Modelizacin matemtica de crecimiento y conversin de sustrato de

Zymomonas
mobilis a los 30 y 35C
1. M. L. Jobses, G. T. C. Egberts, A. Van Baalen, y J. A. Roels
Laboratorio de bioingeniera, Departamento de Qumica e Ingeniera Qumica, la Universidad de Tecnologa de Delft, Delft, Pases Bajos
aceptado para su publicacin el 2 de agosto de 1984
Zymomonas mobilis fue crecido en culturas continua
a los 30 y 35C. Las tasas de consumo de sustrato especfico a 35C
fueron superiores a los 30C. Un modelo matemtico no estructurados
sobre la base de la ecuacin lineal para consumo de sustrato
proporcion una descripcin estadsticamente adecuados para cultivos a
35C, pero no para cultivos
en 30C. Una estructura de dos compartimientos describi el modelo de
crecimiento y consumo de sustrato bien a las dos temperaturas. Algunos
aspectos tericos y prcticos de la dos-compartimiento modelo son
discutidos.
Introduccin
Zymomonas mobilis tiene unas caractersticas interesantes para su
aplicacin en gran escala de la fermentacin de la glucosa en etanol.
Especialmente su alta tasa de consumo de glucosa y su habilidad de crecer
estrictamente anaerbicamente" llevara a una preferencia por la bacterium sobre las especies de levaduras utilizados comnmente. Furthermore Z. mobilis muestra una mayor conversin ef- ficiency de glucosa
en etanol,293, que es el resultado del bajo nivel de formacin de
subproducto4 y el bajo rendimiento de biomasa de las propiedades
fisiolgicas de la bacteria Zymomonas mobilis son ampliamente examinados
por vaivenes y de Ley." Los modelos matemticos de las fermentaciones se
basan generalmente en ecuaciones cinticas y ecuaciones de equilibrio.
El modelo no estructurados utilizado aqu se basa en una ecuacin
cintica de crecimiento, la ecuacin de Monod,8 y en una ecuacin
cintica para sustrato conversin - la ecuacin lineal para consumo de
sustrato. Para la fermentacin anaerbica, la tasa de formacin de
producto puede calcularse a partir de estas ecuaciones y el elemental
balanceS. 5.6.9,10 la ecuacin lineal para consumo de sustrato fue
postulada en 1898 por Ducleaux" y ms tarde restablecido por Pirt." El
modelo de en- dogenuous metabolismo, como propuesto por Herbert,13 es
matemticamente equivalente a la ecuacin lineal para consumo de
sustrato.
Direccin actual: Gistbrocades N.V., investigacin y desarrollo, en
Delft, Holanda. la
biotecnologa y la bioingeniera, vol. XXVII, Pp. 984-995 (1985)
La ecuacin lineal afirma que parte del sustrato es consumida para el
mantenimiento de la actual tasa de crecimiento de la biomasa
(independientes), y que la parte restante se consume para la produccin
de nueva biomasa (tasa de crecimiento dependiente):
rs = (1/Ys,)pCx + msc, (1)
La independencia de la energa de mantenimiento requieren- ment (m), la
tasa de crecimiento (p) ha sido objeto de mucho debate (vase ref. 14).
Neyssel y tempestad" con- cluida desde sus resultados continuos de
cultivos con diferentes tipos de limitacin (sustrato, fuente de
nitrgeno, fosfato y sulfato) que el requisito de energa de

mantenimiento es dependiente de la tasa de crecimiento, en forma lineal.

Westerhoff Hellingwerf'6 y et aI.l7 pro- rectamente un modelo basado en
nonequilibrium namics thermody-, que es capaz de describir, al menos
cualitativamente las diferencias observadas en el mantenimiento factores
encontrados para diferentes limitaciones de nutrientes. Adems, este
modelo tambin contiene un factor de mantenimiento que depende
linealmente de la tasa de crecimiento. Desde el punto de vista
experimentalistas, sin embargo, una tasa de crecimiento linealmente
dependientes del factor de mantenimiento no se pueden distinguir de una
constante sobre la base de constantes experimentos de cultivo continuo
estado solos.
Otra literatura18-22 informa que el requisito de sustrato de
mantenimiento depende de la tasa de crecimiento en una forma no lineal:
el factor de mantenimiento parece disminuir progresivamente con la
disminucin de la tasa de crecimiento.
Para tener en cuenta esta caracterstica, la ecuacin lineal para
consumo de sustrato tiene que ser modificado o un crecimiento
estructurado y fermentacin debe ser ap- modelo para surcado .6,23-2S
alta especificidad del producto, en la que la glucosa es casi
cuantitativamente convertidos a etanol y carbondioxide, hacer Z. mobilis
un organismo adecuado para estudiar diversos modelos matemticos, en el
cual la misa y saldos elemental juegan un papel importante. Asimismo, la
alta exigencia energtica tenance principal- estudios de modelizacin:
puede ser muy ventajoso para
esperar que la exp- imental determinacin es ms precisa para la altade
0 1985 John Wiley & Sons, Inc.
CCC 0006-3592/85/070984-12$04.00

de bajo mantenimiento de los requisitos de energa. Quently conse-, los

efectos de las condiciones del proceso, tales como la tasa de crecimiento
y la temperatura, en el mantenimiento de la demanda de energa pueden ser
detectados con ms claridad.
Teora
Esta seccin proporciona una descripcin de la cintica de equaciones de los modelos estructurados y no estructurados que se utilizaron
en la interpretacin de los resultados. En estado estacionario, la red
culturas continua formacin de cualquier compuesto me ser igual a su
descoloracin:
ri = DCi (2)
Cuando esta ecuacin se aplica a la tasa de formacin de biomasa,
resulta evidente que la tasa de crecimiento especfico, p, es igual a la
tasa de dilucin, D. El modelo no estructurados o el mantenimiento del
modelo se basa en la ecuacin lineal para consumo de sustrato","2 eq.
( 1 ) .
La tasa de consumo de sustrato especfico qs(qs =
rs/Cx) est dada por:
9 s= (1/YSX)D + m, (3)
El modelo de metabolismo endogeneous' es matemticamente equivalente al
modelo de mantenimiento. El mantenimiento de los requisitos de energa de

