Chapter 15

TRUE AND APPARENT YIELDS AND MAINTENANCE

COEFFICIENT AND THEIR SIGNIFICANCE ON
FERMENTATION KINETICS

J. A. RODRIGUEZ-LEON*, G. BUENO, D. E. RODRIGUEZ, G.

DELGADO , P. SERRANO , M. A. BRIZUELA

Instituto Cubano de Investigaciones de Derivados de la Cana de Azucar

(ICIDCA). VIa Blanca 804 y Carretera Central. La Habana. Cuba; e-mnil:
rodleon@icidca.edu.cu

Abstract
Yield based on substrate (Yxis) or oxygen consumption (Y xlo) is a very important
parameter. This parameter indicates how efficient a fermentation iso At the same
time it is very c10sely related with the maintenance coefficient (m). By means of
yield and maintenance coefficient it is possible to estimate the proportion of energy
that cells consume in biomass and metabolites synthesis and the proportion of
energy that allows the cells to maintain their capability for their biological
performance. Unfortunately the maintenance coefficient is not normally considered
due to difficulties in its estimation and therefore the assessment yield could be
disturbed. A method for the estimation of the former parameters is proposed in this
work. The method was applied to a submerged fermentation with Lactobacillus
rhamnosus and compared with results previously obtained for solid state
fermentation of sugar cane and citrus residues with Candida utilis and Aspergillus
niger respectively.
Key words: Lactobacillus rhamnosus, fermentation, sugar consumption balance,
yield, maintenance coefficient.

Introduction
Carbon source or substrate in a heterotrophie fermentation process is the main basis
for energy and elements that growing cells require. At the same time the
measurement of substrate utilization is a very important index that characterizes the
development of the process. Therefore, yield based on substrate consumption is one
of the most common parameters that indicates how efficient the process iso
Yield based (Yxis) on substrate consumption is defined as:
Y xis = dXldS (EI)
Where: X: biomass concentration (g/l) ; S: substrate concentration (g/l)

163
New Horizons in Biotechnology, 163-172, S. Roussos et al. (eds)
2003 Kluwer Academic Publishers.
164 J.A. Rodriguez-Leon et al.

In Equation (EI) there are no distinctions concerning how the substrate is employed
and with what purpose. It is common to postulate that the substrate is consumed in
order to allow cell multiplication, often called "cell growth", and for cell
maintenance (1). A substrate consumption balance that considered the former
implications was therefore proposed as:
dS/dt = (INxis) dXldt + mX (E2)
Where: m: maintenance coefficient (g substrateig biomass h)
The maintenance coefficient represents the energy that the individual cell requires
to maintain its biological performance without considering the energy required for
cell duplication. It is defined through the concept of specific substrate consumption
as:
m = l/X ds/dt (E3)
In the case of an aerobic fermentation and when O2 consumption is considered,
Equation (E2) can be written as:
d0 2/dt = (INxl02) dXldt + mX (E4)
Where: Yxl02 : yield based on O2 consumption ; m: maintenance coefficient (g O2
consumed Ig biomass h)
Equation (E4) keeps the same considerations as in Equation (E2) or in other words,
it represents an O2 consumption balance considering whole cell performance. This
equation was resolved by numerical methods (2) in order to estimate the
synthesized biomass in a solid fermentation process. In this method Equation (E4)
was solved for a particular fermentation considering values of Yxl02 and m that were
estimated from former data available. At the same time, it was proposed that the
parameters inc1uded in the equation could be estimated by a trial and error
procedure (parameter optimization procedure) (3). Then Equation (E4) would
estimate not only the synthesized biomass but also the parameters corresponding to
a particular fermentation without the need of previous values that correspond to
other processes. This procedure was applied previously in several solid state
fermentations (4-6).
The former equations and their solutions and definitions enable the obtaining of a
more precise analysis of the meaning of some parameters that characterize the
development of a fermentation process.
The present work proposes a procedure to analyse a Lactobacillus rhamnosus
fermentation, growing in a submerge culture, to estimate the yield based on sugar
consumption and the maintenance coefficient when sugar cane molasses are
employed as carbon source.
Yields and Maintenance Coefficient on Fermentation 165

Materials and Methods

Strain.
The Lactobacillus rhamnosus strain employed belongs to ICIDCA Microorganism
Collection. This strain is maintained at 4°C in MRS broth (Difco Laboratories,
Detroit, Michigan). Additionally it is conserved as a lyophilized stock.

