Comparative research on the use of methanol, ethanol and methane as electron donors for denitrification
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1 Comparative research on the use of methanol, ethanol and methane as electron donors for denitrification S. G. Santos, M. Zaiat, M. B.Varesche and E. Foresti * Departamento de Hidráulica e Saneamento, Escola de Engenharia de São Carlos, Universidade de São Paulo (USP), Av. Trabalhador SãoCarlense, CEP 559, São Carlos, SP, Brasil Fax () 7955; eforesti@sc.usp.br. Abstract The results of denitrification assays using three electron donor sources methanol, ethanol, and methane are presented and discussed herein, based on the apparent kinetic parameters estimated from the experimental data. The research was carried out in batch anoxic reactors fed with synthetic substrate simulating nitrified effluents from domestic sewage treatment plants. The most effective electron donor was ethanol, which completely removed nitrite and nitrate in 5 minutes. This efficiency was achieved by feeding the reactors with methanol and methane for and 5 minutes, respectively. The two reactions in series kinetic model, having nitrite as the intermediate compound, adequately represented the denitrification process in the reactors fed with methanol and ethanol. To apply this model, the conversions of nitrate to nitrite and of nitrite to molecular nitrogen were represented, respectively, by first and zero order equations. In both the methanol and ethanol experiments, nitrite conversion was the limiting step in the overall process. The model of two reactions in series could not be adjusted to the data from the methanefed reactor, probably due to the significant interference of the mass transfer phenomena on the overall process. However, a good adjustment was achieved with a simple model expressing the conversion of nitrate directly into molecular nitrogen. Keywords Denitrification; ethanol; kinetics; methane; methanol Introduction Denitrification occurs mainly in the absence of dissolved oxygen, under anoxic conditions in the presence of nitrate as electron acceptor. Denitrifying bacteria utilize organic matter as carbon and energy sources. However, effluents from secondary treatment plants normally contain very low concentrations of easily degradable organic matter. Hence, carbon and energy from external sources are commonly supplied to the reactor to enable the biological denitrification process to proceed satisfactorily. Nitrate can be converted into ammonium via assimilative reduction or into gaseous nitrogen byproducts, forming nitrite as an intermediate product in dissimilative reduction (Madigan et al., 997). Methanol, glucose, ethanol and acetate are some of the external electron donors successfully used for denitrification. Methanol is a common carbon source (Zhao et al., 999), mainly in fullscale wastewater treatment plants (Louzeiro et al., ). Particularly in Brazil, ethanol, abundantly produced from sugar cane and usually costing less than other convenient carbon sources, may represent a feasible alternative. Nevertheless, the need for additional exogenous electron donor sources increases operational costs, possibly representing a drawback for the use of innovative anaerobic processbased technologies. That is the reason why the role of methane in denitrification, which constitutes more than 7% of the biogas produced in anaerobic reactors, is investigated. The existence of microorganism consortia that can use methane to produce electron donors for denitrification under certain environmental conditions has been demonstrated (Houbron et al., 999; Costa et al., ). However, the process has not yet been completely established. The denitrification rate depends both on the carbon and energy sources used and on the carbon to nitrogen (C/N) ratio. Low C/N ratios can cause nitrite to accumulate (Bandpi & Elliott, 99), while the dissimilative reduction to ammonium can occur at high C/N ratios (Gylsberg et al. 99), damaging the denitrification process. Callado () obtained very efficient denitrification rates at C/N ratios of.9 to.7.
