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Environmental Technology, Vol. 27. pp 103-108 Selper Ltd., 2006 ESTIMATION OF (C/N) RATIO FOR MICROBIAL DENITRIFICATION P. SOBIESZUK* AND K. W. SZEWCZYK Warsaw University of Technology, Faculty of Chemical and Process Engineering, Warynskiego 1, 00-645 Warsaw, Poland (Received 1 December 2004; Accepted 3 August 2005) ABSTRACT Elemental balances of microbial growth have been applied to calculate (C/N) ratio in a denitrification process. The correlation between biomass yield coefficient and degree of reduction has been used to estimate the critical (C/N) ratio. It has been shown that when the degree of reduction of the substrate is lower than 4.67, the critical value of (COD/N) is almost constant and equals 7.6 g O 2 g -1 N. The batch cultures of denitrifying bacteria Ervinia sp. on glucose, sodium acetate and methanol have been used to verify theoretical relations. The results of experiments generally agree with theoretical considerations. Keywords: INTRODUCTION Mass balances are fundamental for processes design. In the case of microbial processes, mass balances including the four elements: carbon, hydrogen, oxygen and nitrogen are commonly used [1]. The mass balance of microbial growth would give some thermodynamic limits of quantitative relationships of microbial processes. Biological denitrification is an example of such a limitation. Denitrification is carried out by heterotrophic microorganisms which reduce nitrite and nitrate to nitrogen in anoxic conditions [2]. The key factor affecting the efficiency of denitrification is the (C/N) ratio, which describes the amount of available carbon source consumed to the amount of nitrogen compounds reduced. Two different situations may occur in the denitrification process. When the real (C/N) ratio in sewage is lower than the critical value, (C/N) cr the nitrogen present exceeds the available carbon substrate and the denitrification efficiency is less then 1. This situation may take place in waste water with a high concentration of nitrogen compounds. An addition of carbon compounds is then necessary to increase denitrification efficiency [3]. When the (C/N)>(C/N) cr waste water contains a small amount of nitrogen in the presence of the carbon substrate surplus and full denitrification is possible [4]. There are a number of studies investigating experimentally or by stoichiometric analysis the critical value Denitrification, (C/N) ratio, elemental balance of (C/N). In most cases a fixed carbon source and stoichiometry of biomass growth is used. Nurse [5] calculated theoretically the (C/N) ratio for denitrification with methanol as a carbon source. Christensson et al. [3] and Kim et al. [6] presented experimental values of (C/N) ratio for denitrification of waste water by active sludge and pure strains. Values presented in the literature vary and it is difficult to explain observed differences in (C/N). Recently Zhou [7], based on McCarty et al s [8] half reaction concept, has presented a developed stoichiometric equation of denitrification. A fixed formula of biomass composition has been applied. The resulting formula consisting of stoichiometric coefficients are complex and not convenient for use. This approach does not allow a general interpretation of experimental data. In the present work an elemental balance and concept of reduction degree are used to generalize the theoretical calculation of (C/N) ratio and analyze factors affecting the value of critical (C/N). ELEMENTAL BALANCES OF DENITRIFICATION The elemental balances are a simple and powerful tool in microbial growth modelling [1]. A microbial growth system is represented as a semi-stoichiometrical reaction. The mass balance is limited to four elements only: carbon, hydrogen, 103
oxygen and nitrogen. Usually an average composition of the biomass is assumed. Denitrification is an anoxic process in which denitrifying bacteria use nitrates as an electron acceptor. If nitrate is a unique available nitrogen source, then it is simultaneously assimilated for biomass synthesis. It has been assumed that the carbon source does not contain nitrogen. The microbial denitrification may be described in general as the following reaction: The efficiency of nitrogen removal depends on the carbon and nitrogen content ratio (C/N) in the broth. Equation (v) shows the (C/N) ratio in the most general case of microbial denitrification. Commonly, the molecular nitrogen is the denitrification product ( =0), and equation (v) simplifies to: ν s CH as O bs N cs + ν N HNO 3 ν x CH ax O bx N cx + ν p CH ap O bp N cp + ν H2O H 2 O + ν CO2 CO 2 (i) ν N = (vi) where ν is the balance coefficient and the subscripts indicate: S the substrate, N the nitrogen source, X the biomass, P the denitrification product. Some authors have proposed a stoichiometric equation with a specific set of coefficient values which are limited only to a specific