Fermentation and media.

MRS media was modified by substituting the glucose content by cane molasses for
a final content of 20 g/l. Yeast extract was employed as nitrogen source to a final
content of 2g/l of N.
Fermentations were carried in a 5 1 fermenter (Marubishi) with pR and temperature
control at 6.5 and 37°C respectively. Produced biomass was measured by optical
density calibrated with a lineal regression model.

Reduced sugar determinations.

The reduced sugars were determined by the modified Eynon-Lane procedure (7).

Results and Discussion

Results obtained during the fermentation of sugar cane molasses in a submerged
fermentation of Lactobacillus rhamnosus are reported in Table 1.
A lag phase of Ih and a log phase that lasted eight hours were deduced from
Table 1. A specific growth rate (11) of 0,239 h- l was obtained from the log phase.
The yield based on sugar consumption (Yxis) was calculated as 24.4%, considering
data corresponding to the log phase. Other authors reported this yield considering
the sugar consumed from the beginning of the fermentation until the end of the log
phase. In this case the yield was 24.21 % too. In this fermentation it can be seen that
there were no practical differences between both estimations. This may due to the
fact that the lag phase lasted only 1 hour and the accelerated growth phase was
practically non-existent. Considerations for the estimation of this overall yield must
also be kept in mind.
166 J.A. Rodriguez-Leon et al.

Table 1. Variation of sugar and biomass content during a fermentation of sugar cane molasses by
Lactobacillus rhamnosus at an initial concentration of 20 gll.
Time Sugar Biomass Sugar Consumed Sugar rate
(h) concentration dry matter consumed R,
(gIl) (gIl (gll) (gI1/h)
0 20.4 0.62 0 0
1 19.5 0.81 0.9 0.9
2 18.8 1.03 1.6 0.7
3 17.2 1.23 3.2 1.6
4 15.3 1.63 5.1 1.9
5 14.4 1.96 6 0.9
6 8.35 2.63 12.05 6.05
8 5.2 4.30 15.2 1.575
10 3.67 4.49 16.73 0.765
12 1.8 4.84 18.6 0.935

Based on sugar consumption balance when solving Equation (E4) with the data
reported in Table 1, the yield based on sugar consumption (Yxis) and the
maintenance coefficient (m) corresponding to this data could be estimated.
Equation (E2) was written as:
~ S=(I/Yxis) ~X+mX ~ t (ES)
Applying the solution proposed for Equation (ES) and substituting R o by Rb and
Y xlo by Y xis in the original equation (2), the following equation was obtained:
1 ( dR,
Xn = ( Yx 1,I'J.t ( - (-)t 0 0
dR')
+ (-)t 0 n +
'oIn-l(-)t
dR,
0'
) (a)
+ 1- - Xo - a I Xi /(1+ -a)
,on-I) (E6)
2 dt dt '01 dt 2 '01 2

Where for simplicity, it was considered that:

a = mY xlsAt (E7)
In order to solve Equation (E6) an initial biomass concentration of 0.62 g/l and a
final concentration of 4.84 and the rate for sugar consumed at different times, were
considered. The values for the parameters estimated by the trial and error procedure
(parameter optimization) were obtained so that the final concentration predicted by
the Equation (E6) was approximately the same as in the original data. The results
achieved for the parameters were: Yxis = 0.239 and m = 0.0945 g substrate/g
biomass/h. The predicted biomass value for the fermentation with Lactobacillus
rhamnosus at each time was obtained by applying the values determined for Yxis
and m to Equation (E6).
Biomass variation from the data in Table 1 and from the values estimated from the
Equation (E6) was reported. As observed, there was very good correlation between
real and predicted values. The differences between the values were less than 8%
except for two points.
Yields and Maintenance Coefficient on Fermentation 167

There was also good similarity, when comparing the yield based on sugar
consumption (24.21%) taken from the data in Table 1 and the value estimated
(23.9%) from the Equation (E6). One must take into account the values obtained for
Yxis when solving Equation (E6).
From the final values obtained it was possible to estimate sugar quantity employed
in cell duplication or growth and the sugar employed for maintenance. However it
seemed necessary to think about the feasibility of considering the values of Y xis and
m as constant during the fermentation particularly when the evaluation for the
whole balance was made considering the data reported in Table 1. It would
therefore be useful to consider the balance of sugar consumed for each growth
phase.

