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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 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

measurement errors

that the estimates of

rate history of the inoculum.

Rma,x

K5, and Y obtained may be influenced by the growth

Both derivative and integrated forms of equa- tions derived for enzyme-catalyzed reactions

have been used to estimate kinetic parameters for microbially mediated processes. In particu- lar, 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 Michae- lis-Menten equation. However, Michaelis-Men- ten kinetics only describe bacterial substrate

consumption either (i) when this process is un-

linked to growth, such as under resting condi- tions, or (ii) when the amount of growth occur- ring is less than that which gives sigmoidal

substrate depletion. Vm., is no longer a parame-

if its value changes during

ter (i.e., a constant) substrate consumption.

When substrate consumption is linked to

growth, the number of catalytic

units, or activi-

ty, increases with time.

Assuming thatthe initial

substrate concentration is greater than that

which gives one-half of the maximum growth

increase in activity concomitant with

rate, an

substrate consumption yields

an S-shaped sub-

strate depletion curve, or sigmoidal kinetics

(12). As mentioned above,

inconsistent with the Michaelis-Menten model,

sigmoidal kinetics is

but is predicted by

In this paper,

Monod kinetics. 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

Experiment Station.

no. 10579 of the Michigan Agricultural

t Present address: Department of Civil Engineering, Mon-

tana State University, Bozeman, MT 59717.

since estimates of growth kinetic parameters may be calculated from a single progress curve.

K, and

least-

squares analysis, can be calculated by using

linearized, discretized forms of the Monod equa- tions describing the rates of change of biomass

substrate concentrations

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 equa- tion for estimation of Lmax, K, and Y. Finally, we give attention to the limitations of fitting

during

and growth-limiting

Y, which are required for nonlinear

We show how initial estimates of Uma,

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

growth, and Y is the

yield

centration 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,

coefficient. The variable X is the biomass con-

the half-saturation constant for

equation 1 becomes

dS/dt = -[pL,,S/(K, + S)][Y(So - S) + Xo)]/Y (3)

which may be integrated to give

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

1453

1454 ROBINSON AND TIEDJE

where C1 = (KSY + SOY + XO)/(YSO +

K5Y/(YSo + XO). Equation 4 gives the familiar S-

and C2 =

XO)

shaped curve for substrate

growth. A relation similar to

increase of biomass

derived

depletion

equation

batch

during batch

describing the

4

has been

obtained after using the mass

solved for S, and

growth

approximated.

may

during

by integrating the expression

S from

equation 1 by

eliminating

balance relation (equation

Equation 4 cannot be hence its solution must be

2) (11, 12). explicitly

numerically

may

Solution curves (S versus t)

solving equation 3 by numerical

be estimated by

or, alter-

be

integration,

and X

nately, solution curves for both

S

generated by solving equation 1 simultaneously with

dX/dt = [~z,u.S/(K, + S)]X

(5)

again by numerical

integration (4).

analysis

variable to

be

Nonlinear regression sitivity of the dependent

ment (although these

Y

may

requires that the sen-

changes

in each of

derivatives of S

this require-

approximat-

derived from the

the parameters be calculable. The first

with respect to p. ,s K5, and Y

satisfy

numerically

ed), and these

integrated Monod

ation (16). The

expressions can be

equation

by

sensitivity equations

using implicit differenti-

for tmax, KS, and

are given below.

dS/dR,max = t/C4

(6)

dS/dK, = {(Y/C3}Rln(X/XO} - ln(S/So}]}/C4 (7)

dS/dY =

{C1(S0 - S)/X +

C1)SO]

ln(X/XO}/C3[K,

C2SO)}/C4

- ln(S/SO)/C3(K,

-

+

(1

-

(8)

In

the above three

equations the terms C3 and C4 equal

C4/S,

respectively. Note that

By

is

of KS and Y.

equation

YSO + XO and C3Y/X +

equations 6, 7, and 8 are all functions

definition, then, the

nonlinear,

integrated Monod

since its sensitivity

equations are not inde-

for nonlinear

pendent of the In addition to

parameters (1).

