Biochemical Engineering Journal 5 (2000) 29±37. Received 10 June 1999; accepted 25 October 1999

Transcription

1 Biochemical Engineering Journal 5 (2000) 29±37 Effect of C/N values on microbial simultaneous removal of carbonaceous and nitrogenous substances in wastewater by single continuous- ow uidized-bed bioreactor containing porous carrier particles Xin-Hui Xing a, Byong-Hee Jun b, Mari Yanagida b, Yasunori Tanji b, Hajime Unno b,* a Division of Material Science and Chemical Engineering, Yokohama National University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama , Japan b Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama , Japan Received 10 June 1999; accepted 25 October 1999 Abstract A single continuous- ow uidized-bed bioreactor system consisting of porous carrier particles for retaining microbes was constructed to simultaneously remove carbonaceous and nitrogenous substances in wastewater under different C/N (mass ratio) values. The suspended microbial concentration in the bioreactor was extremely low compared with that of retained microbes. A TOC removal of >91% and a maximum total nitrogen removal of 85% were achieved under a moderate C/N value. By using a set of simpli ed reaction kinetics, the multiple microbial reactions involved in the simultaneous removal process of carbonaceous and nitrogenous components were analyzed. The related kinetic parameters in terms of carbonaceous and nitrogenous loadings were obtained. # 2000 Elsevier Science S.A. All rights reserved. Keywords: Biokinetics; Bioreactions; Nitrogen removal; Fluidized-bed bioreactor; Organic removal; Wastewater treatment 1. Introduction In a conventional biological wastewater treatment process carbonaceous substances are mainly removed by aerobic heterotrophs, while nitrogenous compounds are eliminated by a combined microbiological reaction of aerobic nitri cation and anaerobic denitri cation, together with microbial uptake for their growth. The most popular biological treatment process of carbonaceous and nitrogenous substances in wastewater consists of at least two separate reactors under aerobic and anaerobic conditions, respectively. In addition, since the autotrophic nitrifying bacteria grow very slowly and the nitri cation rate is correspondingly slow, the microbial residence time and hydraulic retention time (HRT) of the process must be long enough to avoid the wash-out of the nitri ers from the system. In order to develop a simple process capable of simultaneous removal of carbonaceous and nitrogenous substances, the authors used a batch uidized-bed reactor wherein porous carrier particles (PCPs) of appropriate sizes were * Corresponding author. Tel.: ; fax: address: hunno@bio.titech.ac.jp (H. Unno). uidized [1±4]. The principle for such a system is to utilize the anaerobic environment concurrently appearing in the internal region of a carrier under aerobically operated condition [5,6]. In order to apply the above principle to a largescale wastewater treatment, the authors in a previous study have examined a continuous uidized-bed bioreactor system consisting of two reactors in series with a recycle- ow [7]. In the present paper, by using the PCPs of 15-mm cubes, it is shown that a single-stage reactor is enough to achieve the simultaneous removal of carbonaceous and nitrogenous components. For the optimization of the process, a simple kinetic analysis for describing the microbial reactions pertaining to the process ef ciency is needed. A set of simpli- ed reaction kinetics proposed in the previous study, which was only examined at one C/N value, is applied for the analysis of the present system, and the process variables are the loadings of carbonaceous and nitrogenous compounds. A bio lm formed on a surface of a substratum, such as a rotating contactor and a uidized-bed bioreactor, has been utilized in wastewater treatment [8±10], and the main advantages are achievement of a high biomass concentration and intensi cation of nitri cation by immobilizing the slowly-growing nitri ers. Even though it is also possible to simultaneously remove the carbonaceous and nitrogenous X/00/$ ± see front matter # 2000 Elsevier Science S.A. All rights reserved. PII: S X(99)00056-X

