactors are presented in Table 2. 3.2. Denitrification activities at a C : N ratio of 6 : 1
3. Results and discussion 3.1. Denitrification activities at a C : N ratio of 9 : 1 During the first 12 days, the bioreactors were operated at the high C : N ratio of 9 : 1 in order to provide the immobilized cells with ample reducing power to eliminate nitrate. In this case, the differences between
In the second 12-day period, the C : N ratio was decreased to 6 : 1, while the flow rate was increased. Despite the lower amount of organic substrate, the activity continuously rose in each case, as shown in Fig. 2(a–c). Similarly, as at a C : N ratio of 9 : 1, ethanol and acetic acid led to a faster denitrification than that for succinic acid. The highest of the denitrification rates 3 1 listed in Table 3 (1.63 kg NO d ) was that for 3 -N m
Fig. 1. Denitrification rates when the different carbon sources were used at a C : N ratio of 9 : 1.
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ethanol. The slopes of the straight lines for the ethanol and acetic acid bioreactors did not differ significantly at C : N ratios of 6 : 1 and 9 : 1. The amounts of substrate-C eliminated, in mg l1, and the determined optimum C : N ratios were very similar to the data observed at a C : N ratio of 9 : 1 in all cases (Table 4). This indicates that the increased denitrification rate was strongly connected
with the shortened HRT. The lower HRT (2.12–2.6 h) did not result in a lower activity. 3.3. Denitrification activities at a C : N ratio of 3 : 1 During the last 12 days, the C : N ratio and the HRT were further decreased, to 3 : 1 and 1.5–1.8 h,
Table 1 Ranges of the effects of different organic substrates and HRT on the denitrification rates of the bioreactors at a C : N ratio of 9 : 1 Carbon source
Succinic acid Ethanol Acetic acid a
HRTa (h)
3.1470.25 4.5871.59 4.8171.05
3 1 Denitrification ratea (kg NO d ) 3 -N m
Max.
Min.
0.48 0.73 0.73
0.15 0.16 0.13
Slope of straight line
0.0263 0.0524 0.0513
Calculated as described in the Materials and Methods section.
Table 2 Substrate degradation and the experimentally determined optimum C : N ratios for different carbon sources at a C : N ratio of 9 : 1 Carbon source
Substrate degradation (%)
Eliminated C (mg l1)
Determined C : N ratio
Succinic acid Ethanol Acetic acid
12.5674.90 9.5370.68 11.2873.18
60.15714.80 51.6874.74 76.3174.72
1.7870.23 0.9270.35 1.7770.15
Fig. 2. Denitrification rates when the different carbon sources were used at a C : N ratio of 6 : 1.
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respectively. The results obtained under these conditions are depicted in Fig. 3(a–c). For succinic acid and ethanol, less than half the initial activity was measured at the end of this period. The highest initial denitrification rate was measured for ethanol: 1.62 kg 3 1 NO d ; this value decreased to 0.56 kg NO 3 -N m 33 1 N m d . The acetic acid bioreactor lost only 26% 3 1 of the initial activity, from 1.43 kg NO d 3 -N m 3 1 to 1.06 kg NO -N m d . The data are listed in 3
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Table 5. The effect observed with the acetic acid bioreactor could possibly be explained by the slightly higher HRT. As demonstrated in the second 12-day period, there was no significant difference in the effectiveness between ethanol and acetic acid regarding the denitrification activities of the immobilized cells at the C : N ratios of 9 : 1 and 6 : 1. The amount of substrate-C eliminated, in mg l1, and the determined optimum C : N ratios were very similar to those in the
Table 3 Ranges of effects of the different organic substrates and HRT on the denitrification rates of the bioreactors at a C : N ratio of 6 : 1 Carbon source
Succinic acid Ethanol Acetic acid a
HRTa (h)
2.1270.57 2.5770.87 2.6670.96
3 1 Denitrification ratea (kg NO 3 -N m d )
Max.
Min.
1.17 1.63 1.56
0.49 0.77 0.58
Slope of straight line
0.0521 0.0697 0.0778
Calculated as described in the Materials and Methods section.
