APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1983, p.

1453-1458

Vol. 45, No. 5

0099-2240/83/051453-06$02.00/0 Copyright C 1983, American Society for Microbiology

Nonlinear Estimation of Monod Growth Kinetic Parameters from a Single Substrate Depletion Curvet
JOSEPH A. ROBINSONt AND JAMES M. TIEDJE* Department of Microbiology and Public Health, Michigan State University, East Lansing, Michigan 48824

Received 27 September 1982/Accepted 25 February 1983

Monod growth kinetic parameters were estimated by fitting sigmoidal substrate depletion data to the integrated Monod equation, using nonlinear least-squares analysis. When the initial substrate concentration was in the mixed-order region, nonlinear estimation of simulated data sets containing known measurement errors provided accurate estimates of the Px. Ks, and Y values used to create these data. Nonlinear regression analysis of sigmoidal substrate depletion data was also evaluated for H2-limited batch growth of Desulfovibrio sp. strain Gil. The integrated Monod equation can be more convenient for the estimation of growth kinetic parameters, particularly for gaseous substrates, but it must be recognized that the estimates of Rma,x K5, and Y obtained may be influenced by the growth rate history of the inoculum.

Both derivative and integrated forms of equations derived for enzyme-catalyzed reactions have been used to estimate kinetic parameters for microbially mediated processes. In particular, Vma and Km estimates have been calculated by fitting data to either integrated (2, 5, 6, 8, 14, 15) or derivative (3, 14, 15) forms of the Michaelis-Menten equation. However, Michaelis-Menten kinetics only describe bacterial substrate consumption either (i) when this process is unlinked to growth, such as under resting conditions, or (ii) when the amount of growth occurring is less than that which gives sigmoidal substrate depletion. Vm., is no longer a parameter (i.e., a constant) if its value changes during substrate consumption. When substrate consumption is linked to growth, the number of catalytic units, or activity, increases with time. Assuming that the initial substrate concentration is greater than that which gives one-half of the maximum growth rate, an increase in activity concomitant with substrate consumption yields an S-shaped substrate depletion curve, or sigmoidal kinetics (12). As mentioned above, sigmoidal kinetics is inconsistent with the Michaelis-Menten model, but is predicted by Monod kinetics. In this paper, we demonstrate the fitting of substrate depletion data (i.e., progress curve data) to the integrated Monod equation by using nonlinear regression, which is advantageous
t Journal article
tana State

since estimates of growth kinetic parameters may be calculated from a single progress curve. We show how initial estimates of Uma, K, and Y, which are required for nonlinear leastsquares analysis, can be calculated by using linearized, discretized forms of the Monod equations describing the rates of change of biomass and growth-limiting substrate concentrations during batch growth. Additionally, we discuss the use of sensitivity coefficients for the optimal estimation of growth kinetic parameters and the advantages of using the integrated Monod equation for estimation of Lmax, K, and Y. Finally, we give attention to the limitations of fitting transient-growth data to the Monod model, which describes balanced bacterial growth only

(13).
THEORY AND METHODS The rate of change of substrate consumption by a bacterium growing in batch may be described by dS/dt = -[ m,,.,,S/(K5 + S)]X/Y (1) where ,u, is the maximum specific growth rate, K5 is the half-saturation constant for growth, and Y is the yield coefficient. The variable X is the biomass concentration and may be eliminated from equation 1 by using X = Y(SO - S) + XO (2) which relates X at time t to S. After elimination of X, equation 1 becomes dS/dt = -[pL,,S/(K, + S)][Y(So- S) + Xo)]/Y (3) which may be integrated to give

Experiment Station.

Michigan Agricultural t Present address: Department of Civil Engineering, Monno.

10579 of the

University, Bozeman, MT 59717.