la biomasa es en este modelo proporcionado por una degradacin de la

biomasa endogenuous:
(4)
y el consumo de sustrato est dada por:
rs = (1/Ysx)rx (5)
en el modelo endogenuous, Pmx y Ks son contrarias a Y., no
numricamente iguales a sus homlogos en el modelo de mantenimiento.
Por comparacin, se deduce que el modelo de mantenimiento y el modelo
olism endogenuous metab- son equivalentes, si tiene la siguiente
expresin:
P E= Ysxms (6)
El modelo del compartimento de dos estructurado como introducida por
Roels et al.6323-25 est representado esquemticamente en la figura 1 .
El modelo divide la biomasa en un K- y un G-compartimiento.
Fisiolgicamente, puede ser considerado como una extensin del modelo
de metabolismo endgeno
Figura 1. Representacin esquemtica de la dos-compartimiento modelo.
La biomasa se compone de dos compartimentos, un K- y un G-compartimiento.
El K-compartimiento es sintetizada directamente desde el substrato S.
El G-compartimiento y depolymerized es sintetizada a partir del Kcompartimiento . Para fermentaciones anaerobias, el producto final del
metabolismo de formacin (no se muestra) puede calcularse a partir del
grado de reduccin b a l a n ~ e . ~ ~ ~ ~ ~ ~ ~ '
JOBSES ET AL.:
[EQ. (4)]. Los requerimientos energticos de mantenimiento se explican
en trminos de una tasa cero de G-compartimiento del volumen de
negocios. Generalmente, el K-compartimiento es interpretado a consistir
de ARN, carbohidratos, y monmeros de macromolculas. El Gcompartimiento est identificado con protenas, ADN y lpidos.
El mantenimiento de la demanda de energa aumentar con el aumento de
las tasas de rotacin de G-compartimiento y de arrugar los valores de
los factores de rendimiento de K en G o G en K. El modelo se basa en las
siguientes ecuaciones:
Para consumo de sustrato:
(7)
para la red de informacin de la K-compartimento:
r, + k,GC, (8) = - c - kkKGC prnaxcs, Ks + cs
la conversin del K- en el G-compartimiento se supone que es
proporcional a la biomasa K-contenido como el reactivo, y proporcional a
la G-contenido como el compartimento que contiene las enzimas para la
con- versin. La conversin del G- en el K-com- piso est considerada
como una reaccin depolimerizaci, que slo es proporcional a la
G-fraccin de la biomasa. El rendimiento de K en G puede esperarse que
sea alto, y es en este modelo que es igual a 1 .
La formacin de la red G-compartimiento es
rc = YkgkkKGCx - k,GC, (9)
la formacin neta de la biomasa total es de
r = r + R, (10)
Por supuesto, el K- y la biomasa total:
G-compartimiento juntos hacen K + G = 1 . Para el estado estacionario, el
crecimiento continuo de la cultura de consumo de sustrato especfico
puede ser calculado por la combinacin de eqs., (2), (7), (8) y (9), lo
que se traduce en la ecualizacin. (11):
4 s= 1 /ysk[kg(kk + k g-k g /Ykg)/(kkYkg) - kg

+ (2Kg + k k- 2kg/Ykg)/(kkYkg)D (1 de 1)
- ( 1 - 1 ) ykg //(kk ykg >D21
Nota que hay tres condiciones: la primera es independiente de la tasa de
crecimiento, la segunda es lineal dependiente de p(D), y la tercera es
una funcin lineal de segundo orden de p.
por aplicacin de ecualizacin. (2) en el estado estacionario Gcompartimiento biomasa y sustituyendo en el EQ. (9), el estado
estacionario K-fraccin de la biomasa se calcula:
K = (kg)/D kkykg (12)
MATERIAL Y MTODOS
cepa Zymomonas mobilis L.M.D. 29.34 (obtenidos del Laboratorio de
Microbiologa de la Universidad de Delft
crecimiento/modelo CONVERSIN PARA Z. MOBILIS 985