Table 4 Substrate degradation and the experimentally determined optimum C : N ratios for the different carbon sources at a C : N ratio of 6 : 1 Carbon source
Substrate degradation (%)
Eliminated C (mg l1)
Determined C : N ratio
Succinic acid Ethanol Acetic acid
36.19714.54 19.73711.70 29.67710.13
67.676.94 58.79719.62 72.13715.56
1.7870.38 1.0170.21 1.6370.65
Fig. 3. Denitrification rates when the different carbon sources were used at a C : N ratio of 3 : 1.
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Table 5 Ranges of effects of the different organic substrates and HRT on the denitrification rates of the bioreactors at a C : N ratio of 3 : 1 Carbon source
Succinic acid Ethanol Acetic acid a
HRTa (h)
1.5170.13 1.5970.26 1.8270.32
3 1 Denitrification ratea (kg NO d ) 3 -N m
Max.
Min.
1.01 1.62 1.43
0.39 0.56 0.70
Slope of straight line
0.0330 0.0348 0.0154
Calculated as described in the Materials and Methods section.
Table 6 Substrate degradation and the experimentally determined optimum C : N ratios for the different carbon sources at a C : N ratio of 3 : 1 Carbon source
Substrate degradation %
Eliminated C (mg l1)
Determined C : N ratio
Succinic acid Ethanol Acetic acid
55.19713.05 44.01712.74 61.33711.32
55.57713.65 53.87719.09 71.28715.10
1.7870.31 0.9570.17 1.7670.42
previous periods (Table 6). The experimentally determined optimum C : N ratio for the ethanol bioreactor was similar to the optimum C : N ratio of 1.25 given by Mohseni-Bandpi and Elliott [11] for a rotating biological contactor (RBC) system. The results of Croll et al. [16] and Mohseni-Bandpi and Elliott [11] suggested that the optimum C : N ratio for acetic acid in an RBC and in a fluidized bed reactor was 1.7 and 1.86, respectively. The optimum C : N ratio of 1.7670.42 found for acetic acid in the present study agrees well with their result. Even the C : N ratio of 3 : 1 for the organic substrates is almost double the optimum ratio, and therefore the amount of carbon source available could not be a limiting factor. When all the aspects mentioned above are taken into consideration, the low HRT appears to be the only cause of the activity loss in all cases. Similar to the earlier results [17], the change in the effluent pH was greater for acetic acid than for ethanol, but the pH values remained within the optimum range (7–9) for denitrification [18].
3 1 NO for succinic acid, 1.63 kg NO 3 -N m d 33 1 3 1 N m d for ethanol and 1.53 kg NO -N m d 3 for acetic acid. The nitrate and nitrite ion concentrations of the effluents were below the prescribed limits during denitrification of levels up to 0.870.1 kg NO33 1 N m3 d1 and 170.1 kg NO d for the 3 -N m succinic acid bioreactor, and for the ethanol and acetic acid bioreactors, respectively. 3. At the same C : N ratios, ethanol and acetic acid proved to be better substrates for the reduction of nitrate and nitrite. Although the C : N ratio was continuously decreased, the substrate content of the influent was always higher than the optimum level. The determined optimum C : N ratios in the last 12-day period were 1.7870.31, 0.9570.17 and 1.7670.42 for succinic acid, ethanol and acetic acid, respectively. Apart from the difference in the effectivity between the electron donors, the main influence on the denitrification rate was exerted by the flow rate. 4. The data indicated that a HRT of 1.5–1.8 h was too short for the immobilized cells to achieve a high rate of nitrate reduction during the last 12 days.
4. Conclusions 1. The present study has revealed the denitrification activity of Pseudomonas butanovora as a previously non-characterized Pseudomonas species. The immobilized cells exhibited high denitrification activities with all three carbon sources, i.e. succinic acid, ethanol and acetic acid. 2. The highest denitrification activities were detected at a HRT of 2–3 h and a C : N ratio of 6 : 1 : 1.17 kg
Acknowledgements ! and Aniko! The authors wish to thank M!aria Toth Ga! rgya! n for their excellent technical assistance. This research was supported by a Ph.D. fellowship grant from the Zolt!an Bay Foundation for Applied Research for P!eter Kesseru+ Istv!an Kiss, Zolt!an Bihari and by a PHARE grant (HU9606-02-01-624).
