The intrinsic kinetics of nitrification in a continuous flow suspended growth reactor

D~lter Re,cue~h Vol. 13, pp. 12'73 to 1279

0OJ.3-1354 79 1201-1."3S02.00 0

C Pergamon Press Lid 1979. Printed in Great Britain

THE INTRINSIC KINETICS OF NITRIFICATION IN A CONTINUOUS FLOW SUSPENDED GROWTH REACTOR WE,~ K. SHtEH Dorr-Oliver. inc. Stamford, CT 06904. U.S.A. and ENRIQUE J. LAMOTTA

Department of Civil Engineering. University of Massachusetts. Amherst, MA 01003, U.S.A.

(Receieed 23 May 1977) Abstract--The intrinsic rate of nitrification was observed in a continuous flow reactor by eliminating external and internal diffusional resistances. The former were minimized by means of intense agitation, and the latter by mechanical rupture of the activated sludge floc using high impeller rotational speeds. The experimental results obtained from the continuous flow experiments confirmed the applicability of Michaelis-Menten kinetics to the activated sludge nitrification process. A possible dependency of k on contact time was found, larger values of k being observed under shorter contact times. The Michaelis constant K, was found practically unaffected by the contact times used in this study.

INTRODUCTION

A large amount of research has been recently done in modeling the activated sludge process. When analyzing the kinetics of the biochemical reactions, most investigators (Andrews, 1969; Lawrence & McCarty, 1970; McKinney, 1962; Reynolds & Yang, 1966; Sherrard & Lawrence, 1973) have implicitly assumed that the system is homogeneous; in other words, interand intra-phase transport of substrate have been neglected. Although these models seem to be able to successfully predict the performance of the process, its intrinsic or true kinetics may not have been revealed by them. The effect of mass transfer resistances has been well documented in both catalytic and enzymatic processes. A reduction of the overall efficiency, and the alteration of the true reaction order have been reported. Considering that the activated sludge process is an autocatalytic system, i.e. it creates its own enzymes, it is evident that neglecting the effect of mass transfer resistances may lead to erroneous conclusions when analyzing kinetic information. The overall objective of the investigation reported herein is to observe the rate of nitrification under such conditions that external and internal diffusional resistances are reduced to negligible values. Since most of the kinetic constants published thus far by EPA (1975) were observed under the assumption that the microorganisms and the liquid form a single phase, it is reasonable to surmise that some of these observations may have been masked by diffusionai effects. Evidence that internal diffusional resistances may affect the observed rate of carbonaceous substrate uptake in the suspended growth system was presented by Baillod & Boyle (1970). However, simi-

lar information dealing with the nitrification process is scarce. Specific objectives of this study include: (a) Determination of the appropriate kinetic expression for the suspended growth nitrification process. (b) Evaluation of the intrinsic value of the kinetic parameters under continuous flow conditions. (c) Evaluation of the effect of biomass contact time on the intrinsic value of the kinetic parameters. MATERIALS AND METHODS

All experiments were performed in a Multigen convertible culture apparatus (Model F-1000, NBS Co., Inc.) with a working volume of 600 ml. Mixing was provided by a turbine propeller driven by a heavyduty variable speed motor. Pure oxygen was supplied throughout the experiments, and the DO concentration was kept above 4.69 x 10 -4 moll-* (15mgl-I). Thus, it is reasonable to assume that oxygen was not limiting the rate of nitrification. The composition of the stock solution for the nutrient medium is given in Table 1. The desired ammonium concentration for any specific run was obtained by suitable dilution of the stock solution with tap water. The alkalinity required for the reaction was provided by adding sodium bicarbonate in amounts sufficient to maintain 4 . 5 9 e q l - t of alkalinity for each moll - t of nitrogen (20rag HCO~/ mg N). The pH of the system was kept at the desired level with phosphate buffer. External diffusional resistances can be eliminated by increasing the rate of external mass transport (e.g.. by introducing vigorous agitation).

1273 w,R. 13 12--K

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vCe~ K Sn~EH and EXRIQLF_J k,Morz~

Table I. Composition of stock solution for synthetic medium Constituent (NH.d2SO.~ KHzPO.~ MgSO,, " 7HzO Fe2(SO,~)3 nH,O Distilled water m

Concentration tmol 1- ~) ling 1- t) 1.786 236 0.588 80 0.081 20 -85 to 1.1.