2 30 X.-H. Xing et al. / Biochemical Engineering Journal 5 (2000) 29±37 Fig. 1. Experimental apparatus of continuous-flow wastewater treatment. components by controlling the bio lm thickness, as far as we know, up to now there is no kinetic analysis available for dealing with the combined bioreactions of organic oxidation, nitri cation and denitri cation. 2. Experimental apparatus and methods 2.1. Experimental apparatus and procedure A reactor system consisting of a single reactor with an effective volume of m 3 (Fig. 1) was used. Arti- cial sewage was fed to the reactor continuously. Porous 15- mm polyurethane carrier cubes (average pore size: 1.47 mm; density: 30 kg m 3 ) were placed with a total volume of m 3 [7]. Air was supplied by a sparger at the bottom of the reactor to maintain the dissolved oxygen (DO) at 5± kg m 3. Arti cial sewage with Table 1 Composition of artificial sewage C/N value (kg m 3 /kg m 3 ) Glucose (10 3 kg-toc m 3 ) TOC loading (kg m 3 per day) Urea (10 3 kg m 3 ) KH 2 PO 4 (10 3 kg m 3 ) 12.6 MgSO 4 (10 3 kg m 3 ) 9.50 CaCl 2 2H 2 O (10 3 kg m 3 ) 1.20 FeCl 3 6H 2 O (10 3 kg m 3 ) 0.10 the composition shown in Table 1 was used. The operating conditions were listed in Table 2. The reactor system was operated by a ll-and-draw mode for 2 weeks to enable the inoculated activated sludge to be completely retained into the porous carrier particles [7], then switched to continuous operation. In all the experiments, hydraulic retention time (HRT) of the sewage was controlled at 8 h. During the continuous cultivation, ph, DO, microbial concentration, total organic carbon (TOC) and nitrogen concentrations of various forms were measured Analytical methods The suspended and retained microbial concentrations were measured by the method described previously [7]. Total organic carbon (TOC) was measured by a TOC analyzer (Shimadzu TOC-5000). Ammonia-N, nitrite-n and nitrate-n concentrations were measured by an ionic chromatograp (Shimadzu SCL-10A, Japan). Urea-N con- Table 2 Operating conditions for the reactor system Reactor volume (V) (m 3 ) Porous carriers loading ratio 12.5% pore size (m) cubic size (10 6 ) 1.5 m 1.5 m 1.5 m Aeration rate (vvm) 0.4 (min 1 ) Feed rate (F) (m 3 h 1 ) Hydraulic retention time 8 (h) Temperature 298 (K)

3 X.-H. Xing et al. / Biochemical Engineering Journal 5 (2000) 29±37 31 centration was determined by measuring its ammonia-n enzymatically converted with urease (Sigma). Concentration of DO and ph were monitored by DO and ph sensors (COS, Japan) installed in the bioreactor. 3. Results and discussion 3.1. Daily variations of microbial reactions in the continuously-fed bioreactor Fig. 2 shows daily changes in retained, suspended and total microbial concentrations in the bioreactor after the beginning of continuous operation. In all of the cases, the retained microbial concentration decreased slightly during the early days, and then reached a steady state. At the steady state, the retained microbial concentration at C/N ˆ 300/40 was 15.0 kg m 3 -particles, and, at the other C/N values, in the range of 12.0 to 13.0 kg m 3 -particles. At this state, the whole PCPs were observed to be occupied by the retained microbes. In all the cases, the suspended microbial concentrations were low compared to the retained one, making up only <9±11% of the total biomass concentration which was the sum of suspended and retained concentrations per reactor volume. Fig. 3 shows daily variations in TOC concentration under the different C/N values. The outlet TOC concentrations were as high as ca kg m 3 at C/N ˆ 300/40, and < kg m 3 at the other C/N values. Fig. 4 shows daily changes in concentrations of urea-n, ammonia-n, nitrite-n, nitrate-n and total-n in the bioreactor. After the start of the continuous operation, the microbial reactions of transforming urea-n to ammonia-n, ammonia- N to nitrite-n and nitrite-n to nitrate-n were detected simultaneously. Ammonia-N production was resulted from the reaction of converting urea-n by urease in the microbes [7,11]. The production of nitrogen oxides was dependent on nitri cation, and the total-n change was dependent on the nitrogen uptake into the biomass and denitri cation inside the carriers. In case of C/N ˆ 100/ 40 and 300/40, the total-n concentrations at the steady states were 25 and kg m 3, respectively, while those at C/N ˆ 100/20, 150/20 and 200/20 were around 8, 4 and kg m 3, respectively. These results indicate that the organic oxidation and nitrogen removal reactions were dependent upon the C/N values. Fig. 2. Daily changes in retained, suspended and total microbial concentrations under different C/N values. (A) and (C), retained microbial concentrations; (B) and (D), suspended and total microbial concentrations.