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References [1] Bouchard DC, Williams MK, Surampalli RY. Nitrate contamination of groundwater: source and potential health effects. Journal AWWA 1992;9:85–90. [2] Kiss I, Szekeres Sz, Bejerano TT, Soares MIM. Hydrogendependent denitrification: preliminary assessment of two bio-electrochemical systems. Water Sci Technol 2000;42 (1–2):373–9. [3] Sakakibara Y, Kuroda M. Electric prompting and control of denitrification. Biotechnol Bioeng 1993;42: 535–7. [4] John P, Whatley FR. The bioenergetics of Paracoccus denitrificans. Biochim Biophys Acta 1977;463:129–53. [5] Stouthamer AH. Metabolic regulation including anaerobic metabolism in Paracoccus denitrificans. J Bioeng Biomembr 1990;23:163–85. [6] Bockle R, Rohmann U, Wertz A. A process for restoring nitrate contaminated groundwater by means of heterotrophic denitrification in an activated carbon filter and anaerobic post-treatment underground. Aqua 1986;5: 286–7. [7] Bohler E, Haldenwange L, Schwabe G. Results and experience with the Nebio Tube Reactor process in the water treatment plant Coswing near Dresden. Water Sci Technol 1994;29(10-11):497–508. [8] Dahab M, Young WL. Nitrate removal from water supplies using biological denitrification. J Water Pollut Control Fed 1988;60:1670–8. [9] Liessens J, Germonpre R, Beernaert S, Verstrate W. Removing nitrate with a methylotrophic fluized bed. J Am Water Wks Assoc 1993;85:144–54.
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[10] Bonin P, Gilewicz M, Denis M, Bertrand JC. Salt requirements in the denitrifying bacterium Pseudomonas nautica. Res Microbiol 1989;140:159–69. [11] Mohseni-Bandpi A, Elliott DJ. Groundwater denitrification with alternative carbon sources. Water Sci Technol 1998;38(6):237–43. [12] Samuelsson MO. Dissimilatory nitrate reduction to nitrate, nitrous oxide, and ammonium by Pseudomonas putrefaciens. Appl Environ Microbiol 1985;50:812–5. [13] Thomas NG, Michael RB, James MT. Numerically dominant denitrifying bacteria from world soils. Appl Environ Microbiol 1976;33:926–39. [14] Kov!acs K, Poly!ak B. Hydrogenase reactions and utilisation of hydrogen in biogas production and microbiological denitrification systems. Proceedings of the Fourth IGT Symposium, Colorado Springs, 1991. p. 1–16 [Chapter 5]. [15] Hungarian Standards for Drinking Water Analysis. Determination of nitrate and nitrite ions. 1983. Standard No. MSZ 448/12-82. [16] Croll BT, Green LA, Hall T, Whitford CJ, Zabel TF. Biological fluidised bed denitrification for potable water. Paper presented at the International Conference on Advance in Water Engineering, Birmingham University, 15–19 July 1985. ! ry A, Rdeggard $ [17] Cs$ H, Bach K, Pujol R, Hamon M. Denitrification in a packed bed biofilm reactor (BIOFOR)Fexperiments with different carbon sources. Water Res 1998;32:1463–70. [18] Henze M, Harremo.es P, Jansen J, Arvin E. Wastewater treatment. Biological and Chemical. Polyteknisk Forlag, Lyngby, Denmark, 1992 [in Danish].