being required

regres-

sion, the sensitivity equations predict (i) whether the

parameters may be

uniquely estimated, (ii) the relative

parameters, and (iii) the

independent variable over which the

precision of the estimated

range of the

model is most sensitive to

The last item is useful in

ments for

changes

in the

parameters.

designing optimal experi-

parameter estimation (1). If the sensitivity

multiples of

then it is impossible to obtain unique

equations are proportional (i.e., if they are

one another),

data

similar, but they are highly

of parameter estimates

set, and this is true for

(Fig.

estimates of the parameters from the

least-

squares analysis (1). Unique parameter estimates may

by

be obtained when the sensitivity equations are very

correlated. This situation is

undesirable

since it implies that several combinations

may describe the same data

equation 4, depending on SO.

theone forKS.

The sensitivity equations for Imax, KS, and Y yield

sensitivity equations

similar curves

for m.Lmax and Y numericallydominating

1), with the

In the first-order region it is not possible to obtain

unique estimates of m

sensitivity coefficients for Lmaxand Y areproportional

(Fig. 1A). This is intuitively clear since an increase in

KS, and Y because the

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

APPL. ENVIRON. MICROBIOL.

parameters

over most of

So for the optimal

having nearly proportional sensitivities

the

progress curve

(Fig.

1C). The value of

estimation of

region

(Fig. 1B).

may

techniques

but

we

Lmax, KS, and Y is in

be used to fit data to

chose the Gaussian method

simplicity (1). The Gaussian

the mixed-order

A number of nonlinear models,

because of its relative

method uses the equation

S - ST = AlXraxdS/dp.max + AK5dS/dK, + AYdS/dY

(9)

where ST iS the centration at time

t

and AILmax, AK,,and

parameters.

The

the

the

evaluating

calculating

positive

theoretically

predicted substrate con-

parameter estimates,

proceeds

by

givencurrent

AY are

application

sensitivity

residual

correction terms for these

of

6, 7, and 8 and

equation 9

equations

errors

(ST - S) at the mea-

parame-

parameter estimates.

some

ILmax,

KS,

sured

terestimates. The correction terms are then calculated

and added (they can be

via

then serve as the and this

process

These

initialestimates forthe next

continues untilthe correction terms are less than

small value

0.01). Once the solution has con-

and Y

may be calculated from the variances of their

substrate concentrations for the initial

multiple linear

or

negative)

regression

to the initial

parameter

corrected

(e.g.,

estimates

iteration,

verged, estimates

respective

of the standard errors of

(8).

correction terms

Nonlinear nonlinear in its

regression analysis for any model that is

parameters requires initial estimates or

"'guesses"

done

the

of

the parameters (1, 7). This is

usually

by fitting

model,

transformed data to a linear version

but the integrated Monod equation is

(7) and cannot be transformed

of

intrinsically nonlinear

into a linear expression for the purpose of estimating

P'max,

KS, and Y.

However,

be

provisional

estimates of

these parameters may

obtained by

using

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

(10)

(11)

Equations 10 and 11 are derived by replacing infinitesi-

mal

time(dt)withfinitetime (At)and

ly

correct if At is

are approximate-

relatively small (16). These finite-

difference equations are nonlinear, but may be

converted into the following linearized forms:

-AtX/AS = (KsY4lumax)(1/S) + Y/Imax (12)

and

(13)

which yield straight lines when -AtX/AS and AtX/AX

are plotted against 1/S.