de tecnologa) que tambin se conoce como cepa ATCC 10988, fue

utilizado. La cepa se mantienen en cultivo de agar inclinado que contiene
20 g/l de glucosa, 10 g/L de extracto de levadura, y 20 g/L de CaC03 a
temperatura ambiente y se transfieren cada 1-2 meses.
El medio de cultivo" consisti en g/L; KH2P04, 1 g/L; NH,CL, 2
2H20, 1.5 mg/L; H20, 4.2 mg/L; CuSO, 5H20, 2.0 mg/L; MnSO, CoSO, - 7H20,
de 1,6 mg/L; Nacl, 50 mg/L; KC1, 50 mg/L; y HCL concentrado, 0,25 mL/L.
7H20, 5 mg/L; ZnSO, 7H20, 7.2 mg/L; CaC1, glucosa 50 g/L; MgSO, .
7H20, 0,49 g/L; Ca-panthothenate, 5 mg/L; FeSO,
glucosa fue esterilizada en autoclave a 110C. Soluciones de sal fueron
esterilizadas por filtracin. Las culturas fueron cultivados en
fermentadores de 2 l con un volumen de trabajo de 1- 1,5 L, salvo el
experimento en D = 0,0077 h-' a 30C, que se realiz en un fermentador
de 15 l con
8 l de volumen de trabajo. El medio del inculo consisti de 20 g/l de
glucosa y 10 g/L de extracto de levadura.
Los cultivos fueron sembrados con 60 mL de inculo y la cultura el pH se
mantuvo a pH 5 por un controlador automtico de pH con NaOH. Los estados
estacionarios en continuo culturas se supone que se estableci despus
de cinco veces el tiempo de residencia. Residencia durante 5-10 veces
despus de este perodo, 4-8 muestras de 40 mL fueron tomadas.
Etanol y acetealdehyde fueron determinadas mediante GLC usando una
columna Porapack QS. En la fase de gas Acetealdehyde fue determinada por
el traspaso del gas que sala del fermentor a travs de una solucin
que contiene un complejo formando hydra~.~' de cido actico, cido
frmico, cido succnico, formiate acetoin, lactato, glicerol y se determined por HPLC en un Biorad HPx columna. El glicerol y cido succnico fueron detectados por la refraccin y los dems componentes por
absorcin de UV.
La glucosa se determin con un kit de glucosa oxidasa (Boehringer) o por
HPLC en un p-Bonda- pack/carbohidratos columna (aguas). El CO, la produccin se determin volumtricamente con un medidor de gas hmedo
callibrated (Schlumberger, Dordrecht). Disuelto.
C02 se calcul segn Stumm y Morgan.28 El peso seco de la biomasa se
determin por centrifugacin o filtracin. La biomasa se lava dos
veces con agua desmineralizada y secado a 85C hasta peso constante se
logr. Contenido de cenizas fue de- termined calentando la biomasa seca

a 650C hasta que pro- stant peso. C, H y N El contenido fue determinada

con un autoanalizador (CHN Perkin-Elmer). Concent oxgeno fue calculada a
partir de la diferencia entre el peso seco y la ceniza, C, H y N pesos.
Arn, la cantidad total de carbohidratos, protenas y fsforo concent se
determinaron en un liofilizado de biomasa; la biomasa se obtuvo por
centrifugacin de la fermentacin total contenido en 4C y lavadas dos
veces con de- agua mineralizada. El ARN se determin en un extracto
cido29 por el orcinol rneth~d.~' E. coli RNA (cal- biochem) fue
utilizado como estndar. Se de- termined protena por el mtodo de
biuret3' con suero bovino albumine (Merck) como estndar. Los
carbohidratos totales
986 la biotecnologa y la bioingeniera, VOL. 27, julio
fue determinada por el mtodo anthron3' con la glucosa como estndar.
Contenido de fsforo fue determinado mediante ICP,31despus dos veces
persulfate digestin.32 La prueba de azul de metileno para tincin de
clulas muertas33se realiza de la siguiente forma: una muestra de la
cultura fue diluido con un medio fresco y suspendido en el 0,01% (p/v) de
azul de metileno y despus de 10 min de incubacin amined ex- por
contraste de fase o microscopia de luz. La capacidad de divisin celular
se midi mediante la diapositiva cultura tech- n i q ~ e . ~ ~
diapositivas contena 50 g/l de glucosa, 10 g/L de extracto de levadura,
y 10 g/l de agar y se incubaron ana- erobically (bajo gas N2) 24-48 h a
30C. Desde
Z. mobilis ocurre a menudo en pares, slo los grupos de ms de dos
bacterias y el incremento del porcentaje de pares eran considerados como
viables.
Para el azul de metileno y deslice las pruebas, duplicar los recuentos de
300 sujetos fueron hechas. Polifosfato fue empleado de tincin segn
Layb~urn.~' para el clculo del carbono y el grado de reduccin
generalizada de saldos, la concentracin de los com- libras fueron
expresados en equivalentes de C/L y C02 del flujo de gas en equivalentes
de C/h:
balance de carbono
+cx + +gcc2 + C02 - flujo de gas - +Cp + x 100%
+(Csi - Cs)
El grado generalizado de un compuesto se define segn la reduccin, y,
de los tambores?
YCaHbOcNd = (ayC cYO + + + dYN)/a
yo = -2; yN = -3 ;1 yH = ;4 yc =
reduccin del grado de equilibrio se calcula de la siguiente manera:
grado de reduccin balance
resulta
dos series continuas de culturas fueron dirigidos a los 30
y 35C en tarifas que varan de dilucin D = 0.008 h-' a D = 0,24 h-'.
Los resultados se muestran en la tabla I.
En todas las carreras, los afluentes de la glucosa (50 g/L) fue
convertida cuantitativamente, ya que no fue detectable en el efluente
(menos de 0,01 g/L), excepto para la carrera de d = 0,24 h-' y 35C,
donde el promedio de glucosa centration efluentes- fue de 1,8 g/L. La
biomasa y los rendimientos de etanol se muestran en la figura 2.
El rendimiento de biomasa a 30C es para todos excepto las tasas de
dilucin D = 0,02 h-' superior a 35C, siendo la diferencia mayor en
bajas y altas tasas de dilucin. Tenga en cuenta que el rendimiento
depende en gran medida de la tasa de crecimiento como resultado de alto

mantenimiento requisitos de energa. La eficiencia de conversin de la

glucosa en etanol fue alta en todas las tasas de crecimiento y mostraron
una ligera
1985