4 32 X.-H. Xing et al. / Biochemical Engineering Journal 5 (2000) 29±37 Fig. 3. Daily change in effluent TOC under different C/N values. (A), C/N ˆ 100/40, 100/20 and 150/20; (B), C/N ˆ 200/20 and 300/40. These combined microbial reactions were supposed to contribute to the ph stabilization in the bioreactor, because the nitri cation converting ammonia-n to nitrite-n by, for example, nitrosomonas sp. results in decreasing Ph [12], while ammoni cation and denitri cation reducing the nitrite-n and/or nitrate-n produce alkalinity. Actually, the ph was almost around 7.5 at C/N ˆ 100/40 and 300/40, and kept almost constant around 7.0 at the other C/N values. Table 3 summarizes a comparison of the TOC and total nitrogen removal ratio at the steady state calculated from the data shown in Figs. 3 and 4. The maximum total nitrogen removal was 85%, being achieved at C/N ˆ 200/20. At the other C/N values, except C/N ˆ 300/40, the TOC removal ratio was >91%, and the total nitrogen removal increased in the following order: C/N ˆ 100/40, 100/20, 150/20 and 200/ Kinetic analysis of the simultaneous removal reactions of organic and nitrogenous components Fig. 5 shows a typical example of time-course pro le of TOC and nitrogen concentrations of various forms by a Table 3 Summary of TOC and total nitrogen removal ratios under different C/N values C/N value (kg m 3 /kg m 3 ) TOC removal (%) 100/ / / / / Total nitrogen removal (%) Fig. 4. Daily changes in various nitrogen concentrations under different C/N values. (A), C/N ˆ 100/40; (B), C/N ˆ 100/20; (C), C/N ˆ 150/20; and (D), C/N ˆ 300/40; (E), C/N ˆ 200/20. batch-wise operation to which the reactor was switched from the continuous operation at the C/N value of 150/20 after reaching the steady state. Total-N concentration decreased rapidly in response to TOC removal. This gure

5 X.-H. Xing et al. / Biochemical Engineering Journal 5 (2000) 29±37 33 Fig. 5. Time course of concentrations of TOC, ammonia-n, nitrite-n, nitrate-n and total-n in a batch culture at the C/N value of 150/20. supports the coexistence of organic oxidation, nitri cation and denitri cation. The main form of nitrogen oxides was nitrate-n, implying that nitri cation converting ammonia-n to nitrite-n is a limiting step. Thus, the denitri cation can be assumed only from nitrate-n, which is similar to the results reported previously [1,7]. Mass balances for the carbonaceous and nitrogenous components in a steady state can be expressed by Eqs. (1)±(5) under the following assumptions [7]. Assumptions: 1. Microbial reactions are expressed by rst-order reactions with respect to both the substrate and microbial concentrations. 2. Contribution of the suspended microbes to organic oxidation is small compared with that of the retained microbes and is neglected. 3. Heterotrophic bacteria mainly contribute to the microbial growth because of the much smaller growth rate of autotrophic nitrifiers [12]. 4. Ammonia-N is the sole nitrogen source for microbial growth [13]. [Carbon balance] FC s0 FC s VK s C s X avk DNO3 C NO3 X ˆ 0 (1) [Urea-N balance] FC u0 FC u VK u C u X ˆ 0 (2) [Ammonia-N balance] FC N VK u C u X Y cvk s C s X VK N C N X ˆ 0 (3) Y N [Nitrite-N balance] FC NO2 VK N C N X VK NO C NO2 X ˆ 0 (4) [Nitrate-N balance] FC NO3 VK NO C NO2 X VK DNO3 C NO3 X ˆ 0 (5) Since F, V, X, Y c,y N, a,organic and nitrogen concentrations in the above five equations are known, only the five reaction-constants are unknown, which can be obtained from the solutions of these simultaneous equations by utilizing the values of carbon and nitrogen concentrations at the steady state and the other measured values shown in Table 4. Values of Y c and Y N in Table 4 are the same as those used in our previous study [7]. Stoichiometric coefficient, a, is obtained from the following denitrification equation as a ˆ /(24 14). 