biomass yield. The balance of nitrogen can be written as: c S + ν N c N = ν X c X + ν P (ii) The available electrons balance leads to: + ν N = ν X + ν P (iii) where Γ is the absolute degree of reduction (it is equal to the number of electrons transferred from the substance during complete oxidation to CO 2, H 2 O and N 2 ). For a substance of formula CH a O b N c the absolute degree of reduction is equal to: Γ = 4+a-2b. Combining equation (ii) and equation (iii), the linear relationship for nitrogen removal and biomass growth can be derived: ν N = c X 1 c N c P ν X (iv) In the equation (iv) is the biomass yield coefficient on substrate with respect to C-mole ( =ν X / ). derived: From equation (iv) the critical ration (C/N) would be The weight ratio (C/N) cr is calculated from: (C / N) cr = 12 14 g C g N (vii) Equation (vii) presents a general relationship of denitrification stoichiometry. Nurse [5] has proposed a particular stoichiometry for denitrification on methanol as a carbon source. The biomass yield coefficient was set to 0.24 C-mole of biomass on C-mole of methanol and (C/N) ratio was 0.88 g C g -1 N. These values satisfy the equation (vii). In the practice of waste water treatment the organic carbon concentration is represented by Chemical Oxygen Demand: COD g O 2 dm -3. The COD equivalent of C-moles of carbon source is [1]: COD = 8 (viii) The critical (COD/N) cr ratio is obtained from equation (vii): COD N 8 Γ = S cr 14 ( ) (ix) The equation (ix) presents a general relationship. Zhou [7] has obtained the particular form of this equation as a fixed stoichiometric biomass formula (CH 1.4 O 0.4 N 0.2 ). Table 1 presents a comparison of experimental values from continuous cultures of activated sludge and pure strain of Hyphomicrobium investigated by Christensson et al. [3] with calculation based on equation (ix). The calculated values agree with experimental results. Some discrepancies may result from measurement accuracies. Roels [1] proposed a simple correlation between biomass yield and reduction degree of the carbon source: ν N = c X c N Γ + c S S (v) 4.67 = 0.13 > 4.67 = 0.6 (x) 104
Table 1. The comparison of experimental critical (COD/N) cr ratio [3] and the values calculated from equation (ix). Microorganisms Carbon source Y XS experimental (C-mole of biomass C-mole -1 of substrate) experimental (COD/N) cr (g O 2 g -1 N) equation (ix) Activated sludge methanol 0.27 4.45 3.64 ethanol 0.27 3.85 3.64 Pure culture (25ºC) methanol 0.44 4.16 4.41 ethanol 0.48 5.81 4.64 Applying the Roels correlation (equation (x)) to equation (ix) and taking into account that average degree of reduction of biomass is equal to 4.8, the following estimation of the critical (COD/N) ratio is obtained: 4.67 > 4.67 COD N COD N = 1.52 ( ) cr = 8 Γ ( Γ ) S N cr 14 ( 2.88) (xi) For substrates with an absolute degree of reduction 4.67, the critical (COD/N) ratio is constant and for nitrates ( = -5) is equal to 7.6 g O 2 g -1 N. For nitrites ( = -3) the critical (COD/N) ratio is 4.6 g O 2 g -1 N. When the absolute degree of reduction of the carbon source is greater than 4.67, the critical (COD/N) ratio depends on the degree of reduction of the carbon substrate. For ethanol or methanol ( = 6) the critical (COD/N) ratio is equal to 5.5 for nitrate and 3.3 for nitrite. Kim et al. [6] investigated (COD/N) ratio for waste waters and reported a value of 7.06, which is very close to the one calculated from equation (xi). Blaszczyk et al. [9] investigated the growth of denitrifying bacteria on acetic acid and reported (COD/N) ratio equal to 5.74. The authors investigated denitrification on ethanol or methanol as the carbon source reported and a value from 3.78 [10] to 6.85 [8, 11], which is close to the value estimated from equation (xi). The low (COD/N) ratio in the Timmermans and Van Haute [10] study is a result of low biomass yield ( = 0.31 C-mole of biomass on C-mole of methanol). Reasons for the discrepancies between data presented in literature are not clear. The experimental study has been undertaken to verify the dependency described by equation (xi). Microorganisms MATERIALS AND METHODS Pure culture of bacteria Ervinia sp. was used in experiments. This strain was a gift from Prof. A. Grabinska- Loniewska, Faculty of Environmental Engineering, Warsaw University of Technology. The strain was maintained aerobically on nutrient agar slants at 4 C. Nutrient agar was supplemented with 1 g dm -3 of potassium nitrate. The bacteria were washed from three nutrient agar slants into 300 ml Erlenmeyer flask containing 100 ml of sterile broth. The broth contained 15 g dm -3 of aminobac, 10 g dm -3 of glucose and 10 g dm -3 of potassium nitrate. This culture was incubated 48 h at 30 C. Then the liquid was centrifuged at 6000 rpm for 20 minutes. A supernatant was poured out and the biomass was washed with sterile water and then centrifuged again. After 20 minutes the supernatant was poured