2 It is well known that kinetic parameters are useful tools for comparative process analysis. Their evaluation for different electron donors, therefore, plays an important role in denitrification studies. Some previous studies have presented the denitrification kinetics as following a single zeroorder reaction (Vieira, ). However, the process can be better represented if the sequence of reactions is considered and the concept of multiple reactions in series is applied (Levenspiel, 999). This approach can lead to a better understanding of process development and control, thus permitting some operating variables to be monitored to avoid intermediate product accumulation or poor denitrification performance. The results of experiments using different electron donors for denitrification (ethanol, methanol, and methane) are presented herein. The discussion that follows is based on the kinetic parameters derived from the data obtained throughout the batch reactor operation. Methods Figure shows a diagram of the labscale batch reactor utilized in this research. Three identical flasks were inoculated with.5 liters of granulated sludge from a poultry slaughterhouse wastewater treatment plant and. liters of synthetic substrate with a composition similar to nitrified effluent (Table ). A headspace of.5 liters was maintained in each flask and the reactors were kept in a controlled temperature chamber at ± ºC. Each reactor received a different carbon source: methanol (5. mg.l ), ethanol (. mg.l ) or methane, besides the synthetic substrate. The initial concentrations of ethanol and methanol resulted in a C/N ratio of. and the reactors were operated simultaneously. It was not possible to establish the C/N ratio for the methane reactor. Two liters of the liquid phase were removed daily from the reactors for analysis and were replaced with the same volume of synthetic substrate containing the corresponding electron donor. Nitrogen was fluxed for 5 minutes in the reactors fed with methanol and ethanol, while a mixture of synthetic air (5%) and methane (5%) was fluxed for minutes in the third reactor. The assays to estimate the kinetic parameters were conducted after two (Phase A) and four (Phase B) months of operation. Samples were taken regularly to obtain temporal profiles of nitrate and nitrite, and the results analyzed by adjusting kinetic models to the temporal profiles of nitrate and nitrite concentrations. The mixed liquor total solids concentration (MLSTV) was measured two and four months after startingup the experiment, when the temporal profiles were obtained. Table Synthetic substrate composition simulating nitrified effluent Constituent Concentration (mg.l ) NNTK. N. SO 7. PO 7. Alkalinity (CaCO ). Volatile acids (as acetic acid). Sodium bicarbonate. NaCl 5. MgCl. H O. CaCl. H O.9 The ph was buffered in 7.5 Figure Batch reactors utilized for denitrification assays. Sample characterization (nitrate, nitrite, alkalinity, MLSTV) followed the Standard Methods for the Examination of Water and Wastewater (99). Cell quantification was evaluated by the MPN method described by Tiedje (9), adapted to liquid samples. Observations of microorganism
3 morphology were performed under common and phase contrast microscopy using an Olympus (BX ) microscope. Results and Discussion Figure shows the temporal profiles of nitrate and nitrite for two distinct conditions: A after two months, and B after four months of operation. The analyses of temporal profiles of nitrite and nitrate concentrations in the ethanol and methanol experiments allowed for adjustment of the irreversible firstorder followed by zeroorder reactions in series model. According to the adjusted model, the first reaction transforms nitrate into nitrite as the sole intermediary product. The second one converts nitrite into N, as follows: k k N Thus, the mass balance in a batch reactor (equations and ) resulted in the nitrate and nitrite concentration profiles (equations and ). dc dt dc dt = k C = k C k kt C = C e o () k.t C = C ( e ) k.t o () () () and In equations () to (), C is the nitrate concentration; C, the nitrite