24NO 3 5C 6H 12 O 6! 30CO 2 18H 2 O 24OH 12N 2 (6) The reaction rate constants obtained from the above equations are summarized in Table 5. In each C/N value, Table 4 Values of the parameters used in the calculation C/N value (kg m 3 /kg m 3 ) 100/40 100/20 150/20 200/20 300/40 C s0 (10 3 kg m 3 ) C u0 (10 3 kg m 3 ) C s (10 3 kg m 3 ) C u (10 3 kg m 3 ) C N (10 3 kg m 3 ) C NO2 (10 3 kg m 3 ) C NO3 (10 3 kg m 3 ) X (kg MLSS m 3 ) F (m 3 h 1 ) V (m 3 ) Y c (kg MLSS kg C 1 ) 1.25 Y N (kg MLSS kg N 1 ) 15.0 a (kg C kg N 1 ) 1.07

6 34 X.-H. Xing et al. / Biochemical Engineering Journal 5 (2000) 29±37 Table 5 Summary of the calculated rate constants C/N value 100/40 100/20 150/20 200/20 300/40 K u (m 3 kg MLSS 1 h 1 ) K NH4 (m 3 kg MLSS 1 h 1 ) K NO2 (m 3 kg MLSS 1 h 1 ) K DNO3 (10 2 m 3 kg MLSS 1 h 1 ) K s (m 3 kg MLSS 1 h 1 ) there was only one set of positive solutions present for the various reaction rate constants. From Eq. (1), the growth rate of retained microbes is given by Eq. (7a). FY c C s0 C s ˆVY c K s C s X VY c ak DNO3 C NO3 X (7a) As indicated by the author's previous study on a model analysis of microbial retainment into PCPs [2], the suspended microbes come from two parts after the retained microbes have occupied the whole PCPs, one being the microbial growths and the another being the detachment of the retained microbes. Taking the above Assumption (2) and the steady states for both the retained and suspended microbes into consideration, the retained microbes growing by utilization of the organic carbon have been detached and turned into the suspended microbes in the bioreactor. A biomass balance in the reactor is thus given by Eq. (7b), resulting in Eq. (7c). FY c C s0 C s FX s ˆ 0 (7b) i. e. X s ˆ Y c C s0 C s (7c) Fig. 6 shows a comparison of the experimental data and result of calculation using Eq. (7c). Taking the fact that, more or less, the suspended microbes would have been Fig. 6. Relationship between X s and (C s0 C s ). The data obtained from the present and previous researches were used. Symbols, experimental data; solid line, calculation by Eq. (7c). related to the organic oxidation into consideration, the calculation by Eq. (7c) agrees well with the experimental data. This result also supports the validity of the above Assumption (2) and Eq. (7). In order to make clear the dependency of various nitrogen conversion rates on TOC loading, the relative nitrogen conversion rates de ned by Eqs. (8)±(11) are evaluated. Relative ammoni cation rate: C u r u ˆ K u X (8) C u0 Relative ammonia oxidation rate: C N r N ˆ K N X (9) C u0 Relative nitrite oxidation rate: r NO2 ˆ K NO2 C NO2 C u0 Relative denitrification rate: r DNO3 ˆ K DNO3 C NO3 C u0 X (10) X (11) Fig. 7 shows effect of TOC loading on the relative nitrogen conversion rates calculated by the above equations. The relative nitrogen conversion rates were independent of the initial urea-n concentration, and were a function of TOC loading. Relative ammonification rate was independent of TOC loading. This was considered reasonable because the ammonification of urea-n was not affected by the organic oxidation as can be seen from Eq. (2). According to Eqs. (2) and (8), the relative ammonification rate was equal to F/V(1 C u /C u0 ), namely dependent