AtX/AX = (Ks4Imax)(1/S) + l4lmax

RESULTS AND DISCUSSION

To evaluate nonlinear

regression analysis for

data containing

equation 4, we fitted simulated

known errors to this

ian method. Theoretically, estimates of Imax,

equation, using the Gauss-

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

B

C

0

la 0

'a

0

a

0

-X

'0

10-

0

0

o 0 '0

NONLINEAR REGRESSION ANALYSIS OF MONOD DATA

1455

between these values, and provisional

linear-

ized, 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

fitting the S-t and X-t data pairs to cubic splines

by

(4) and then evaluating the first derivatives of

examples of

fitting transformed data having either relative or

simple measurement errors are

the fitted cubic polynomials. Two

Ks estimates can be calculated by using a

pm. and

depicted in Fig.

pmae were

of the

2. For both cases (A and B), Ks and

calculated from the y intercepts and slopes

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 (MON-

ODCRV) that fits S

Gaussian method. In addition to estimating the

data to equation 4 by the

MONODCRV ap-

growth kinetic parameters,

proximates (i) the standard errors of FLm., K5,

and Y, assuming no correlation among measure- ment errors, and (ii) the absolute residual sum- of-squares at each iteration. The pattern of pa-

Time

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.

correctly derived, since incorrect

pressions can (i) prevent fitting data to a given model altogether or (ii) lead to dramatic errors in the estimated parameters. Twelve simulated

sensitivity ex-

data sets were created by

numerically integrat-

ingequation 2forthefollowingparameter values

and initial conditions: m

0.2, So = 20, and Xo = 1. Measurement errors of the relative type (constant coefficient of varia-

tion) were added to six of the error-free data

sets, using a pseudo-random number

according to the procedure described by Har- baugh and Bonham-Carter (10). Similarly, sim-

ple errors (constant standard deviation) were introduced into the remaining six data sets.

generator

= 0.1, Ks = 5, Y =

Initial

,

K5, and Y estimates were ob-

tained from linear regression analysis of the

simulated S-t and X-t data pairs transformed

according to equations 12 and 13. (Actually,

only initial and final estimates of the biomass

concentration are required; a provisional esti-

mate of Y can be obtained from the difference

A

An

so6

0

la 40

20

0

 

o

2

 

I

B

12

9I

Go

10

x

 

0

 

3

0

 

0

0.2

 

400

 

0

 

300

 

x

 

200x

SD-=0.01

100

4

6

a

1/8

 

ist~~~~~~~~~~~~~~~~~~~~

0

Uvv

x

a

cv- 0.005

0.4

118

0.6

40

30X

x

20 0

10

n

0.8

FIG. 2. Linearized, discretized Monod data. Simu-

lated data containing errors having a constant coeffi-

cient of variation (CV) of 0.5% (A) and a constant standard deviation (SD) of 0.01 (B) were transformed

according to equations 12 and 13. The values of lmax, K5, and Y are the same as those used in Fig. 1.

1456 ROBINSON AND TIEDJE

rameter updating for data fitted to equation 4 is illustrated in Table 1. In both cases, five itera-

tions were required before the correction terms

were less than 0.01 and the residual sum-of-

squares was minimized. The updating of initial

parameter estimates was usually complete after four or five iterations when a convergence crite- rion 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 analy- sis 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 (simula- tion 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 resid- ual 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 fittingdata 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 iJmax, Ks, and Y

may be in substantial error (e.g., 500%), al- though the residual sum-of-squares has been

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

APPL. ENVIRON. MICROBIOL.

and Tiedje, manuscript in preparation). The growth of this organism was monitored by fol- lowing the protein content. Initial estimates of the growth kinetic parame-

ters were calculated according to the above- described 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,

h-1, 2.4

and Y,

respectively, were 5.6 x 10-2

and 0.99 g of protein

jxM dissolved

H2,

per mol of H2. Others have determined bacterial growth pa- rameters from batch data, but generally they have used computationally inefficient proce- dures 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 esti- mates. Before this, investigators typically fitted

biomass formation data to the integrated version

of the Monod equation describing biomass pro- duction, 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 coeffi- cients 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

Estimateb

Relative

error'

Simple error'

 
 

IJLmax

K5

Y

RSS'

ILmax

Ks

Y

RSS

Initialf

0.14

10.5

0.31

80.9

0.11

5.99

0.18

46.70

Iteration 1

0.12

7.26

0.21

8.9

0.12

7.40

0.21

0.83

Iteration 2

0.10

4.58

0.20

1.1

0.10

4.49

0.20

0.16

Iteration 3

0.10

5.02

0.21

0.09

0.10

5.00

0.20

0.004

Iteration 4

0.10

4.95

0.21

0.08

0.10

5.04

0.20

0.003

Final (iteration 5)