concentration; C, the initial nitrate and nitrite concentrations; t, the time; k and k are, respectively, the o C firstorder and zero order kinetic constants. When methane was used as electron donor, the abovedescribed kinetic model did not fit the experimental data well. In this case, the nitrite concentration profile did not follow the temporal behavior observed in the experiments with ethanol and methanol. Therefore, a single zeroorder reaction model was proposed, considering the direct conversion of nitrate into N. The mass balance in a batch reactor resulted in: o C = C o k o t (5) In equation (5), k o is the zeroorder kinetic constant. It is worth noting that mass transfer phenomena had influence on the apparent kinetic parameter values determined here. In the methane assays, this influence was undoubtedly considerable. Table lists the kinetic parameters derived from the expressions adjusted for ethanol and methanol assays, while Table gives the parameters obtained for methane. After the acclimatization period, the reactor performance improved when ethanol and methanol were the electron donors, showing increasing denitrification rates throughout the experiment. Moreover, the time for complete denitrification was significantly shortened in phase B compared to phase A. For ethanol, k increased twofold in the second phase, while k increased approximately threefold. For methanol, k was about 5% higher in phase B. Since k remained unaltered with time in this case, it was assumed that the microbial community converting nitrite to N had a greater affinity with methanol from the beginning of the experiment. The zeroorder kinetic constant increased 5% from phase A to phase B in the reactor fed with
4 methane. Moreover, the conversion of nitrite to N was incomplete in phase A. In Phase B, the ethanolfed reactor took 5 minutes, the methanolfed reactor minutes, and the methanefed reactor 5 minutes to achieve % of nitrogen removal. (mg.l ) N Electron Donor: Ethanol Phase A, Electron Donor: Methanol Phase A,,5,,5,,5,,5,,5 (mg.l ) N (mg. l ) N Electron Donor: Ethanol Phase B 5 Electron Donor: Methanol Phase B,,5,,5,,5,,, (mg. l ) N (a) (mg.l ) N,,5, (mg. l ) N (mg. l ) N,,, (mg. l ) N,5, (mg. l ) N, Electron Donor: Methane Phase A 5 (mg. l ) N (mg. l ) N, Electron Donor: Methane Phase B 9 5 7,,5,,,,, (mg. l ) N (b) (c) Nitrate Experimental Data Nitrite Experimental Data Kinetic Model (Eq. ) Kinetic Model (Eq. ) Kinetic Model (Eq. 5) Figure Nitrate and nitrite concentration profiles in phases (A) and (B) for three electron donors: (a) Ethanol, (b) Methanol, and (c) Methane The firstorder kinetic constant for nitrate consumption was considerably higher for ethanol than for methanol. Similarly, the kinetic constant for N formation was higher in the presence of ethanol, thus confirming the best performance indicated by the denitrification rates observed in the ethanol
5 fed reactor. It is important to stress that, in both cases, the conversion rate of nitrite to N was constant and independent of the nitrite concentration (zero order model). The data indicated that the nitrite to N conversion reaction was the limiting step in the two reactors overall reaction rates. Table Kinetic parameters of the multiple irreversible reactions model obtained from the data of phases A and B using ethanol and methanol as electron donors Electron donors Apparent kinetic parameters Ethanol Methanol Phase A Phase B Phase A Phase B k (h ). ±.. ±..9 ±.. ±.5 k x (mol.l.h ).59 ±.7.7 ±.. ±..5 ±. R for first order reaction R for zero order reaction Table Kinetic parameters of the zeroorder reaction model in phases A and B based on the data of experiments using methane as electron donor Apparent kinetic parameters Electron donor: Methane Phase A Phase B k o x (mol.l.h ).7 ±.9.9 ±. R Figure presents the temporal profiles of conversion rates calculated for the batch systems (equations and ), using the estimated kinetic parameters (Table ). As discussed earlier herein, the conversion of nitrite into N was the limiting step of the overall conversion process for the systems containing methanol and ethanol