on conversion ratio of urea-n. The relative ammonia oxidation and nitrite oxidation rate decreased identically with an increase in TOC loading, which coincided with the result that oxidation of ammonia-n to nitrite was a limiting step for nitrification. Denitrification rate was lower than that of nitrification and showed a decrease with an increase in TOC loading. As the denitrification was supposed to occur in the inner anaerobic region into which the nitrate produced by the nitrification in the outer aerobic layer must be transferred by a diffusion, the observed denitrification rate was considered to be limited by the nitrification rate and lower than the latter. In addition, compared with the relative denitrification rate,

7 X.-H. Xing et al. / Biochemical Engineering Journal 5 (2000) 29±37 35 Fig. 7. Effect of TOC loading on nitrogen conversion rates. the decrease in the relative nitrification rate with an increase in TOC loading was rapid. The reason was unclear, but it was considered to be related to the following events: 1. An increase in TOC loading would have increased the nitrogen uptake for bacterial growth which consequently have affected the ammonia-n available to the nitri cation inside the PCPs; and 2. the increase in TOC loading would also have raised the oxygen consumption by organic oxidation as described latter which would also have limited the nitrification. For evaluating contributions of the organic oxidation or bacterial growth and the denitri cation to TOC and nitrogen removal under the different experimental conditions, the following equations are used. Overall TOC removal rate: R TC ˆ F C s0 C s (12) Overall nitrogen removal rate: R TN ˆ F C TN0 C TN (13) TOC removal rate by bacterial organic oxidation: R GC ˆ Y c K s C s Y c XV (14) Nitrogen uptake rate by bacterial growth: XV R GN ˆ Y c K s C s (15) Y N TOC consumption rate by denitrification: R DC ˆ ak DNO3 C NO3 XV (16) Fig. 8. Effect of TOC loading on TOC removal rates by organic oxidation, denitrification and the total one under different initial urea-n concentrations. Nitrogen removal rate by denitrification: R DN ˆ K DNO3 C NO3 XV (17) Fig. 8 shows the result of TOC removal rates by bacterial organic oxidation and denitrification and the overall TOC removal rate calculated from Eqs. (14), (16) and (12), respectively. TOC removal rate by bacterial organic oxidation approached closely to the overall rate, indicating organic oxidation contributing mainly to the TOC removal. At the TOC loading less than about 0.75 kg m 3 per day, the overall TOC removal rate increased linearly with the increase of TOC loading, approximating to the 100% removal line. Whereas, at the TOC loading higher than about 0.75 kg m 3 per day, the overall TOC removal rate went beyond the line, implying the maximum treatment ability of the present system. Table 6 summarizes a comparison of TOC and nitrogen removal fractions contributed by organic oxidation or biomass growth and denitri cation under different conditions. The TOC required for denitri cation was scarcely noticeable, being only 9% of the overall TOC removal at the TOC loading of 0.3 kg m 3 per day and, nally, decreased to 0.2% at the TOC loading of 0.9 kg m 3 per day. TOC removal by bacterial organic oxidation was raised from 91 to 99.8% of the overall TOC removal with an increase in TOC loading. For the nitrogen removal as shown in Table 6, nitrogen removed by the denitri cation at the TOC loading of 0.3 kg m 3 per day was 52% and 39% of the overall total-n removal at the inlet urea-n concentrations of