Tabla I. cultura continua datos de Z. mobilis a 30C y 35C. Qs y C, se

basa en la biomasa sin cenizas. Las culturas se ejecute a pH 5.
;4 Las cifras entre parntesis representan los intervalos de confianza
del 95%. El grado de reduccin se define, de acuerdo con la ref. 36,
como YSL,,,,, =
y yco2 = 0. Los valores promedio son de 30 C, el C-balance es del 92,4%
(2,7%), y-balance es de 93,4% (2,8%), y la brecha es de 4,5 (0,6); en
35"C, el C-balance es el 91,9% (2,3%), y el saldo es de 92,0% (3,3%), y
la y-gap es de 3,8 (0,6). ;6 yblornalr 1 = 4,2; ycthano, =
Temperatura q s C, C
("C) D-7 (g/g h) (g/L) (g/L)
30C 0.0078 1,75 50,0 24,3 (0,0002) (0.19) 0.0096 2.17 49,4 23,9 0,22
C, C, C-balance y equilibrio
(g/U (g/L) (%) (%) Y-WP
0.217 nd 95,7 94,7 4,9
(0,0001) (0.12) 0,020 3,82 50,5 24,3 (0.001) (0,21) 0.032 4.484 49,9 24,2
(0.002) (0,56) 0,035 3,69 49,0 23,3 0,47
nd 94,0 95,4 2,9
0,26 nd 96,4 97,8 5,7 0,36 nd 96,9 97,7 5,4
(0.001) (0,17) 0.039 4.18 50.0 22.2
94.3 98.4 4.0 0.47 Nd
nd 88,0 93,4 4,0 (0.003) (0,54) 0,049 4,31 48,6 20,8
(0.005) (1,22) 0,049 4,55 52,2 25,5 (0.001) (0,96)
0,075 5,99 51,0 22,1 (0.004 (0.40) 0.122 7.09 50. Yo 23,2 (0.001) (0,10)
0,133 6,91 49,8 21,4
(0.001) (0,10) 0,237 8,44 51,4 22,1
35C 0,0106 3,52 49,4 22,1 (0,0002) (0.22) 0.0200 3.43 50,8 23,8
(0,0002) (0.02) 0.0321 4,65 50,1 23,9 (0.000.5) (0,11) 0.0526 5.92 50,7
24,9 (0,0002) (0.32) 0.079 6.72 50.2 23.8 0.57
0.49 Nd 85,2 83,0 4,0 0,58 nd 97,0 99,0 4,7 86,5 93,6 4,0 0,65 nd 0.86 Nd
94,2 94,2 4,0 90,5 85,8 6,0 0,96 nd
I 0,44 nd 89,5 87,4 4,8
0,15 nd 87,8 89,2 3,5 0,27 nd 90,9 92,5 3,3 94,4 95,7 3,1 0,31 nd 0.40 Nd
94,9 97,2 2,2
(0.002) 0.117 (0,18) 6,98 50,3 23,6 (0.001) (0,25), 0,134 8,26 50,6 20,7
(0,0005) (0.14) 0,137 9,24 50,3 24,0 (0,0003) (0.27) 0,162 9,08 49,8 23,0
0,89
Nd 92,6 92,7 4,0
94,3 94,4 3,9 0,75 nd 0.91 Nd 84,9 80,7 5,2 0,67 nd 94,5 95,1 3,6
(0.001) (0.35) 9.71 0.191 49,7 23,5 (0.001) (0,46) 0.238 10,86 49,9 21,1
(0.001) (0,36),
n.d., no detectables (< 0,01 g/L).
Con el aumento de la tasa de crecimiento de tendencia como se puede
esperar sobre la base de la media no estructurados
e~ para todos los datos fue cp = 0,46 g de etanol/g de glucosa, que es el
90% de la capacidad mxima terica (0,51 g/g). Desde el carbono y el

grado de reduccin de saldos (vase la seccin de Materiales y

mtodos) calculado a partir de la glucosa, etanol, biomasa y C02 Flujos
cerrados por slo el 92%, hemos buscado varias posibles subproductos.
Los resultados se muestran en la Tabla 11. La cantidad total de
subproductos representaron entre el 2% y el 3% del total de carbono,
JOBSES ET AL.:
nd 93,6 92,5 4,7
0,87 nd - - 1,07 1,8 91,5 90,1 4,7
que es comparable con los resultados de Amin y co- trabajador~.~ hemos
encontrado, sin embargo, menos glicerol y acetoin, que podra estar
correlacionada con el nivel de etanol en el lquido de la cultura; en
nuestros experimentos, etanol concen- trations fueron alrededor de 23 g/L
en contraste a 70 g/L en sus experimentos.
A partir de los subproductos, succinato aumenta con el aumento en la tasa
de crecimiento o la concentracin de biomasa, mientras que el lactato es
relativamente alto a bajas tasas de crecimiento. El acetal- dehyde
represent en el caldo de fermentacin para slo el
crecimiento/CONVERSIN DE MODELO DE Z. MOBILIS 987

t 0'03 f
9/9
0.02 X
D 1 h 0"
0
d ln m
- m eo ".
Xx 0 X
I
I I 0 1 I
0,1 0,2
D i'/hr) __c
Figura 2. Rendimiento de biomasa y etanol en glucosa
0 x .
X X
I I 0 0.1
D ('/hr)
en continua culturas se ejecute a pH 5. Los afluentes de la glucosa (50
g/L) fue convertida cuantitativamente,
I
0.2 a los 30 y 35C. Las culturas excepto a D = 0,24 h-'
a 35C. Otros datos que se muestran en la tabla I. El promedio de
rendimiento de producto fue E~ = 0,46 g/g (lmites de confianza del 95%:
0.45-047). Los smbolos muestran (A) rendimientos de biomasa, (B) el
rendimiento de etanol, ( X) 30C. y (0) 35C.