0.10

4.95

0.21

0.08

0.10

5.04

0.20

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.

passes were required before the solution converged. For these simulated data, a

convergence

b

Generally, four or five

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

Simulation

of errors in parameter estimates and residual sums-of-squares for linear versus nonlinear regression analysis of simulated Monod dataa

Initial estimates

% Error in:

Final estimates

RSSj/RSSf'

i-Mx K,nK Y

>

1

12.2

29.8

14

2

2

29

80.4

37

9

3

41

110

56.5

1

4

1.2

10.4

8.5

3

5

7.1

10.8

5.5

5

6

22.5

52.4

20

1

7

7

12.8

5.5

1

8

16

26.6

22

0

9

0

5.6

23.5

1

10

21

33

10

0.5

11

5.2

7.2

2

2

12

4.8

4.2

5

0

K,

Y

1.2

3.5

71.8

12.8

9.5

337

1

2.5

974

2.4

4

144

5.2

6.5

4.01

0.4

2.5

69.5

1.2

1

15.8

0.2

0

1,150

1.6

1

22,220

1

0

3,430

2.6

1.5

46.1

0

0

75.4

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.

possible to uniquely estimate parameters for a

given nonlinearmodel when the sensitivitycoef-

ficients predict otherwise.

(particularly for gaseous

from some limitations. It is advantageous since

substrates) but suffers

estimates of growth kinetic parameters may be

obtainedfrom a single substrate depletioncurve.

Further, estimating

by using the integrated Monod equation offers an alternative to estimating these parameters for

growth kinetic parameters

gaseous substrates by using

in which mass transport

chemostat cultures

limitations may yield

unrealistically high

the integrated Monod equation cause the derivative form, from

K, estimates (14). The use of

is limited be-

which the inte-

version is derived, describes balanced

grated

(steady-state) bacterial growth

describe transient (batch)

and may fail to

bacterial growth (13).

Powell has shown that apparent Monod parame-

The integrated Monod equation is advanta-

geous for the estimation of Umax, K,, and Y

A

c

0

-

O-

C

e

c

C

0

dU e 0

U-

S1

0o:

B

20C

Time

c

0

a

16

Final estimates

4-

C0

e0

C

12

12~~~CV=

cvo.o

0.005

0

U

e

8

0- a

a,.

4 . Initial estimates ',

O1

.

5

.

10

*i

15

-m

20

2S5

Time

3

FIG. 3. Comparison of theoretical curves calculat-

ed by using initial versus final (best) ,

K,, and Y

estimates with simulated data points. The data are

those that were transformed and plotted in Fig. 2.

cO

I

co

0 am

0

0

8

16

Hour

24

32

40

 

FIG. 4.

Comparison

of theoretical curve with mea-

sured H2 concentrations (O) for

growth

of Desulfovib-

rio 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

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 chemo- stat studies forestimating growth kinetic param- eters, but it should be used only after its limita- tions 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 ac- knowledge the expert technical assistance of Walter Smo- lenski 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 explana- tion for accumulation of nitrite, nitric oxide, and nitrous oxide during bacterial denitrification. Appl. Environ. Mi- crobiol. 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.

APPL. ENVIRON. MICROBIOL.

5. Cornish-Bowden, A. 1979. Fundamentals of enzyme kinet-

ics, 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 481:297-

312.

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. Tem- pest (ed.), Microbial physiology and continuous culture.

Her Majesty's Stationery Office, London, United King-

dom.

14.

Robinson, J. A., and J. M. Tiedje. 1982. Kinetics of

hydrogen consumption by rumen fluid, anaerobic digestor sludge, and sediment. Appl. Environ. Microbiol. 44:1374-

1384.

15.

Strayer, R.

F., and J. M. TiedUe. 1978. Kineticparameters

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 geome- try. p. 76-81.