8 36 X.-H. Xing et al. / Biochemical Engineering Journal 5 (2000) 29±37 Table 6 Contribution of organic oxidation and denitrification to overall removal of TOC and total nitrogen C/N value (kg m 3 /kg m 3 ) TOC loading (kg m 3 per day) With respect to TOC (%) With respect to total-n (%) R GC /R TC R DC /R TC R GN /R TN R DN /R TN 100/ / / / / and kg m 3, respectively, which declined greatly with an increase in TOC loading. This trend was similar to the other research on immobilized activated sludge [14]. A set of simpli ed reaction kinetics proposed in this study was veri ed experimentally by using the arti cial sewage with different C/N values. The obtained bioreaction rate constants pertaining to the simultaneous removal of organic and nitrogenous substances were dependent on mass transfer of the substrates and the intrinsic reactions occurring inside the PCPs under the different conditions, which will be analyzed latter. This analysis, after being examined by treating a real wastewater under different conditions including C/N value and DO concentration, is expected to be applicable to a process design and optimization. 4. Conclusions A single continuous- ow uidized-bed bioreactor system consisting of PCPs could be operated to remove simultaneously carbonaceous and nitrogenous substances at a hydraulic retention time of 8 h. C/N values markedly affected the TOC and nitrogen removal ef ciency. TOC removal ratio was >91% at all the C/N values, except 300/ 40. The total nitrogen removal ratio reached maximum value of 85% at the C/N value of 200/20. By using a set of simpli ed reaction kinetics, the multiple microbial reactions occurring in the bioreactors were well analyzed. The rate constants of organic oxidation and ammoni cation of urea, nitri cation and denitri cation were closely dependent on the C/N values of the fed sewage. From the bioreaction rate constants, contributions of organic oxidation, microbial growth and denitri cation to the removal of carbonaceous and nitrogenous components were estimated which can be used for the process optimization. 5. Nomenclature C s organic carbon concentration (kg m 3 ) C u urea-n concentration (kg m 3 ) C N ammonia-n concentration (kg m 3 ) C NO2 nitrite-n concentration (kg m 3 ) C NO3 nitrate-n concentration (kg m 3 ) F flow rate of fed artificial sewage (m 3 h 1 ) K s organic oxidation rate constant (m 3 kg MLSS 1 h 1 ) K u ammonification rate constant from urea-n (m 3 kg MLSS 1 h 1 ) K N nitrification rate constant from ammonia-n (m 3 kg MLSS 1 h 1 ) K NO nitrification rate constant from nitrite-n (m 3 kg MLSS 1 h 1 ) K DNO3 denitrification rate constant from nitrate-n (m 3 kg MLSS 1 h 1 ) r u ammonification rate based on relative urea-n concentration (h 1 ) r N ammonia oxidation rate based on relative ammonia-n concentration (h 1 ) nitrite oxidation rate based on relative nitrite-n r NO2 r DNO3 concentration (h 1 ) denitrification rate based on relative nitrite-n concentration (h 1 ) R microbial reaction rate (kg h 1 ) V reactor volume (m 3 ) X retained microbial concentration (kg MLSS m 3 ) X s Y c Y N a 6. Subscripts Suspended microbial concentration (kg MLSS m 3 ) yield coefficient of microbes related to the consumption of organic carbon (kg MLSS kg C 1 ) yield coefficient of microbes related to the consumption of nitrogen (kg MLSS kg N 1 ) stoichiometric coefficient related to glucose-c consumption in denitrification of nitrate-n (kg C kg N 1 ) 0 influent of reactor DC TOC consumed by denitrification DN total-n consumed by denitrification GC TOC consumed by organic oxidation GN total-n consumed by cell growth TC overall TOC removal TN overall total-n removal