0,3% y en la fase gaseosa por slo 0,004% del total en el curso de

carbono. Este ltimo valor es muy bajo, con- sidering que el punto de
ebullicin del acetealdehyde es de 21C.
Sin embargo, tericamente el acetaldehdo contenido en fase gas
ascendera a 0,001% del balance de carbono, cuando se calcula a partir de
su concentracin en la fase lquida, su presin de vapor, y su
actividad coef- ficient en agua.37 Cuando los productos son tomados en
cuenta, el carbono y el grado de reduccin de saldos para cerrar la
Tabla 11. Subproducto de formacin continua en las culturas a los 30 y
35C. Afluentes de la concentracin de glucosa fue de 50 g/L. Otros
datos que se muestran en la tabla I. Para determinaciones, consulte la
seccin de Materiales y mtodos. Debido a las bajas concentraciones de
los compuestos, las cifras slo representan una estimacin global. El
grado de reduccin generalizada y se define de acuerdo con la ref. 36.
Temperatura 30 C a 35 C y
tasa de dilucin (h-') 0,01 0,13 0,24 0,01 0,13 0,24
acetato (g/L) 0,5 0,1 0,4 0,4 0,25 0,5 4,0
lactato (g/L) 0,05 0,05 0,04 0,4 0,1 0,09 4,0
succinato (g/L) 0,06 0,14 0,24 0,05 0,14 0,28 3,5
Acetoin (g/L) <0,05" 5.0
propionato (g/L) <0,05 4.1
Formiate (g/L) <0,05
glicerol (2.0 g/L) 4.67
Acetealdehyde (g/L) 5.0
~~~~
&lt;0.05 <o. 1
~ ~ ~
a The values for the following compounds are equal for all dilution
rates and both temperatures.
988 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 27, JULY
for some unknown product(s) with a generalized degree of reduction of 44.5. There are many biological com- pounds in this range of generalized
degree of reduction, including carbohydrates. From this class of
compounds, levan and sorbitol are reported as by-products in sucrose
fermentations. 38 5 about 95 (Tables I and II), leaving a gap of
The growth constants, Y, and m,, of the unstructured model can be
estimated by plotting the specific glucose consumption rate, qs, vs. the
specific growth rate p,
which equals D in the continuous cultures. According to eq. (2), this
should yield a straight line with slope 1 / Y, and intercept m, . From
the experimental results (Fig. 3), it is obvious that the relationship
between q5
and D at 30C is not linear. The data points at D =
0.008 h-' and D = 0.01 h-' are statistical anomalies from the linear
regression line on all data points.39 Although a slight deflection of qs
vs. D at 35C can be noticed, a linear relationship between these
variables can not be rejected statistically.
In spite of the fact that the unstructured model does not provide an
adequate description of the experimental results, at least at 30"C, it
was applied to the nearly linear part of the curve for comparison with
literature.
Linear regression was applied to the data at 30 and
35"C, both between D = 0.02 h-' and D = 0.014 h-'.

In Figure 3 the data points are shown with their 95 confidence limits
in q, and D separately. Since these variances are obtained from replicate
samples (see the Materials and Methods section) of one particular steady
1985

l4 1 independent variable D to the dependent variable

q, standard linear regression weighted to the variance in q, was
applied.39 This method yielded essentially the same results as methods in
which the variances
0 s 8 of both variables were taken in account separately.4034
Consequently, the joint confidence regions of the es6 t timated parameters were also calculated by standard
4 { !ti methods.39 The result is shown in Figure 4. The es- timated
parameters with their separate 95 confidence intervals are:
0.05 0.25 for 35C, 010 0.15 0.20
O(l/hr) - rn, = 3.1 (2.2-3.9) g/g h and
14 - 1/Y, = 40.0 (29.7-50.3) g/g
B 12 - for 30C m, = 3.0 (2.3-3.7) g/g h and 1 lo- +
0 s 8 - I/Y, = 31.6 (25.3-38.0) g/g.
g/g.hr
+ t4

These values are comparable to values reported in literature (see Table

111). According to the literature, higher ethanol concentrations result
in increased values +
6 2 of m,. This is not apparent from the data presented in Table 111. It
must be recognized, however, that the
0 0.05 010 0.25 effect may be masked by the influence of medium
D(/hr) composition on m,.
Although the separate parameters do not differ sig- Figure 3. Q7 vs. D
plot of continuous cultures
0.15 0.20 grown at 35 and
30C. Data are shown with their 95 confidence limits calculated
nificantly and lie within each others confidence limits,
for D and q. separately. The temperatures were (A) 30C and (B) the
joint confidence regions overlap each other only
35C. partly. The point estimates of Y,, and m, suggest that m, does
not vary and Y, decreases with increasing state, they represent the
minimum real variance. As temperature. The overlap of the joint
confidence regions the variances differ mutually, weighted linear
regression indicates that a unique conclusion regarding the tem- was
used. After transformation of the variance of the
14 - I
12 10 -7 4< - $ 2 $ 0 I I
perature dependence of Y, and m, is impaired.

50 40 30 20 - t
10- - g
X zm -b
JOBSES ET AL.: GROWTH/CONVERSION MODEL FOR Z. MOBILIS 989

Table 111. Comparison of Y,, and m, values with the literature. The
presented figures are based on ash-containing dry weights
Ethanol concentration Temperature Medium (g/L)
30C 30C complex complex 45 40 30C complex 50 30C minimal 27
30C complex 23 35C complex 23
a The complex is a medium containing yeast extract minimal refers to a
~~
K I m,
k/g) (g/g h) Reference
2.9 3 0.06 - 1.6 66 0.03 1.6 69 0.01 2.1 0.035 2.7 45
this work
0.03 2.8 this work
medium without yeast extract.
A higher q, at elevated temperatures is in agreement with batch where
increasing temperature 2. mobilis,
Since the qs vs. D line is not linear, but deflects for at least at 3WC,
the linear equation for lowers the overall yield of biomass on substrate