The lag phase balance

The lag phase is characterized by the fact that there is no cell duplication or growth.
That means that the specific growth rate ([1.) was zero and therefore it was feasible
to assurne that Ll X = O. Then, the expression for Equation (E5) that corresponded to
this phase was reduced to:
m = Ll S/X Ll t (E8)
The maintenance coefficient corresponding to the lag phase as 1.452g sugar/g
biomasslh was estimated from Equation (E8). Comparing this value with the value
obtained in Equation (E6), it was not possible to consider the last (O.0945g
substrate/g biomasslh) constant during the whole process. In this case the increase
of the maintenance coefficient was in the order of 15. At the same time, the yield
based on sugar consumption was zero. This fact indicated that parameter validity
must always be checked very closely during the different stages ofthe process.
After these considerations it seemed obvious that it was very important to establish
fermentation conditions that avoid long lag phases or at least the shortest lag phase
could be obtained. This fact may seem trivial but could result in a loss of material,
energy and time in any fermentation process.

The log or exponential phase balance

This phase is characterized by a constant specific growth rate.
Taking into account the definition of growth rate:
[1. = (l/X) dX/dt (E9)
and from Equations (E2) and (E9) and rearranging, the following was obtained:
dS = (INxls+m1 [1.) dx (ElO)
Equation (ElO) is very useful when considering the log phase because it represents
the complete relation between sugar consumption and biomass production and
maintenance in a very simple manner. Expressing this equation as finite
differentials was achieved by:
Ll S = (IN xls+m1 [1.) Ll X (Ell)
168 J .A. Rodriguez-Leon et af.

The value for sugar consumption (~ S) through Equation (EIl) was estimated
considering Yxls=0.239, m=0.0945 g substrate Ig biomass I h, !l=0.238 hol and
~X = 3.03 g. The predicted consumed substrate by Equation (EIl) during the log
phase was 13.9 g. From Table 1 data, the value obtained for sugar consumption in
the log phase was 14.3 g.
The proportion of sugar that was employed in growth and maintenance could be
calculated forthwith considering the fIrst and the second term of the right side in
Equation (EIl). They were 14.6 and 1.4 g respectively, which meant that 8,7% of
sugar consumption was employed in maintenance.
When considering the proportion of sugar employed for growth and maintenance
one should not forget that in the present case the fermentation with Lactobacillus
rhamnosus was a homolactic fermentation in which lactic acid was produced as
result of a main metabolie pathway characteristic of this strain (8). The lactic acid
produced was associated with growth such that for each synthesized cell an exact
quantity of acid was produced. That implied that sugar related to the acid synthesis
was already considered during microbial growth. This was why, in this process, the
yield based on sugar consumption was lower when compared to those processes in
which only produced biomass and CO 2 were present. Meanwhile, taking into
account these considerations it was necessary to report the yield of lactic acid
produced on basis of biomass synthesized and not on yield based on sugar
consumed.
If considered in Equation (E5), the proportion of energy or substrate employed for
lactic acid synthesized could be re-written as:
~ S = (11Y xis) ~ X + mX ~ t + CI> (EI 2)
Where <I> is the fraction of sugar consumed for a particular metabolite synthesis as g
substrate consumed.
DefIning the specifIc metabolite production as:
CI> = (l/X) MIM (E13)
Where: CI> : g metaboliteig biomass h ; ~p : grams of the particular metabolite
produced in the time interval analyzed.
Considering the yield ofproduct based on sugar consumption (Yp/s ) as:
Y pis =~P/~S (E14)
Equation (EI2) could be written as:
~S = (11Y xis) ~ + (m + Cl>/Ypls)X ~t (EI5)
The term (m + Cl>/Yp/s ) in Equation (EI5) is a constant that considers not only the
maintenance coeffIcient, but the specifIc metabolite production too. So it can be
postulated that:
m I = m + Cl>lY p/s (E16)
As a matter of fact, the previous value obtained for m (0.0945g substrate/g
biomasslh) was actually an estimation of the value of mI .
Yields and Maintenance Coefficient on Fermentation 169