Investigation of the denitrification activity of immobilized Pseudomonas butanovora cells in the presence of different organic substrates + Istva! n Kiss, Zolta! n Bihari, Be! la Polya! k Pe! ter Kesseru*, ! Bay Foundation for Applied Research, Institute for Biotechnology, Derkovits fasor 2, Szeged, H-6726, Hungary Zoltan Received 18 January 2001; received in revised form 2 July 2001; accepted 16 July 2001
Abstract An investigation was made of the effects of variation of the hydraulic retention time (HRT) and the concentrations of three different carbon sources (succinic acid, ethanol and acetic acid) on the denitrification activity of immobilized Pseudomonas butanovora cells. The highest denitrification activity was in all cases measured at a C : N ratio of 6 : 1 and a 3 1 relatively low HRT (2.5 h). The highest denitrification rates were 1.17 kg NO d for succinic acid, 1.63 kg 3 -N m 3 1 3 1 NO3 -N m d for ethanol and 1.53 kg NO3 -N m d for acetic acid. At the same C : N ratios, ethanol and acetic acid proved to be better substrates for the reduction of nitrate and nitrite. The determined optimum C : N ratios were 1.7870.31, 0.9570.17, 1.7670.42 for succinic acid, ethanol and acetic acid, respectively. The optimum C : N ratios for the different substrates did not change in response to an increased flow rate. At a C : N ratio of 3 : 1 and a HRT of 1.5 h, the immobilized cells did not retain their activity. Apart from the difference in the effectivity between the electron donors, the main influence on the denitrification rate was exerted by the flow rate The results of this study demonstrated that Pseudomonas butanovora can utilize all three of these carbon sources to achieve a high rate of denitrification. r 2002 Elsevier Science Ltd. All rights reserved. Keywords: Acetic acid; Cell immobilization; Denitrification; Ethanol; Pseudomonas butanovora; Succinic acid
1. Introduction As a consequence of the intensive industrialized agricultural production during the past 50 years, the residual ions of fertilizers and chemicals spread onto the land now pose ever-increasing problems both in the soil and in the groundwater, which is used as drinking water in certain areas. As in most European countries, nitrate contamination is a problem even in Hungary. In many areas, the nitrate concentration in the groundwater exceeds the limit of 50 mg l1 NO 3 set by the World Health Organization and the European Community.
*Corresponding author. Tel.: +36-62-432-252; fax: +36-62432-250. + E-mail address: [email protected] (P. Kesseru).
Although the primary toxicity is low, the consumption of drinking water contaminated with nitrate causes serious health risks because of the secondary and tertiary effects, i.e. the reduction to nitrite and the formation of nitrosamines [1]. Both chemical/physical and biological processes have been developed for the removal of nitrate from water. Although reverse osmosis, electrodialysis and ionexchange are very effective in removing nitrate from contaminated water, they also result in a concentrated contaminated waste brine, which must still be disposed of. In biological treatment procedures, the denitrifying bacteria make use of the chemically bound oxygen in nitrate as a terminal electron acceptor, and nitrogen is released as gaseous N2. Various biological denitrification technologies have been established in order to solve the problem of nitrate
0043-1354/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 3 - 1 3 5 4 ( 0 1 ) 0 0 3 6 4 - 5
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P. Kesseru+ et al. / Water Research 36 (2002) 1565–1571
in drinking water and groundwater. The existing technologies differ in the nature of the bacterium used for this purpose and in the technical setup of the equipment. Depending on the biochemical properties of the nitrateutilizing bacteria, they can be divided into two main groups: chemolithotrophic and heterotrophic bacteria. The chemolithotrophic cells are able to grow on the HCO 3 content of water and use H2 to transform nitrate into N2. They play an important role in bio-electrochemical systems developed for the elimination of nitrate [2,3]. In this approach, nitrate is reduced by bacteria immobilized on the surface of the cathode inside an electrochemical cell. Paracoccus denitrificans is the wellknown chemolithotrophic bacteria [4,5]. Most denitrifying bacteria, however, are heterotrophic. They require an organic carbon source for cell growth and nitrate reduction. Methanol, ethanol and acetic acid are commonly used as organic substrates to provide the reducing power for nitrate elimination [6–9]. The best-characterized heterotrophic bacteria are from the Pseudomonas genus. Following the discovery that Pseudomonas denitrificans is able to achieve complete denitrification, other species of Pseudomonas have also been reported to be capable of reduction of nitrate and nitrite [10–13]. The present investigation was undertaken in order to characterize the denitrification ability of Pseudomonas butanovora. A study was carried out for the effects of the variation of the hydraulic retention time (HRT) and the concentrations of succinic acid, ethanol and acetic acid as substrates on the operation of bioreactors containing immobilized Pseudomonas butanovora cells.