9 X.-H. Xing et al. / Biochemical Engineering Journal 5 (2000) 29± Abbreviations Ammonia-N DO Nitrate-N Nitrite-N TOC Urea-N Total-N Acknowledgements ammonia nitrogen dissolved oxygen nitrate nitrogen nitrite nitrogen total organic carbon urea nitrogen total nitrogen The authors are grateful to INOAC Corporation for supplying the polyurethane. This research is supported by a Grant-in-Aid for Scienti c Research from the Japanese Ministry of Education, Culture and Sports (No ). References [1] X.-H. Xing, H. Honda, N. Shiragami, H. Unno, Characteristics of microbial community retained in porous support particles for degradation of organic wastewater (in Japanese), Kagaku Kogaku Ronbunshu 17 (1991) 524 ±530. [2] X.-H. Xing, H. Honda, N. Shiragami, H. Unno, A model analysis of microbial retainment process in porous support particles in a fluidized-bed wastewater treatment reactor, J. Chem. Eng. Jpn. 25 (1992) 89±95. [3] X.-H. Xing, H. Unno, Simultaneous removal system of organic and nitrogenous substances from wastewater by using aerobic denitrification phenomenon (in Japanese), Mizushori Gijutu 34 (1993) 429± 437. [4] X.-H. Xing, T. Inoue, Y. Tanji, H. Unno, Enhanced microbial adaptation to p-nitrophenol by using activated sludge retained in porous carrier particles and simultaneous removal of nitrite released from degradation of p-nitrophenol, J. Biosci. Bioeng. 87(3) (1999) 372±377. [5] D. Agustiyani, X.-H. Xing, N. Shiragami, H. Unno, Distribution of microbial species retained in porous substratum capable of simultaneous removal of organic and nitrogenous substances in wastewater, in: W.K. Teo, M.G.S. Yap, S.K.W. Oh (Eds.); Better Living Through Innovative Biochemical Engineering, Continental Press, Singapore, 1994, pp. 835±837. [6] M. Yanagida, X.-H. Xing, Y. Tanji, H. Unno, Microbial population distribution in porous carrier in fluidized-bed bioreactor for simultaneous removal of carbonaceous and nitrogenous compounds: numerical simulation based on experimental distributions, Proceedings of APBioCHEC'97, 1997, pp. 1085±1088. [7] X.-H. Xing, N. Shiragami, H. Unno, Simultaneous removal of carbonaceous and nitrogenous substances in wastewater by a continuous-flow fluidized-bed bioreactor, J. Chem. Eng. Jpn. 28 (1995) 525±530. [8] S. Masuda, Y. Watanabe, M. Ishiguro, Biofilm properties and simultaneous nitrification and denitrification in aerobic rotating biological contactors, Wat. Sci. Technol. 23 (1991) 1355±1363. [9] P.F. Cooper, The use of biological fluidised beds for the treatment of domestic and industrial wastewaters, The Chemical Engineer, August/September, 1981, 373±376. [10] I. Walker, E.P. Austin, The use of plastic, porous biomass supports in a pseudo-fluidised bed for effluent treatment, in: P.F. Cooper, B. Atkinson (Eds.), Biological Fluidised Bed Treatment of Water and Wastewater, Ellis Horwood, Chichester, [11] C.H. Collins, P.M. Lyne, J.M. Grange, Urease test, in: Microbiological Methods, 6th Edition, Butterworths, London, 1984, 107 pp. [12] M.A. Winkler, Nitrogen and phosphorus removal, in: Biological Treatment of Wastewater, 1st Edition, Ellishorwood, New York, 1981, 226±260 pp. [13] H. Takahashi, H. Saito, Y. Tedsuka, S. Mizushima, H. Yamaguti; Introduction to the Microbial World, 14th Edition, Baifukan, Japan, 1990, pp. 76±234. [14] S. Hashimoto, K. Furukawa, H. Hama, Study on activated sludge immobilization and its function (in Japanese), Gesuido Kyokaishi 23 (1985) 42±50.