Cjln{[Y(SO - S) + XO]/XO} - C21n(S/So} = Lmaxt (4)

1453

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ROBINSON AND TIEDJE

APPL. ENVIRON. MICROBIOL.

nately, solution curves for both S and X may be generated by solving equation 1 simultaneously with dX/dt = [~z,u.S/(K, + S)]X (5) again by numerical integration (4). Nonlinear regression analysis requires that the sensitivity of the dependent variable to changes in each of the parameters be calculable. The first derivatives of S with respect to p. ,s K5, and Y satisfy this requirement (although these may be numerically approximated), and these expressions can be derived from the integrated Monod equation by using implicit differentiation (16). The sensitivity equations for tmax, KS, and Y are given below. (6) dS/dR,max = t/C4 = dS/dK, {(Y/C3}Rln(X/XO} - ln(S/So}]}/C4 (7) dS/dY = {C1(S0 - S)/X + ln(X/XO}/C3[K, + (1 -

where C1 = (KSY + SOY + XO)/(YSO + XO) and C2 = K5Y/(YSo + XO). Equation 4 gives the familiar Sshaped curve for substrate depletion during batch growth. A relation similar to equation 4 describing the increase of biomass during batch growth has been derived by integrating the expression obtained after eliminating S from equation 1 by using the mass balance relation (equation 2) (11, 12). Equation 4 cannot be explicitly solved for S, and hence its solution must be numerically approximated. Solution curves (S versus t) may be estimated by solving equation 3 by numerical integration, or, alter-

parameters having nearly proportional sensitivities 1C). The value of So for the optimal estimation of Lmax, KS, and Y is in the mixed-order region (Fig. 1B). A number of techniques may be used to fit data to nonlinear models, but we chose the Gaussian method because of its relative simplicity (1). The Gaussian method uses the equation
over most of the progress curve (Fig.

C1)SO] - ln(S/SO)/C3(K, - C2SO)}/C4

(8)

In the above three equations the terms C3 and C4 equal YSO + XO and C3Y/X + C4/S, respectively. Note that equations 6, 7, and 8 are all functions of KS and Y. By definition, then, the integrated Monod equation is nonlinear, since its sensitivity equations are not independent of the parameters (1). In addition to being required for nonlinear regression, the sensitivity equations predict (i) whether the parameters may be uniquely estimated, (ii) the relative precision of the estimated parameters, and (iii) the range of the independent variable over which the model is most sensitive to changes in the parameters. The last item is useful in designing optimal experiments for parameter estimation (1). If the sensitivity equations are proportional (i.e., if they are multiples of one another), then it is impossible to obtain unique estimates of the parameters from the data by leastsquares analysis (1). Unique parameter estimates may be obtained when the sensitivity equations are very similar, but they are highly correlated. This situation is undesirable since it implies that several combinations of parameter estimates may describe the same data set, and this is true for equation 4, depending on SO. The sensitivity equations for Imax, KS, and Y yield similar curves (Fig. 1), with the sensitivity equations for m.Lmax and Y numerically dominating the one for KS. In the first-order region it is not possible to obtain unique estimates of m KS, and Y because the sensitivity coefficients for Lmax and Y are proportional (Fig. 1A). This is intuitively clear since an increase in IL,,I is equivalent to a decrease in Y in the first-order region. The sensitivity equations also predict that relatively poor estimates of .,max, KS, and Y will be obtained when So is saturating. This results from the

S - ST = AlXraxdS/dp.max + AK5dS/dK, + AYdS/dY (9) where ST iS the theoretically predicted substrate concentration at time t given current parameter estimates, and AILmax, AK,, and AY are correction terms for these parameters. The application of equation 9 proceeds by evaluating the sensitivity equations 6, 7, and 8 and calculating the residual errors (ST - S) at the measured substrate concentrations for the initial parameter estimates. The correction terms are then calculated via multiple linear regression and added (they can be positive or negative) to the initial parameter estimates. These corrected parameter estimates then serve as the initial estimates for the next iteration, and this process continues until the correction terms are less than some small value (e.g., 0.01). Once the solution has converged, estimates of the standard errors of ILmax, KS, and Y may be calculated from the variances of their respective correction terms (8). Nonlinear regression analysis for any model that is nonlinear in its parameters requires initial estimates or "'guesses" of the parameters (1, 7). This is usually done by fitting transformed data to a linear version of the model, but the integrated Monod equation is intrinsically nonlinear (7) and cannot be transformed into a linear expression for the purpose of estimating P'max, KS, and Y. However, provisional estimates of these parameters may be obtained by using