On the other hand, internal diffusional resistances can be minimized by decreasing floc size, thus yielding an effectiveness factor close to 1.0. In this case there will be no significant concentration differences inside the floc and all microorganisms in the matrix will be able to consume substrate at the same rate. However, the mechanical reduction of the floc particles, which is required to achieve this objective, will significantly affect floc settleability, making it necessary to use an unreasonably large settling unit to allow enough time for particle reflocculation. Thus a conventional chemostat with cell recycle was deemed inappropriate for this study. A reasonable alternative, which allows maintaining a constant biomass in the reactor, is feeding continuously a suspension of microorganisms of known concentration, and simultaneously, another stream containing the medium. In this way, the detention time can be varied at will, regardless of whether the microorganisms can grow under the selected dilution rate. Also important is that a constant physiological

Feed 'lank

state of the biomass is provided by this practice. Sundstrom et aL (19761 have adopted this method in their study of the response of biological reactors t~ sinusoidal variations of substrate concentrations. Following this reasoning, activated sludge from a 28-1. continuous flow conventional unit (seed reactor) was added continuously to the influent of the Multigen unit as a biomass source. This simulated, to a certain extent, sludge recycling from a final clarifier. The ammonium concentration in the mixed liquor of the seed unit was always less than 7.4 x 10 -6 tool N 1- ~, so that this stream did not contribute any sig. nificant amount of substrate to the Multigen unit. A schematic diagram of the continuous flow setup is shown in Fig. I. The operation characteristics of the seed reactor are shown in Table 2. To study the intrinsic kinetics of nitrification, five different liquid detention times were used. Under a fixed flow rate several influent substrate concentrations, ranging from 5.43 x 10 -~" to 6.86 x 10-3 tool • N1-1 (7.6 to 9 6 m g N I -~) were used. The effluent substrate concentration, S~, and the MLVSS concentration, X~ were determined at each influent substrate concentration when steady state was reached. At least three samples were collected and the average values of S~ and Xe were used as the representative values for that specific run. In each case, steady state conditions were reached when S, and X~ attained constant values. The concentration of ammonium in solution was determined by using an Orion specific electrode. The concentration of cells was estimated by determining the volatile suspended solids. A pH of &0 and

Seed m~h)r (CF3TR)[

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Fig. 1. Schematic diagram of experimental setup.

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Continuous flow suspended growth reactor Table 2. The operation characteristics of the seed reactor 28 L I day 20 days 22 + 2 C around 8.0 120.-140 mg I -~ >6mgl -t 1.429 × 10- -' tool 1- t (200mgl -I1 as N <7.143 x 10-6 moll -I (<0.1mgl - t l a s N

Reactor volume: Liquid retention time: Solid retention time: Temperature: pH: MLVSS: Dissolved oxygen concentration in the mixed liquor: Inttuent ammonium concentration: Effluent ammonium concentration:

a temperature of 30~C, which were found Shieh & LaMotta, 1978a) to be the optimum values, were selected for this investigation. The pH of the reactor was continuously monitored and phosphate buffer solution was added if necessary. The dissolved ogygen concentration was checked three times a day to assure it was always kept above 4.69 x 10 -'= tool I- 1 (15 mg ]-i). The impeller rotational speed was selected by measuring the rate of nitrification under different speeds. As demonstrated by Shieh & LaMotta (1978bk who used the same reactor under batch conditions, speeds larger than 500rev rain- 1 allow the observation of the intrinsic rate; a speed of 900rev rain- I was selected for this study to ensure that both external and internal diffusional resistances were minimized.

R E S U L T S AND DISCUSSION

A mass balance of substrat¢ (ammonia) around the Multigen unit with constant volume yields: V dS, = Q'S' + QS~ - (Q + Q')S, - v~X, v dt

(I)

where V = volume of the Multigen unit, ml; Q' = seed flow rate, ml m i n - t ; Q = influent flow rate, ml r a i n - I ; S' --" substrate concentration in seed input flow -,,0 mol I- I; S~ = influent substrate concentration, tool I- 1. S,---effluent (or mixed liquor) substrate concentration of the Multigen. unit, tool I- t ; t = time, rain; c~ = intrinsic rate of substrate removal per unit biomass, tool rag- I day" 1; X, = concentration of cells in the reactor and in the effluent stream mgl "1. Under steady-state conditions dS,/dt = O, and the im trinsic rate is found to be (Q/O. + O.')s, - s , I?i

where

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t - Q + Qm. is the liquid retention time. The applicability of the Michaelis-Menten equation, namely

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(3)

where k = maximum rate of substrate utilization per unit mass of floe, tool rag- t day - t K, = Michaelis constant, tool I- ' c"an now be tested, as described below.