implying that m, is elevated or Y,, is lowered. Fieschko and Humphrey4
observed in continuous cultures of Z.
mobilis an increase in m, at higher temperatures, whereas Y, did not
change. This was also observed for other microorganisms (see ref.
14).20946 A decrease in Y, at higher temperatures together with an
increase in m, , however, has also been An effect of temperature on Y, is
theoretically not obvious. On first sight, Y,, appears as a conversion
yield of glucose into biomass and will not likely be affected by
temperature. This parameter is, however, not a simple stoichiometric
constant, as is illustrated by the fact that experimentally determined
YATp values are very often lower than the calculated theoretical
stroichiometric values (see Table V).I4 Parameter Y,,
covers numerous reactions correlated with the specific growth rate. As
these reaction rates may vary with temperature, the value of Y,, may also
vary with temperature.
Table IV. Elemental composition of 2. mobilis at various growth rates.
The
substrate consumption seems not to be valid. The unstructured model
regards biomass as a simple chem- ical compound, and therefore the
nonlinearity may be due to the state of the biomass: the biomass
composition and activity.
The biomass activity was determined by means of two viability
tests. Samples were taken from con- tinuous cultures grown at 30 and
35C at dilution rates from D = 0.008 to D = 0.12 h- and the
viability was tested by the slide culture technique. The viability did

not change with growth rate ca. 30 of the biomass was able to divide
in this test at all dilution rates.
~ and negati~e~~~.~ support of these results is reported in
literature. Both p o ~ i t i v e ~ ~ ~ ~ ~
The viability was also measured by methylene blue staining of the
biomass in samples from all dilution rates up to D = 0.24 h-.
According to this test, more than 95 of the biomass at all dilution
rates was viable.
The difference in the two tests was also noticed by viability between
other author^.^^.'^
elements were determined in dried biomass samples of continuous cultures
grown at various dilution rates at 30 and 35C. For the determinations,
see the Materials and Methods section. Oxygen content was calculated by
subtraction of C, H, and N from the ash-free dry ash-free biomass formula
is CHI 720041N023 and the mean generalized degree of reduction is 4.2,
calculated from all weight. The mean data (30 + 35C).
Percentage of ash-free dried Percentage of total biomass dried biomass
Dilution rate C H
Temperature (h I (I ()
30C 0.08 54.7 7.6 30C 0.01 50.6 7.2 30C 0.02 50.3 7.7 30C 0.03
51.4 8.0 30C 0.12 48.6 7.2 30C 0.13 51.4 7.1 30C 0.24 50.9 6.9
35C 0.01 51.1 7.2 35C 0.02 3 5C 0.03 53.0 7.9 35C 0.05 50.0 7.6 35C 0.13 50.9 7.2 35C 0.16 48.9 7.1 35C 0.24
51.3 7.1
a Figures in parentheses are estimated by interpolation.
990 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 27, JULY
0 N ash P
() (I () (I
22.4 15.2 12.5 28.1 14.1 (1 1) 29.0 13.0 (1 1)
5.1 27.8 13.2 10.9 30.7 13.5 8.7
- 27.5 14.0 (8.5) 2.0 28.0 14.2 7.1 I .8 27.7 13.9 (10.6) 6.2 25.6 10.6 13.5 - 10.6 29.1 13.2 10.7 27.6 14.4 (8.9) 30.2 13.8 8.3 2.2
- - 26.9 14.7 6.9 1.8
1985

The elemental composition of the biomass differed elevation of the growth

rate (cf. ref. 20) and temperature somewhat at various growth rates
(Table IV), but a results in an increased size of the bacteria.
general trend as reported by Esene? for Klebsiellu The macromolecular
composition at various growth
aerogenes was not observed. The ash content and rates is shown in Table
V. The results are in close phosphorous content however declined
systematically agreement with values reported by Dawes and Large with
increasing growth rate both at 30 and 35C. No for Z. mobilis and Z.
unuerobiu. The low carbohydrate polyphosphate is reported for batch-grown

Z. mobili~.~ content correlates to the absence of glycogen. In In

our experiments, however, organisms grown at low agreement with other r e
p ~ r t s , ~ ~ ~ ~ ~ ~ ~ ~ the RNA content growth rates showed
on staining for polyphosphate so increases with increasing growth rate.
At 35C the called metachromatic granules (cf. ref. 7) at both tem- RNA
and carbohydrates content is lower and the protein peratures, as shown in
Figure 5. The metabolic role content is slightly increased (compare refs.
57 and 58)
of polyphosphate is not yet clear the formation requires compared to
30C.
ATP and phosphate and will probably occur when one Table V shows the
theoretical (stoichiometric) amount or both compounds are in excess. The
formation does of ATP necessary for the formation of one gram dried not
occur during fast growth but at nutrient deprivation biomass and the
theoretical Y, calculated from this like sulphur or other salt
depri~ation.~~ Polyphosphate ATP requirement. Under the assumption that
the ef- may serve as an energy or phosphate storage. The ficiency of
biomass formation on glucose is the same phosphorous content at D = 0.01
h- at 30C was ca. for all growth rates, these theoretical yields
predict
20 lower than at 35C, suggesting that at 35C more an increase in
Y,, by 13 at the lowest growth rate.
polyphosphate is stored, which would cause a higher This increase is too
small to account for the strong
qs at this temperature. Polyphosphate accumulation deviation of q, from
the linear relationship at low growth is, however, not in line with the
observed deflection rates at 30C.
of q, at low growth rates. Figures 5(A)-5(D) show that The results
clearly indicate that the biomass comA C
B D
Figure 5. Light microscopy pictures of biomass stained on polyphosphate.
Biomass growth at various dilution rates was stained according
to ref. 35. Magnification was 1000x. The rates were (A) D = 0.14 h-I,