out, water was added to the biomass and the inoculum was transferred into Erlenmeyer flasks. Medium Mineral medium contained (per litre): 4.5 g of MgCl 2 x 6H 2 O, 0.8 g of K 2 HPO 4, 0.01 g of (NH 4 ) 6 Mo 7 O 24 x 4H 2 O. Methanol, glucose and sodium acetate were used as the carbon source. The concentration of carbon substrate was chosen to achieve the required (COD/N) ratios. Potassium nitrate was the only nitrogen source. All media were flushed with nitrogen to ensure anoxic conditions. Culture Conditions The batch cultures of Ervinia sp. were run in 300 ml Erlenmeyer flasks containing 100 ml of mineral medium. The inoculum from three agar slants was added to one flask. All batch cultures were incubated at 28 C. Analytical Methods The concentrations of nitrates, nitrites, glucose, acetate and methanol were analyzed by liquid chromatography HPLC (Waters). The column IC-PakTM Anion HR, 4.6mm 50mm was used to analyse the concentration of nitrates and nitrites. The column Sugar-PakTM, 6.5mm 3000mm was used to analyse the concentration of glucose and methanol. The column Rspak KC-811, 8mm 300mm and the precolumn 6mm 50mm were used to analyse the concentration of acetate. The concentration of biomass was determined by gravimetric and nephelometric methods [12]. 105
RESULTS AND DISCUSSION Figure 1 shows concentration profiles in an exemplary culture with methanol as the carbon source with initial (C/N) in the medium equal to 1.5 g C g -1 N-NO - 3. Methanol concentration decreases due to the assimilation by microorganisms, while the concentration of biomass increases at the same time. The average biomass yield for growth on methanol was 0.59. The experimental value is very close to the one calculated from Roels correlation (equation (x)). For growth on glucose and sodium acetate, the growth yields were 0.55 and 0.56 respectively. The measured values are also very close to the 0.52 predicted by correlation (equation (x)). The nitrates concentration decreases as a result of respiration. The experimental data were used to calculate the ratio of the amount of nitrate removed to the biomass increase. Figure 2 presents the results of the calculations. According to the elemental balance (equation (iv)), a linear relation between Figure 1. Concentration profiles for a culture, carbon source: methanol, (C/N)=1.5. Figure 2. The dependence of biomass growth on total nitrogen consumption, for methanol as a carbon source, (C/N)=1.5. 106
biomass growth and total nitrates consumption is expected. The experimental data confirm theoretical predictions. The theoretical value of ν N /ν X calculated from equation (iv) for methanol is 1.04. In the experiments, for the initial values of (C/N) equal to: 0.4, 1.5, 3.8 g C g -1 N-NO - 3, the experimental values of ν N /ν X were: 1, 1.02, and 1 mole C- mole -1 respectively. All values are very close to the theoretical value Figure 3 shows the relation between the efficiency of nitrogen removal and the initial (COD/N) ratio. The critical ratio (COD/N) cr, calculated from equation (xi) for sodium acetate and glucose, equals 7.6 g O 2 g -1 N; the one for methanol (COD/N) cr is lower and equals 5.5 g O 2 g -1 N. Both values are marked in Figure 3. If either glucose or sodium acetate is the unique carbon source and (COD/N)>(COD/N) cr, a 100% efficiency of the denitrification is obtained. In the case of methanol as the sole carbon source, no more than 50% of nitrate was reduced. A similar effect was noticed by Akunna et al [13]. It is worth noting that in the cultures with methanol and sodium acetate as a mixed carbon source, full denitrification was observed (Figure 3). CONCLUSIONS The application of elemental balances to the denitrification process allows for a generalized estimation of the critical (C/N) ratio. The ratio of amount of reduced nitrate to the amount of consumed carbon source depends on the degree of reduction of carbon source and biomass yield coefficient. It was found that for carbon source reduction degree less that 4.67 the critical (COD/N) may be assumed to be constant and equals 7.6 g O 2 g -1 N. The experimental investigations of Ervinia sp. cultured on different carbon sources confirm the theoretical calculations. When methanol was used as a sole carbon source a lower limit of denitrification efficiency was observed. For a mixture of methanol and acetate, complete denitrification was obtained. Figure 3. Influence of (COD/N) ratio on efficiency of nitrogen removal for various carbon sources. REFERENCES 1. Roels, J.A., Energetics and Kinetics in Biotechnology, Elsevier Biomedical Press, Amsterdam, The Netherlands (1983). 2. Ferguson, S.J., Denitrification and its control. A. van Leeuw., 66, 89-110 (1994). 3. Christensson, M., Lie, E. and Welander, T., A comparison between ethanol and methanol as carbon sources for denitrification. Water Sci. Technol., 30, 83-90 (1994). 107
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