Determination of k and K, According to equation (3), evaluation of k and K, requires observing the intrinsic uptake rate, vl, under several different emuent concentrations, S,. This can be done by two procedures, namely, by keeping a constant influent concentration and varying the detention time, or by keeping a constant detention time and varying the influent substrate concentration. The first approach has been used by several researchers (Gates et al.. 1967; Lawrence & McCarty, 1970; Middlebrooks & Garland, 1968; Reynolds & Yang, 1966; Stensel, 1971) based on the argument that current kinetic models predict that the effluent organic concentration is not influenced by the influent concentration. However, recent studies (Benefield & Randall, 1977; Grady & Williams, 1975; Grau et al., 1975; LaMotta, 1976)based on the second approach, have demonstrated that reactor performance could be significantly affected by the substrate level in the influent stream. Thus it seemed necessary to use both experimental procedures to observe the effect of both parameters (holding time and influent substrate concentration), on the value of the kinetic constants k and K=. Several sets of continuous flow experiments were performed; each set was run under a constant detention time, while the influent ammonium concentration in each run was different. In this way, intrinsic rates were observed under different ammonium concentrations for each set of constant detention time.

1276

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Fig. 2a--e. Intrinsic kinetics of nitrification under different liquid retention times. Figure 2a-e display the results; it can be seen that in each case of constant contact time, the effluent substrate concentration could be varied by c h a n g the influent substrate level. The resulting emu=nt n i t r o g ~ levels could be described reasonably well by the Micbaelis-Menten equation. Evaluation of the kinetic constants was performed graphically using three different plots. The first one is based on the following transformation of the Michaelis-Menten equation:

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Continuous flow suspended growth reactor

1277

Table 3. Values of k and K, obtained from linearization of equation (31 t rain

l0 t k Equation tool rag- t day- t

100 120 150 200 300 ~.?,

104 K, tool I- t

Correlation coefficient

4 5 6 4 5 6 4 5 6 4 5 6 4 5 6

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proximately 7 x 10- 5 mol m g - t day- t under batch conditions (infinite detention time). In order to include all data from Table 3 in the same graph a plot of k vs l/i was prepared (Fig. 3); the line o[ best fit has a correlation coefficient of 0.95 which indicates that there is a statistical relationship between k and i. A physiological explanation for this dependency of k on the ceU-substrate contact time could be advanced as follows. The levels of cell mass, cellular RNA, and enzymes involved in the nitrification protess were originally defined in the seed reactor, in which steady state conditions had been attained under particular operating conditions (namely, continuous flow with cell recycle, a cell retention time of 30 days, and low ambient ammonium concentration). A continuous stream of cells from this reactor was fed to the Multigen unit, where the microorganisms were suddenly exposed to much higher levels of substrate under much shorter contact times. This stress must have provided sufficient stimulus for the cells to establish new levels of RNA and enzymes,

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Table 3 summarizes the values of the kinetic constants obtained from each plot, as well as the correlation coefficient of the respective lines of best fit. Also included are the values of k and K, which were obtained under batch conditions at high initial ammonium concentrations. These experiments are reported elsewhere (Shieh & LaMotta, 1978b). It can be seen that the first plot consistently gave the best correlation coefficient. However, in two cases, the line of best fit had negative intercepts, which in turn, yielded negative values of K,. In these cases, the second best correlation was. selected, i.e. the Lineweaver-Burk plot. Close examination of the values of k and K, which were selected from Table 3 reveals an unusual variability of these constants with detention time. The coefficient k is seen to decrease from about 5 x I 0 - " mol rag- t day- t observed at short detention times to ap-

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Fig. 4. Re.lationship between the kinetic constant K~ and contact time. which in turn resulted in higher uptake rates, as reflected by the increasing values of k. It appears that the cells adjusted their metabolism to the existing environmental conditions; whether they could have continued to increase the maximum uptake rate under contact times less than I00 min cannot be ascertained due to the limited available experimental data. A similar plot was prepared in the case of Ks. Figure 4 shows K, plotted versus lfi, so that all values of K, listed in Table 3 could be included in the same graph. Although the line of best fit (dashed line) would seem to indicate a relationship between K, and t, the correlation coefficient is so low (r = -0.38) that the statistical hypothesis of no correlation between the two variables could not be rejected. Therefore, it can be concluded that in these experiments K, is not statistically dependent on t, as indicated by the solid line in Fig. 4. The analysis presented above might be criticized on the grounds that insufficient data was collected at detention times of 200 and 300min. If Table 3 is reexamined it can be seen that the values of k corresponding to I00, 120 and 150min are very similar, whereas those corresponding to 200 and 300 rain are