-AS/At = [(LmaxS/(Ks + S)]X/Y AX/At = [jmaxS/(Ks + S)]X

(10) (11)

Equations 10 and 11 are derived by replacing infinitesimal time (dt) with finite time (At) and are approximately correct if At is relatively small (16). These finitedifference equations are nonlinear, but may be converted into the following linearized forms:
-AtX/AS
and
=

(KsY4lumax)(1/S)

Y/Imax

(12)

AtX/AX = (Ks4Imax)(1/S) + l4lmax (13) which yield straight lines when -AtX/AS and AtX/AX are plotted against 1/S.

RESULTS AND DISCUSSION To evaluate nonlinear regression analysis for equation 4, we fitted simulated data containing known errors to this equation, using the Gaussian method. Theoretically, estimates of Imax, K, and Y should be close to those parameter values used to generate the simulated data. By analyzing simulated data it is possible to check whether the sensitivity coefficients have been

VOL. 45, 1983

A

NONLINEAR REGRESSION ANALYSIS OF MONOD DATA

1455

la 'a

B
0 a
0

-X

'0

10-

C
0

0
o

'0 0

between these values, and provisional pm. and Ks estimates can be calculated by using a linearized, discretized version of equation 3.) To fit data to equations 12 and 13, estimates of -dS/dt and dX/dt are required. These were obtained by fitting the S-t and X-t data pairs to cubic splines (4) and then evaluating the first derivatives of the fitted cubic polynomials. Two examples of fitting transformed data having either relative or simple measurement errors are depicted in Fig. 2. For both cases (A and B), Ks and pmae were calculated from the y intercepts and slopes of the straight lines fitted to the transformed biomass data. The two Y estimates were calculated by dividing the y intercepts of the best-fit lines for transformed substrate data by the y intercepts of the linear equations fitted to the transformed biomass data. The initial estimates of ium., K5, and Y were entered into a computer program (MONODCRV) that fits S data to equation 4 by the Gaussian method. In addition to estimating the growth kinetic parameters, MONODCRV approximates (i) the standard errors of FLm., K5, and Y, assuming no correlation among measurement errors, and (ii) the absolute residual sumof-squares at each iteration. The pattern of paA
Time

An
0

400

FIG. 1. Sensitivity coefficients for Prax (dS/diL,,), Ks (dS/dK5), and Y (dS/dY). For all three panels, A,,, = 0.1, Ks = 5, Y = 0.2, and XO = 1. SO = 0.1, 20, and 250 for panels A, B, and C, respectively.

so6
0

300
x

la

40
20

200x

SD-=0.0 1

100

correctly derived, since incorrect sensitivity expressions can (i) prevent fitting data to a given 2 a o 4 6 model altogether or (ii) lead to dramatic errors in 1/8 ist~~~~~~~~~~~~~~~~~~~~~ the estimated parameters. Twelve simulated data sets were created by numerically integrat0 ing equation 2 for the following parameter values B 12 Uvv and initial conditions: m = 0.1, Ks = 5, Y = 40 0.2, So = 20, and Xo = 1. Measurement errors of x 9I the relative type (constant coefficient of variaGo 30X tion) were added to six of the error-free data x x sets, using a pseudo-random number generator 0 a 20 0 according to the procedure described by Harcv- 0.005 3 baugh and Bonham-Carter (10). Similarly, sim10 ple errors (constant standard deviation) were n introduced into the remaining six data sets. 0.6 0 0.2 0.8 0.4 Initial , K5, and Y estimates were ob118 tained from linear regression analysis of the FIG. 2. Linearized, discretized Monod data. Simusimulated S-t and X-t data pairs transformed lated data containing errors having a constant coeffiaccording to equations 12 and 13. (Actually, cient of variation (CV) of 0.5% (A) and a constant only initial and final estimates of the biomass standard deviation (SD) of 0.01 (B) were transformed concentration are required; a provisional esti- according to equations 12 and 13. The values of lmax,
0
I
10

mate of Y can be obtained from the difference

K5, and Y are the

same as those used in

Fig. 1.