2. Materials and methods 2.1. Culturing conditions The Pseudomonas butanovora strain was isolated from the soil of highly contaminated land formerly used for military purposes. The isolate was maintained on DSM 1 agar plates (5 g peptone, 3 g meat extract and 15 g agar in 1000 ml distilled water). The bacterium was enriched in shaken cultures in DSM 1 broth, pH=7.0 (200 rpm, at 371C). The bacterial biomass for cell immobilization was fermented in the 80 l New Brunswick Mobile Pilot Plant fermentation system. The bacterium was identified by the staff of the National Collection of Agricultural and Industrial Microorganisms, University of Horticulture and Food Industry, Budapest, Hungary. The identification was based on the results of the API-20 NE test series. 2.2. Cell immobilization The pure grown culture was collected by centrifugation (Sorvall RC 3B, 2600 g, 30 min, 101C). The pellet
was resuspended in composite gel solution [14] at a final cell concentration of 5% (w/w). The composite solution was prepared by mixing 1.6% Na-alginate (Sigma) in 200 mg l1 Na2-EDTA solution and 1.6% Gelrite (Sigma) in 200 mg l1 Na2-EDTA solution in a volumetric ratio of 1 : 1. The two solutions were sterilized separately and mixed when hot. After the composite solution had cooled down, the collected bacterial cells were added. The mixed gel solution was added in drops to 1% CaCl2 solution. The average diameter of the resulting beads was 2 mm. The beads were stirred at 251C for 40 min and were then incubated at 41C for 12 h. 2.3. Analysis Nitrate and nitrite analyses were performed in accordance with Hungarian Standard No. 448/12-82 [15]. For nitrate concentration determination, the water sample and fresh 5 g l1 Na-salicylate solution were mixed in a volumetric ratio of 5 : 1. The samples were evaporated to dryness and dehydrated at 2001C for 30 min. Concentrated H2SO4 was then added in a volume equal to that of the initial Na-salicylate solution. After standing for 10 min at room temperature, the acidic solution was neutralized by the addition of 10 N NaOH solution and the optical density (OD) was measured at 410 nm on a Unicam Helios a spectrophotometer. Nitrite ion concentration was determined via reaction with sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride and the OD was measured at 540 nm. The measurements of succinic acid, ethanol and acetic acid were carried out on a Gynkotek HPLC instrument, with a SARASEP CAR-H column. Detection: with a refractive index detector and UV detector. Eluent: 0.01 N H2SO4, flow rate: 0.9 ml/min. Column temperature: 501C. Data evaluation: with CHROMELEON software. 2.4. Bioreactor operation The composite beads were placed in three 700 ml columns (46 cm long by 5 cm diameter). The volume of the beads was 510 cm3 in the case of succinate, and 300 cm3 for the ethanol and acetic acid bioreactors. The beads in the reactors were supported on the surface of hollow disks. Artificial nitrate-contaminated water was prepared by adding KNO3 to the ion-exchanged water 1 to attain a final NO 3 -N concentration of 45 mg l . The vertical flow in the columns was laminar flow. The pH of the water entering the bioreactors was adjusted to 7.0 and this water in all cases contained 0.05% CaCl2 in order to preserve the stability of the composite beads. The average effluent pH values were 7.4870.13, 7.6870.22 and 7.6470.14 for ethanol, acetic acid and succinic acid, respectively. The bioreactors were operated continuously for 36 days at 251C in three periods of
P. Kesseru+ et al. / Water Research 36 (2002) 1565–1571
12 days, in which the C : N ratio was initially 9 : 1, then 6 : 1 and finally 3 : 1. The initial HRT was decreased throughout the operation time so as to reach the maximum denitrification rates for the immobilized cells, and to investigate the effects of variation of the HRT and the concentration of the organic substrate in the medium on the activities of the bioreactors. 2.5. Calculation of HRT and denitrification rate In the determination of the average values of the HRT, the total volume of the bioreactors (700 ml) was taken into consideration. Denitrification rates, in 3 1 eliminated NO d , were calculated as 3 -N kg m ðinput NO 3 -N conc:2output NO3 -N conc:Þ flowrate : volume of the beads
The beads serve as the biologically active volume of the given bioreactor. T and F probes were used for the statistical analysis of the data.