significantly different. Thus. it would have been desirable to have additional data to strongly support to the contention that k depends on contact time. This is more clearly seen when all data points {with the exception of those obtained under batch conditions} are plotted in the same graph. Figure 5 shows there is a lack of critical data for 200 min ( n ) and for 300 min (O) at high substrate concentrations. In fact, if the observation at S~ = 2.64 x 10 -J mol N I- ' is removed from the graph, a single curve, indicated by the dashed line, would provide a fair prediction of intrinsic rate of nitrification with k = 5.26 x I O - ' * m o l m g - ' day -t and K, = 2.13 x 10-'*moll -u. However, it must be emphasized that Fig. 5 is applicable only to continuous flow conditions: it does not include the entirely different results obtained with the same system under batch conditions, which are reported elsewhere (Shieh & LaMotta, 1978b). The authors recognize that additional research is needed to strengthen the contention that the maximum uptake rate, k, is dependent on contact time. Nevertheless, Fig. 3 suggests that if it were desired to obtain kinetic information for plant scale up, a bench-scale continuous flow apparatus with a cell

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Continuous flow suspended growth reactor contact time as close as to the plant detention time as possible would be desirable. Otherwise, different kinetic constants might be obtained, and plant performance might differ from that of the laboratory unit. CONCLUSIONS The following conclusions can be drawn from the observations presented in this paper: (1) The Michaelis-Menten equation is an appropriate expression for describin~ the intrinsic kinetics of nitrification in continuous flow reactors. (2) The data presented in Fig. 3 suggest a possible dependency of the maximum uptake rate, k, on the biomass-substrate contact time. This finding must be confirmed with additional experimental data. The Michaelis constant, K~, seems to bear no relationship with contact time.

Acknowledyements--This research was performed with support from the Massachusetts Division of Water Pollution Control, Research and Demonstration Project Number 76-1001. The senior author also received support from the Civil Engineering Department, University of Massachusetts, through a teaching associateship.

REFERENCES

Andrews J. F. (1969) Dynamic model of the digestion process. J. Sanit. Engny Div., ASCE 9(3, 95-116. Baillod C. R. & Boyle W. C. (1970) Mass transfer limitations in substrate removal. J, Sanit. Engng Div., ASCE 96, 525. Benefield L. D. & Randall C. W. (1977) Evaluation of a comprehensive kinetic model for the activated sludge process, d. Wat. Pollut. Control Fed. 49, 1636. Environmental Protection Agency (1975) Process Desiyn

1279

Manual of Nitrogen Control. USEPA Technology Transfer. Gates W. E. et al. (1967) A rational method for the anaerobic contact process. J. War. Pollut. Control Fed. 39, 1951. Grady C. P. & Williams D. R. 11975) Effects of influent substrate concentration on the kinetics of natural microbial populations in continuous culture. Water Res. 9, 171. Grau P. et al. (1975) Kinetics of multicomponent substrate removal by Activated Sludge. Water Res. 9, 637. LaMotta E. J. (1974) Evaluation of diffusional resistances in steostrate utilization by biological films, Ph.D. Dissertation, Univ. of North Carolina at Chapel Hill. LaMotta E. J. (19761 Kintetics of growth and substrate uptake in a biological film system. Appl. Envir. Microbiol. 31,286. Lawrence A. W. & McCarty P. L. (1970) Unified basis for biological treatment design and operation. J. Sanit. Enyny Di~'., ASCE 96, 757. McKinney R. E. (1963) Mathematics of complete mixing activated sludge. J. Sanit. Engn9 Div., ASCE 88, 87. Middlebrooks E. J. & Garland C. F. (1968) Kinetics of model and field extended-aeration wastewater treatment units. J. Wat. Pollut. Control Fed. 40, 586. Reynolds T. D. & Yang J. T. (1966) Model of the completely mixed activated sludge process. Proc. 21st Ind. Waste Conf., Purdue Univ., 696. Sherrard J. H. & Lawrence A, W. 11973) Design and operation model of activated sludge. J. Envir. En9ny Div., ASCE 99, 773. Shieh W. K. & LaMotta E. J. (1978a) Kinetics of simultaneous diffusion and reaction for the nitrification process in suspended growth systems, Univ. of Massachusetts, Env. Eng. Program, Dept. of Civil Engineering, Report No. Env. E. 58-78-1. Shieh W. K. & LaMotta E. J. (1978b) Effect of initial substrate concentration on the intrinsic rate of nitrification in a batch experiment. Biotechnol. Bioengn.q 21,201. Stensel H. D. (1971) biological kinetics of the suspended growth denitrification process, Ph.D. Dissertation, Cornell Univ. Sundstrom D. W. et al. (1976) Response of biological reactors to sinusoidal variations of substrate concentration. Biotechnol. Bioengng 18, I.