30C (B) D = 0.01 h-, 30C (C) D = 0.14 h-, 3SC and
(D) D = 0.01 h-, 35C.
JOBSES ET AL.: GROWTH/CONVERSION MODEL FOR Z. MOBILIS 99 1

Table V. Macromolecular composition of Z. mobilis at various growth rates

at 30 and 35C. Macromolecular content of freeze-dried biomass from
continuous cultures was determined as described in the Materials and
Methods section. The percentages (w/w) are based on ash-free dry weight.
The ATP requirement for biomass formation is based on the figures shown
below and the DNA content is assumed to be 2.7 (ref. 59) the
remaining biomass is assumed to be lipids. When the sum of compounds
exceeds 100, the calculated value is normalized to 100. ATP values
for macromolecule formation are obtained from ref. 14. The calculated
theoretical Y,, is based on the formation of 1 ATP/mol glucose
metabolized. Glucose incorporation in the compound's carbon skeleton is
neglected.
ATP required Theotical

D Protein ( m m o 1 / g y, Temperature V ' ) RNA (I ()

Carbohydrates
(I
Sum (I dry wt)
30C 0.01 15.7 68.1 3.8 87.6 33 0.17 0.13 23.4 70.0 7.9 101.3 37 0.15
0.24 29.7 68.8 7.3 105.8 38 0.15
30C 0.01 10.5 69.6 3.4 83.5 32 0.17 0.16 18.8 75.0 4.7 98.5 37 0.15
0.24 26.4 72.3 5.3 104.0 37 0.15
position is not constant but a function of the growth remainder are
allowed to adapt. Therefore, for the rate and temperature. Bringing the
biomass composition description of the data at 35"C, the values of Ysk
and or state in the growth model, leads automatically to
a structured model. The simplest structured model is the two compartment
model as proposed by Roels et
Ykg were adopted from the dataset at 30C and kk and
k, were estimated using the same algorithm. The sim- ulation describes
the experimental results quite well.
a1.6,23-25 The model predicts a linear increase in the The model also
predicts a lower K-compartment (see K-compartment on increasing growth
rates [eq. (12), RNA and carbohydrates contents in Fig. 6), and therefore
it is logical to ascribe the K- higher turnover rate of compartment to
the RNA or RNA and carbohydrates
Table III), and a this compartment (kk and k,)
at 35C compared to 30C.
content of the biomass. Although the model describes the observed
features Figures 6(A) and 6(B) show simulations of qs at qualitatively
well, the absolute amount of the K-com- various D, calculated with the
partment and the relative increase are much different [eq. (ll)]. The
parameters two-compartment model
Ysk, Ykg, kk, and k, were from the observed RNA contents. This might be
adapted estimated from the data at 30C by least-square ap- by choosing
alternative kinetic equations, containing proximation using Marquardt's
Compromise algorithm more complex relationships and/or more constants for
in a computer program. Because of the scatter in the the formation of the
K- and G-compartments, but it data points and the almost linear
relationship between is questionable whether such a model is useful.
Exactly
qs and D at 35"C, the fit of this data set is rather insensitive to
variations in one parameter when the the same fit to the qs data can be
obtained by a second- order polynomial in D: qs = a + bD - cD2 [compare
t lo
8 Qs g/g.hr
6
4
I
0 0
Figure 6. Description of Q$ vs. D relation by
30C
2
l , , 1 " " l "
0 t0.1 o. 2 0 0.1 0.2
Dllhr 1 - D I ' h r )-

the two-compartment model: (A) description of results at (9) description

of results at 35C. The symbols show (0) data points, (-) simulation
based on estimated parameters, and (---) calculated K-compartment. The
estimated parameters are at 30C: k, = 3.75 h-', k,
= 0.036 h-I, Y,, = 0.123 g/g, Yk, = 0.118 g/g at 35C: kk = 4.80 h-',
k, = 0.047 h-'. Both Ys, and Y,,
were adopted from 30C. The estimated K-compartment for 30C is D =
0.02 h-', 0.125, and D = 0.24 h-',
0.621 for 35T, D = 0.02 h-', 0.118, and D = 0.24 h-', 0.506.
992 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 27, JULY 1985

this to eq. (ll)]. The constants of this polynomial, however, have no

physical or physiological meaning at all.
DISCUSSION
The high conversion efficiency of glucose into ethanol by Z. mobilis is
due to the low level of by-product formation and the low biomass yield.
The low biomass yield is only partly due to the unfavorable catabolism5:
glucose is converted by the Entner-Doudoroff route which yields only 1
ATP/mol glucose. Irrespective of what kind of model is used for the
description of growth and fermentation, this low yield implies that a
large part of the substrate is not conserved in biomass, but is obviously
spilled or used for maintenance functions.
The difference between substrate consumption for energy spilling
reactions and true maintenance reactions is often not clear and remains
of phylosophical interest.
For most technological applications, there is no need to distinguish
between these processes in a mathematical modelling exercise, and both
can be captured in the maintenance matabolism.
Examples of processes which are believed to con- tribute to the
maintenance metabolism are turnover of macromolecules, occurrence
of an ATPase activity and/or futile cycles, inefficient synthetic routes,
support of the membrane potential and chemical gradients over the
membrane, and motility of the organism. Some attempts have been made to
estimate the magnitude of the contribution of several processes to the
main- tenance metabolism.
The ATPase activity of crude homogenates of Z.
mobilis is 7.5 mmol phosphate/g dry wt h,59 which is equivalent to 1.3 g
glucose/g dry wt h. This activity is 2.5-4 times higher than the ATPase
activity of S.
faecalis,@- which can be calculated from the experi- mental data to
be 3-6.5 mmol P/g dry wt h. The yield of biomass on ATP under the