as electron donors. The rates observed in phase A (after two months) were significantly lower than those observed in phase B (after four months) when ethanol was used. The rates varied slightly in the methanol experiment. Owing to the higher conversion rates attained, these results indicated ethanol as the most suitable electron donor. However, they also indicated that the biomass acclimatized more rapidly to methanol. The kinetic advantage of using ethanol instead of methanol is clearly illustrated in Figure. The conversion rates in the second phase, in which the biomass was acclimatized, were approximately twofold higher when ethanol was used. Moreover, less time was required for a batch cycle in the ethanol experiment. Low conversion rates (close to zero) were obtained after minutes with ethanol, while minutes were necessary when methanol was the electron donor. Louzeiro et al. () found a maximum denitrification rate of 9 mg N/gMLVSS/day in a methanolfed (. mg.l ) sequencing bath reactor. This rate was estimated based on the concentration of mixed liquor volatile suspended solids (MLVSS). They also observed distinct rates in the system with and without methanol, at the beginning and at the end of the cycle. In both cases, the intermediate (nitrite) was not considered and a zeroorder kinetic was adjusted for the conversion of nitrate into N. In this work, a denitrification rate of mg N N/gMLVTS/day was obtained for the system with methanol in phase B, i.e., 7% higher than that obtained by Louzeiro et al. (), probably due to the availability of methanol throughout the denitrification process. Table presents the theoretical alkalinity generated during the denitrification process for each electron donor tested. The production of one mol of OH or 5 g of alkalinity as CaCO is expected for each mol of nitratenitrogen reduced. For methane, the solubility of the gas was considered as.5 ml/ ml at 7 C. Table 5 presents the alkalinity values produced in each phase. The ratio between the bicarbonate alkalinity generated and nitrogen reduced (BA prod / N red ) was determined from the data of the oxidized nitrogen forms (nitrate and nitrite). For methanol and ethanol the results obtained for this ratio were close to the theoretical value of.57 mg CaCO /mg N, despite the deviation of the experimental values of bicarbonate alkalinity from the theoretical 5
6 ones (around %). However, higher deviations were found in the system containing methane, probably due to solubility problems and mass transfer phenomena., Electron Donor: Ethanol Phase A, Electron Donor: Ethanol Phase B Conversion rate (mol. l.h ),5,,5,,5 Nitrate ( ) Nitrite ( ) Conversion rate (mol. l.h ),5,,,, Nitrate ( ) Nitrite ( ),, Electron Donor: Methanol Phase A, , Electron Donor: Methanol Phase B,, Conversion rate (mol. l.h ),,,, Nitrate ( ) Nitrite ( ) Conversion rate (mol. l.h ),,,, Nitrate ( ) Nitrite ( ),,,, Figure Temporal profiles of nitrate and nitrite consumption rates in phases A and B Table Theoretical alkalinity produced during the denitrification process Bicarbonate Electron Alkalinity donor (mg CaCO.l ) Simplified stoichiometric equation Ethanol CH CH OH N + CO + 9H O + OH Methanol CH OH N + 5CO + 7H O + OH Methane 5 + 5CH N + 5CO + H O + OH Apparently abnormal BA prod /N red values were observed in the two phases when methane was used as electron donor. In phase A, the high value of bicarbonate alkalinity generated was incompatible with the amount of nitrogen (as nitrate and nitrite) reduced. This may be attributed to the growth of methanogenic organisms that use intermediate methanotrophic products, such as methanol (Madigan et al., 997) or acetic acid (Costa et al., ). Methanogenic organisms, therefore, may have had a competitive advantage over the denitrifiers in the acclimatization phase, thus generating bicarbonate alkalinity. In fact, Methanosarcinalike archaea that use methanol and acetate were observed by optical microscopy in sludge samples taken in phase A. These organisms were absent in phase B, in which the alkalinity produced was lower than that expected from a stoichiometric relation.