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the activities of the bioreactors originated only from the degrees of effectiveness of the individual substrates. The denitrification rates increased in all cases, as may be seen in Fig. 1(a–c). The ethanol and the acetic acid bioreactors had attained higher denitrification rates than that for the succinic acid bioreactor by the end of this period. The final denitrification rate for the succinic acid bioreactor was similar to half those for the other two substrates (Table 1). The slopes of the straight lines fitted to the experimentally measured points differed significantly for the succinic acid and ethanol bioreactors (P=2 ¼ 0:0279) and for the succinic acid and acetic acid bioreactors (P=2 ¼ 0:0428). There was not a significant difference between the slopes of the straight lines fitted to the points for the ethanol and acetic acid bioreactors (Table 1). The substrate degradation activity, the amount of substrate-C eliminated, in mg l1, and the determined optimum C : N ratio for the different bioreactors are presented in Table 2. 3.2. Denitrification activities at a C : N ratio of 6 : 1
3. Results and discussion 3.1. Denitrification activities at a C : N ratio of 9 : 1 During the first 12 days, the bioreactors were operated at the high C : N ratio of 9 : 1 in order to provide the immobilized cells with ample reducing power to eliminate nitrate. In this case, the differences between
In the second 12-day period, the C : N ratio was decreased to 6 : 1, while the flow rate was increased. Despite the lower amount of organic substrate, the activity continuously rose in each case, as shown in Fig. 2(a–c). Similarly, as at a C : N ratio of 9 : 1, ethanol and acetic acid led to a faster denitrification than that for succinic acid. The highest of the denitrification rates 3 1 listed in Table 3 (1.63 kg NO d ) was that for 3 -N m
Fig. 1. Denitrification rates when the different carbon sources were used at a C : N ratio of 9 : 1.
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ethanol. The slopes of the straight lines for the ethanol and acetic acid bioreactors did not differ significantly at C : N ratios of 6 : 1 and 9 : 1. The amounts of substrate-C eliminated, in mg l1, and the determined optimum C : N ratios were very similar to the data observed at a C : N ratio of 9 : 1 in all cases (Table 4). This indicates that the increased denitrification rate was strongly connected
with the shortened HRT. The lower HRT (2.12–2.6 h) did not result in a lower activity. 3.3. Denitrification activities at a C : N ratio of 3 : 1 During the last 12 days, the C : N ratio and the HRT were further decreased, to 3 : 1 and 1.5–1.8 h,
Table 1 Ranges of the effects of different organic substrates and HRT on the denitrification rates of the bioreactors at a C : N ratio of 9 : 1 Carbon source
Succinic acid Ethanol Acetic acid a
HRTa (h)
3.1470.25 4.5871.59 4.8171.05
3 1 Denitrification ratea (kg NO d ) 3 -N m
Max.
Min.
0.48 0.73 0.73
0.15 0.16 0.13
Slope of straight line
0.0263 0.0524 0.0513
Calculated as described in the Materials and Methods section.
Table 2 Substrate degradation and the experimentally determined optimum C : N ratios for different carbon sources at a C : N ratio of 9 : 1 Carbon source
Substrate degradation (%)
Eliminated C (mg l1)
Determined C : N ratio
Succinic acid Ethanol Acetic acid
12.5674.90 9.5370.68 11.2873.18
60.15714.80 51.6874.74 76.3174.72
1.7870.23 0.9270.35 1.7770.15
Fig. 2. Denitrification rates when the different carbon sources were used at a C : N ratio of 6 : 1.
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respectively. The results obtained under these conditions are depicted in Fig. 3(a–c). For succinic acid and ethanol, less than half the initial activity was measured at the end of this period. The highest initial denitrification rate was measured for ethanol: 1.62 kg 3 1 NO d ; this value decreased to 0.56 kg NO 3 -N m 33 1 N m d . The acetic acid bioreactor lost only 26% 3 1 of the initial activity, from 1.43 kg NO d 3 -N m 3 1 to 1.06 kg NO -N m d . The data are listed in 3
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Table 5. The effect observed with the acetic acid bioreactor could possibly be explained by the slightly higher HRT. As demonstrated in the second 12-day period, there was no significant difference in the effectiveness between ethanol and acetic acid regarding the denitrification activities of the immobilized cells at the C : N ratios of 9 : 1 and 6 : 1. The amount of substrate-C eliminated, in mg l1, and the determined optimum C : N ratios were very similar to those in the
Table 3 Ranges of effects of the different organic substrates and HRT on the denitrification rates of the bioreactors at a C : N ratio of 6 : 1 Carbon source
Succinic acid Ethanol Acetic acid a
HRTa (h)
2.1270.57 2.5770.87 2.6670.96
3 1 Denitrification ratea (kg NO 3 -N m d )
Max.
Min.
1.17 1.63 1.56
0.49 0.77 0.58
Slope of straight line
0.0521 0.0697 0.0778
Calculated as described in the Materials and Methods section.