1456

ROBINSON AND TIEDJE

APPL. ENVIRON. MICROBIOL.

rameter updating for data fitted to equation 4 is

illustrated in Table 1. In both cases, five iterations were required before the correction terms were less than 0.01 and the residual sum-ofsquares was minimized. The updating of initial parameter estimates was usually complete after four or five iterations when a convergence criterion of 0.01 was used. Nonlinear regression analysis of the simulated Monod data generally provided better estimates of FLmax, Ks, and Y than did least-squares analysis of the linearized data (Table 2, Fig. 3). This was always true for Ks, but in a few cases the final Rmax (simulations 4 and 9) and Y (simulation 5) estimates were slightly more in error than the initial estimates of these two parameters. Nonlinear regression analysis always increased the overall goodness-of-fit by reducing the residual sum-of-squares, in some cases by as much as 104-fold (simulation 9). In addition to the results presented above, we have found that fitting data to equation 4 by using nonlinear regression analysis is far superior to linear analysis of transformed data for several combinations of parameter values and error levels. However, when the relative error level is greater than or equal to 5%, then estimates of Ks, and Y may be in substantial error (e.g., 500%), although the residual sum-of-squares has been
iJmax,

minimized.

To demonstrate the fitting of biological data to equation 4 by using nonlinear regression, we obtained H2 depletion and biomass formation data for the sulfate reducer Desulfovibrio sp. strain Gl1 growing on H2 as the sole electron donor. The culture conditions and experimental details will be described elsewhere (Robinson

and Tiedje, manuscript in preparation). The growth of this organism was monitored by following the protein content. Initial estimates of the growth kinetic parameters were calculated according to the abovedescribed procedure, and these were updated by MONODCRV. The goodness-of-fit for the H2 concentration data versus the theoretical curve calculated from the best parameter estimates is shown in Fig. 4; for these data, estimates of lLmax, Ks, and Y, respectively, were 5.6 x 10-2 h-1, 2.4 jxM dissolved H2, and 0.99 g of protein per mol of H2. Others have determined bacterial growth parameters from batch data, but generally they have used computationally inefficient procedures or paid little attention to the limitations of estimating these parameters under transient or nonsteady-state conditions. Graham and Canale (9), in a recent investigation on the kinetics of a four-trophic level predator-prey system, fitted batch data to Monod growth models without considering the influence that the growth rate histories of their predator and prey cultures might have had on the optimal parameter estimates. Before this, investigators typically fitted biomass formation data to the integrated version of the Monod equation describing biomass production, using a systematic procedure (11) which involves changing Ptmax, Ks, and Y until the theoretical biomass formation curve agrees with the experimental points. Systematic (or random) search techniques are inefficient and should be avoided if possible (1). The larger danger, however, is that since sensitivity coefficients are not required for systematic fitting procedures, the researcher may believe that it is

TABLE 1. Course of parameter updating during nonlinear regression analysis of simulated Monod dataa
Estimate b Relative error' _____________________ IJLmax
K5

____________________
ILmax Ks
Y

Simple error'

RSS'

RSS

Initialf
Iteration 1 Iteration 2 Iteration 3 Iteration 4 Final (iteration 5)