7 The mixed liquor total solids concentration presented no representative variation along the experimental time in the three reactors. Values of 9.5 g.l,. g.l and 7. g.l were observed for the systems with ethanol, methanol and methane, respectively. Table 5 Bicarbonate alkalinity produced during the denitrification process Parameter Ethanol Methanol Methane Phase A Phase B Phase A Phase B Phase A Phase B Produced Bicarbonate Alkalinity BA (mg CaCO.l ) Reduced Nitrogen N red (mg N.l ) * BA prod /N red (mg CaCO /mg N) Initial ph Final ph *Ratio between produced bicarbonate alkalinity and reduced nitrogen The estimated number of denitrifying microorganisms also confirmed the improvement of the denitrification process along time. In the inoculum, this number was. x 7 MPN/g TVS (Total Volatile Solids) with the predominance of cocorods and straight rods. The values in the adapted biomass were 5. x 9 MPN/g TVS,.7 x 9 MPN/g TVS and.7 x 9 MPN/g TVS for ethanol, methanol and methane reactors, respectively. The higher dilutions in phase B showed an expressive predominance of rods in the three reactors. These data confirmed the predominance of denitrifying microorganisms after the acclimatization period. Conclusions The following conclusions can be drawn from the data obtained from the experiments: Among the three electron donor sources, ethanol was found to be the most appropriate for the denitrification process, considering the high values of its kinetic parameters. The denitrification biomass in the three systems showed significant performance improvements during the fourmonth acclimatization period. The acclimatization phase of the methanolfed system was shorter than the others. Complete denitrification took place in 5, and 5 minutes, with ethanol, methanol and methane, respectively. Using ethanol and methanol as carbon and energy sources, an irreversible firstorder followed by zeroorder reactions in series model, with nitrite as intermediate, provided a good representation of the denitrification process. The kinetic constants obtained in the system with ethanol were considerably higher than those obtained for the system with methanol, resulting in higher denitrification rates for ethanol. Nitrite conversion was the limiting step in the overall reaction rate of methanol and ethanolfed systems. The denitrification kinetics in the presence of methane differed significantly and a reaction in series model failed to represent the process adequately. A simple zeroorder kinetics was therefore used to represent nitrate conversion into N. The behavior of methane in the experiments was clearly affected by solubility problems and mass transfer phenomena. However, the experiments confirmed the viability of using methane as electron donor for denitrification. Physical problems of the nature described herein can be better solved using properly designed reactors. 7
8 References Bandpi, A. M.; Elliott. (99). Groundwater denitrification with alternative carbon source. Water Science technology. Vol. No., pp. 7. Callado, N. H. (). Anaerobic/aerobic SBR system treating synthetic substrate simulating domestic sewage (in portuguese). p. Doctoral thesis Escola de Engenharia de São Carlos, Universidade de São Paulo. Costa, C.; Dijkema, C.; Friedrich, M.; Encina, P. G.; Polanco, F. F.; Stams, A. J. M. (). Denitrification with methane as electron donor in oxygenlimited bioreactors. Applied Microbiology and Biotechnology. 5: 757. Gylsberg, B.; Frette, L.; Westermann, P. (99). Dynamics of N O production from activated sludge. Water Research. Vol., N 7, pp. Houbron, E.; Torrijos, M.; Capdeville, B. (999). An alternative use of biogas applied in water denitrification. Water Science and Technology. Vol. No., pp. 5. Louzeiro, N. R.; Mavinic, D. S.; Oldham, W. K.; Meisen, A.; Gardner, I. S. (). Methanolinduced biological nutrient removal kinetics in a fullscale sequencing batch reactor. Water Research. pp. 77. Madigan, M. T.; Martinko, J. M.; Parker, J. (997). Brock Biology of Microorganisms. th edition. Prentice Hall. Levenspiel, O. (999) Chemical Engineering Reactor. rd edition. John Wiley & Sons, New York. Standard Methods for the Examination of Water and Wastewater (99). 9 th ed. Amer. Public Health Assoc., Americ. Water Works Association, Water Pollution Control Federation, Washington, D.C., p. Tiedje, J. M. Denitrification. In: Page, A. L., Miller, R. H. ; Keeney, D. R. (9). Methods of Soil Analysis. Part. Chemical and Microbiological Properties. ed. Madson, Wisconsin: USA, p. Vieira, L. G. T. (). Development of a combined fixedfilm anaerobic/aerobic system for nitrogen removal from domestic sewage pretreated by anaerobic reactor (in protuguese). p. Doctoral thesis Escola de Engenharia de São Carlos, Universidade de São Paulo. Zhao, H. W.; Mavinic, D. S.; Oldham, W. K.; Koch, F. A. (999). Controlling factors for simultaneous nitrification and denitrification in twostage intermittent aeration process treating domestic sewage. Water research. Vol., No., pp Acknowledgments This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo FAPESP (São Paulo, Brazil). We also thank Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
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