Table 4 Substrate degradation and the experimentally determined optimum C : N ratios for the different carbon sources at a C : N ratio of 6 : 1 Carbon source
Substrate degradation (%)
Eliminated C (mg l1)
Determined C : N ratio
Succinic acid Ethanol Acetic acid
36.19714.54 19.73711.70 29.67710.13
67.676.94 58.79719.62 72.13715.56
1.7870.38 1.0170.21 1.6370.65
Fig. 3. Denitrification rates when the different carbon sources were used at a C : N ratio of 3 : 1.
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Table 5 Ranges of effects of the different organic substrates and HRT on the denitrification rates of the bioreactors at a C : N ratio of 3 : 1 Carbon source
Succinic acid Ethanol Acetic acid a
HRTa (h)
1.5170.13 1.5970.26 1.8270.32
3 1 Denitrification ratea (kg NO d ) 3 -N m
Max.
Min.
1.01 1.62 1.43
0.39 0.56 0.70
Slope of straight line
0.0330 0.0348 0.0154
Calculated as described in the Materials and Methods section.
Table 6 Substrate degradation and the experimentally determined optimum C : N ratios for the different carbon sources at a C : N ratio of 3 : 1 Carbon source
Substrate degradation %
Eliminated C (mg l1)
Determined C : N ratio
Succinic acid Ethanol Acetic acid
55.19713.05 44.01712.74 61.33711.32
55.57713.65 53.87719.09 71.28715.10
1.7870.31 0.9570.17 1.7670.42
previous periods (Table 6). The experimentally determined optimum C : N ratio for the ethanol bioreactor was similar to the optimum C : N ratio of 1.25 given by Mohseni-Bandpi and Elliott [11] for a rotating biological contactor (RBC) system. The results of Croll et al. [16] and Mohseni-Bandpi and Elliott [11] suggested that the optimum C : N ratio for acetic acid in an RBC and in a fluidized bed reactor was 1.7 and 1.86, respectively. The optimum C : N ratio of 1.7670.42 found for acetic acid in the present study agrees well with their result. Even the C : N ratio of 3 : 1 for the organic substrates is almost double the optimum ratio, and therefore the amount of carbon source available could not be a limiting factor. When all the aspects mentioned above are taken into consideration, the low HRT appears to be the only cause of the activity loss in all cases. Similar to the earlier results [17], the change in the effluent pH was greater for acetic acid than for ethanol, but the pH values remained within the optimum range (7–9) for denitrification [18].
3 1 NO for succinic acid, 1.63 kg NO 3 -N m d 33 1 3 1 N m d for ethanol and 1.53 kg NO -N m d 3 for acetic acid. The nitrate and nitrite ion concentrations of the effluents were below the prescribed limits during denitrification of levels up to 0.870.1 kg NO33 1 N m3 d1 and 170.1 kg NO d for the 3 -N m succinic acid bioreactor, and for the ethanol and acetic acid bioreactors, respectively. 3. At the same C : N ratios, ethanol and acetic acid proved to be better substrates for the reduction of nitrate and nitrite. Although the C : N ratio was continuously decreased, the substrate content of the influent was always higher than the optimum level. The determined optimum C : N ratios in the last 12-day period were 1.7870.31, 0.9570.17 and 1.7670.42 for succinic acid, ethanol and acetic acid, respectively. Apart from the difference in the effectivity between the electron donors, the main influence on the denitrification rate was exerted by the flow rate. 4. The data indicated that a HRT of 1.5–1.8 h was too short for the immobilized cells to achieve a high rate of nitrate reduction during the last 12 days.
4. Conclusions 1. The present study has revealed the denitrification activity of Pseudomonas butanovora as a previously non-characterized Pseudomonas species. The immobilized cells exhibited high denitrification activities with all three carbon sources, i.e. succinic acid, ethanol and acetic acid. 2. The highest denitrification activities were detected at a HRT of 2–3 h and a C : N ratio of 6 : 1 : 1.17 kg
Acknowledgements ! and Aniko! The authors wish to thank M!aria Toth Ga! rgya! n for their excellent technical assistance. This research was supported by a Ph.D. fellowship grant from the Zolt!an Bay Foundation for Applied Research for P!eter Kesseru+ Istv!an Kiss, Zolt!an Bihari and by a PHARE grant (HU9606-02-01-624).
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