particular growth con- ditions was 3-6.5 g dry wt/mmol for 2. mobilis and
10.9 g dry wt/mmol for S.fae~a1is.I~ In intact organisms, the ATPase
activity will probably be suppressed by the existing ion and proton
gradients over the membrane
(see ref. 61). The turnover rate of RNA and protein is ca. 3-6/h under
starvation and substrate-limited the RNA degradation gro~th.~.~~ For
Z. anaerobia,
rate was 2/h on tarv vat ion.^' Assuming that protein and RNA account
for 90 of the biomass of 2. mobilis,

the turnover of these molecules would require 1 1.7 mmol ATP/h g dry
wt,I4 which is equivalent to 0.2- 0.3 g glucose/g dry wt h. Obviously,
the turnover rates are underestimated or other energy requiring processes
must occur.
Stouthamer and Bettenhausen@ concluded by com- paring YATp of wild type
and an ATPase negative that sustaining the membrane
E. coli (YATp = 8.5 g/mol) mutant (YATP = 17.6 g/mol), potential
requires half
JOBSES ET AL.:
of the energy produced by catabolism under anaerobic conditions.
Curiously, although the results are computed in view of the linear
equation for substrate consumption, which implies that membrane
energetization is expected to be growth rate independent and should be
reflected in the maintenance factor, the reported maintenance factors are
equal for both organisms (7 and 5 mmol/g h, respectively). Neysse16
observed an increase of the maintenance factor of 1 mmol glucose/g dry wt
h when the membrane potential of K1. aerogenes was depleted by addition
of an uncoupler under aerobic conditions.
This would be equivalent to a maximum of 6.5 g glu- cose/ dry wt h for
Z. mobilis.
From the foregoing, it is evident that the quantitative contribution of
several processes to the maintenance energy requirement of the intact and
normal functioning organisms is very difficult to estimate.
Anyway, since the model based on the linear equation for substrate
consumption is not valid for 2. mobilis
at least at 30C, it is also not valid to explain its growth behavior
in terms of m, and Y,. The two-compartment model offers a better
description of the growth behavior, which can be expected from a
mathematical point of view since the model is more complex.
Mathematically, it provides the same description of the q5 vs. growth
rate relationship as a second-order polynomial in D.
The two-compartment model offers, however, the possibility to be more
physiologically descriptive. In this model, the maintenance energy demand
is ex- pressed in the turnover of G-compartment. Equation
(11) shows that the maintenance energy demand is
partly growth rate independent, partly linearly growth rate dependent,
and partly nonlinearly growth rate dependent (0). As mentioned in the
Introduction, experimental results indicate that the maintenance substrate demand is partly growth rate dependent,I4.l5and in some cases also
partly in a nonlinear In this regard, the two-compartment model can be
pre- ferred over other approaches.
When the K-compartment is ascribed to the RNA (and carbohydrate) content
of the biomass, the model predicts the effect of growth rate and
temperature on the RNA content qualitatively well. Furthermore, a higher
turnover rate of the compartments at 35C com- pared to 30C is
predicted as can be expected for chemical reactions.
The K-compartment and its increase with growth rate is, however,
quantitatively different from the es- timated RNA contents. These
discrepancies may stem from the fact that the K turnover rate in the
model is linearly proportional to the concentration of K (and G)
compartment. It is quite possible that in reality the RNA turnover rate
is not only proportional to the RNA content but might be controlled by
growth-rate- dependent factor~.~~ This reasoning also applies to
other macromolecules and energy requiring compounds

or systems, whose content need not to be altered but whose turnover rate
can be a function of growth rate.
GROWTH/CONVERSION MODEL FOR Z. MOBILIS 993

Furthermore, other maintenance metabolisms such as for sustaining

membrane potentials, ion gradients, mo- tility, etc., in this model are
also represented in the K and G turnover, and will not be reflected in
ex- perimentally determined macromolecular contents or turnover rates.
Because of the complexicity of metabolism associated with growth and
maintenance of organisms, one can conclude that a simple, practically
useful mathematical model will never reflect quantitatively the real
microbial composition or state. Therefore, such a model should be tested
on its descriptive power of the features one is interested in, and on its
qualitative implications regarding other important properties.
The authors wish to thank Professor K. Ch. A. M. Luyben for discussing
the manuscript and Ir. B. Ramakers for providing the computer program for
the parameter estimation of the two-com- partment model.
NOMENCLATURE
concentration of compound i (g/L) flow (L/h) conversion rate of compound
i (CJh) generalized degree of reduction of compound i
(dimensionless).
yield factor of biomass on substrate (g/g)
yield factor of biomass on ATP (g/mol) yield factor of K-biomass on
substrate (g/g) yield factor of G-biomass on K-biomass (g/g) maintenance
factor based on substrate requirement (g/g h) maintenance factor based on
ATP requirement (mmol/g hr) dilution rate (h-I) specific growth rate
(h-') maximal specific growth rate (h-I) specific endogenous biomass
degradation rate (h- ')
specific substrate consumption rate (g/g h) Monod constant (g/L)
fraction of biomass (g/g)
fraction of biomass (g/g)
reaction rate constant of G-biomass formation (h-') reaction rate
constant of G-biomass degradation (h-')
Subscripts
s substrate
si influent substrate
x biomass
p product(s)
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