0.14 0.12 0.10 0.10 0.10 0.10

10.5 7.26 4.58 5.02 4.95 4.95

0.31 0.21 0.20 0.21 0.21 0.21

80.9 8.9 1.1 0.09 0.08 0.08

0.11 0.12 0.10 0.10 0.10 0.10

5.99 7.40 4.49 5.00 5.04 5.04

0.18 0.21 0.20 0.20 0.20 0.20

46.70 0.83 0.16 0.004 0.003 0.003

True value 0.10 5.00 0.20 0.10 5.00 0.20 a Untransformed data were directly fitted to equation 4 by using nonlinear regression analysis, given the initial parameter estimates. b Generally, four or five passes were required before the solution converged. For these simulated data, a convergence criterion of 0.01 was used. c The relative errors have a constant coefficient of variation (0.5%). d The simple errors have a constant standard deviation (0.01). e RSS, Residual sum-of-squares. f Initial estimates of fLmax, Ks, and Y were calculated by fitting transformed S-t and X-t data pairs to equations 12 and 13.

VOL. 45, 1983

NONLINEAR REGRESSION ANALYSIS OF MONOD DATA

1457

TABLE 2. Comparison of errors in parameter estimates and residual sums-of-squares for linear versus nonlinear regression analysis of simulated Monod dataa % Error in: Final estimates Initial estimates Simulation RSSj/RSSf'
Y K, 71.8 3.5 1.2 2 14 29.8 12.2 1 337 9.5 12.8 9 37 80.4 29 2 974 2.5 1 1 56.5 110 41 3 144 4 2.4 3 8.5 10.4 1.2 4 4.01 6.5 5.2 5 5.5 10.8 7.1 5 69.5 2.5 0.4 1 20 52.4 22.5 6 15.8 1 1.2 1 5.5 7 12.8 7 1,150 0 0.2 0 22 26.6 16 8 22,220 1 1.6 1 23.5 5.6 0 9 3,430 0 1 0.5 10 33 21 10 46.1 1.5 2.6 2 2 7.2 5.2 11 75.4 0 0 0 5 4.2 4.8 12 a True parameter values were identical for all 12 simulations: eLmax = 0.1, K, = 5, and Y = 0.2. b Simulations 1 through 6 contain errors having a constant coefficient of variation (0.5%) (relative errors); simulations 7 through 12 contain errors with a constant standard deviation (0.01) (simple errors). I Ratio of residual sum-of-squares for initial parameter estimates to residual sum-of-squares for final parameter estimates.

i-Mx

Y > K,nK

possible to uniquely estimate parameters for a given nonlinear model when the sensitivity coefficients predict otherwise. The integrated Monod equation is advantageous for the estimation of Umax, K,, and Y
A
c

0
-

O-

e
C

dU
e

0 0

US1

(particularly for gaseous substrates) but suffers from some limitations. It is advantageous since estimates of growth kinetic parameters may be obtained from a single substrate depletion curve. Further, estimating growth kinetic parameters by using the integrated Monod equation offers an alternative to estimating these parameters for gaseous substrates by using chemostat cultures in which mass transport limitations may yield unrealistically high K, estimates (14). The use of the integrated Monod equation is limited because the derivative form, from which the integrated version is derived, describes balanced (steady-state) bacterial growth and may fail to describe transient (batch) bacterial growth (13). Powell has shown that apparent Monod parame-

0o:
Time

B
c

20C
0
a
4-

I
Final estimates

cO

16

C0 0 12 e C
0
U

CV= 0.005 12~~~

cvo.o

co 0 am
0
._

e
0a
a,.

8
4 .

Initial estimates
5
.

',

16

O1

10

-m 20 3 2S5 15 Time
*i
curves

Hour

24

32

40

FIG.

3.

Comparison of theoretical

calculat-

K,, and Y ed by using initial versus final (best) , estimates with simulated data points. The data are those that were transformed and plotted in Fig. 2.

FIG. 4. Comparison of theoretical curve with measured H2 concentrations (O) for growth of Desulfovibrio sp. strain Gll on H2 as the sole electron donor. The initial substrate concentration (SO) was estimated by extrapolating back to t = 0 on the H2 concentration axis.

1458

ROBINSON AND TIEDJE

APPL. ENVIRON. MICROBIOL.

5. Cornish-Bowden, A. 1979. Fundamentals of enzyme kinetics, p. 200-210. Butterworth, Inc., Boston, Mass. 6. Counotte, G. H. M., and R. A. Prins. 1979. Calculation of Km and V,,,0, from substrate concentration versus time plot. Appl. Environ. Microbiol. 38:758-760. 7. Draper, N. R., and H. Smith. 1981. Applied regression analysis, p. 459. John Wiley & Sons, Inc., New York. 8. Duggleby, R. G., and J. F. Morrison. 1977. The analysis of progress curves for enzyme-catalyzed reactions by nonlinear regression. Biochim. Biophys. Acta 481:297312. 9. Graham, J. M., and R. P. Canale. 1982. Experimental and modeling studies of a four-trophic level predator-prey system. Microb. Ecol. 8:217-232. 10. Harbaugh, J., and G. Bonham-Carter. 1970. Computer simulation in geology, p. 61-97. John Wiley & Sons, Inc., New York. 11. Knowles, G., A. L. Downing, and M. J. Barre"t. 1965. Determination of kinetic constants for nitrifying bacteria in mixed culture, with the aid of an electronic computer. J. Gen. Microbiol. 38:263-278. 12. Pirt, S. J. 1975. Principles of microbe and cell cultivation, p. 22-28. John Wiley & Sons, Inc., New York. 13. Powell, E. 0. 1967. The growth rate of microorganisms as a function of substrate concentration, p. 34-56. In E. 0. Powell, C. G. T. Evans, R. E. Strange, and D. W. Tempest (ed.), Microbial physiology and continuous culture. Her Majesty's Stationery Office, London, United Kingdom. 14. Robinson, J. A., and J. M. Tiedje. 1982. Kinetics of hydrogen consumption by rumen fluid, anaerobic digestor sludge, and sediment. Appl. Environ. Microbiol. 44:13741384. 15. Strayer, R. F., and J. M. TiedUe. 1978. Kinetic parameters of the conversion of methane precursors to methane in a hypereutrophic lake sediment. Appl. Environ. Microbiol. 36:330-340. 16. Thomas, G. B., Jr. 1972. Calculus and analytical geometry. p. 76-81. Addison-Wesley, Inc., Reading, Mass.

ters determined by using batch growth data may depend on the growth rate history of the orga-

nism (13). Lastly, maintenance requirements, which we neglected for strain Gil, may produce erroneous parameter estimates (particularly of Ks) if ignored. In summary, the integrated Monod equation offers an alternative to chemostat studies for estimating growth kinetic parameters, but it should be used only after its limitations have been considered.
ACKNOWLEDGMENTS We thank Dave Myrold for use of a computer program that fits cubic splines to data and Dan Shelton for many helpful discussions on bacterial growth kinetics. We gratefully acknowledge the expert technical assistance of Walter Smolenski and thank James Beck for a critical review of the manuscript. A copy of MONODCRV is available from us
upon

request.

This work was supported by National Science Foundation grants DEB 78-05321 and DEB 81-09994.
LITERATURE CITED 1. Beck, J. V., and K. J. Arnold. 1976. Parameter estimation in engineering and science, p. 334-350. John Wiley & Sons, Inc., New York. 2. Betlach, M. R., and J. M. Tiedje. 1981. Kinetic explanation for accumulation of nitrite, nitric oxide, and nitrous oxide during bacterial denitrification. Appl. Environ. Microbiol. 42:1074-1084. 3. Betlach, M. R., J. M. Tiedje, and R. B. Firestone. 1981. Assimilatory nitrate uptake in Pseudomonas fluorescens studied using nitrogen-13. Arch. Microbiol. 129:135-140. 4. Burden, R. L., J. D. Faires, and A. C. Reynolds. 1978. Numerical analysis, p. 116-128 and 239-245. Prindle, Weber and Schmidt, Boston, Mass.