From Equation (E13) and the definition for the maintenance coefficient in Equation
(E3), the relation between m and <I> was obtained as:
<l>/m = Yp/s (EI7)
Then Equation (EI6) was reduced to:
m'=2m (EI8)
Taking into account the value obtained for m' as the value for m reported
previously, the real value for m could be estimated as O.0473g substrateig
biomass Ih if lactic acid produced is taken into account.
The value for specific metabolite production of lactic acid was already obtained
from Equation (EI7) taking into account the value of Yp/s ' Unfortunately the
production of lactic acid was not reported in the data analyzed.
The method here proposed could be generalized. If more metabolites are
considered, the Equation (EI2) could be written as:
AS=(1/Y xis) AX+mXAt+<I>, +<1>2 + ..... <1>0 (E19)
Specific metabolite production for the different metabolites considered were
defined as:
<1>0 = (I/X) Al' o/At (E20)
Then Equation (EI5) was written as:
AS = (I/Yxis) AX + (m +<1>1 /Y pUs +<1>2 /Yp2/s + ... <1>0 /Ypnls) XAt
(E2I)
The particular relation between specific metabolite production of each metabolite
with the maintenance coefficient was already obtained as:
<l>ofm = Ypnls (E22)
Then Equation (EI8) could be postulated as:
m'= (n+I) m (E23)
where n is the number of metabolites considered in the process. It must be kept in
mind that m' was calculated as a first approximation considering only the biomass
produced.
From the results achieved with Equation (E23) it could be postulated that the
maintenance coefficient was an index not only of the substrate consumed for
maintenance of individual cells and the metabolites synthesis but a measure of the
metabolites that are synthesized and need to be considered. However, if production
of any metabolite were partially associated with growth, then the maintenance
coefficient and the different yields may be no longer constant during the log phase
and should be considered.

The negative accelerated growth phase balance

Negative accelerated growth phase occurs immediately after the log or exponential
phase. In this phase the specific growth rate is lower than the specific growth rate
attained at the log phase and changes with time. Although this is a phase in which
170 J.A. Rodriguez-Leon et al.

specific growth is not a constant, it may be assumed that the yield based on sugar
consumption and the maintenance coefficient are not constant either. However,
Equation E6 was employed to give a first estimate for these values considering a
period time between 8 and 12 h. The values obtained are reported in Table 2.
The values for X predicted by Equation (6) for 8-12 interval time considering the
corresponding values of Yxis and m the values differ by less than 1% in relation to
the biomass reported. An overall specific growth rate (constant) for this phase was
estimated as 0.026 h- 1 taking these values as reference.

Table 2. Values estimated trough Equation 6 for different time intervals.

Time interval Y>.1s maintenance coefficient : m
(h) (g sugar/g biomasslh)
2-8 0.239 0.0945
8-12 0.207 0.0714

To determine how far this assumption was from reality, an estimation of the
specific growth rate employing Equation (E9) was made for specific time intervals
namely 8-10 and 10-12 h using data reported in Table 1. The values obtained for
both intervals were 0.022 and 0.036 h- 1 respectively. These values indicate a
variation that seemed to be under the influence of the measurement employed. A
smaller time interval should therefore be considered. However, the dec1ine in the
specific growth phase which is characteristic of this phase, was obvious when
compared with values obtained from the specific growth rate at log or exponential
phase.
When considering this phase it was very important to determine whether other
metabolites not associated with growth were synthesized because this phase was
c10sely related with secondary metabolites. This is not the case of the present
fermentation.
The values of the maintenance coefficient for Lactobacillus rhamnosus and the
values obtained in solid state fermentation of Candida utitis grown in a molasses
medium and Aspergillus niger growing on citrus waste (9) were compared. The
values of the maintenance coefficient reported for Candida utitis and Aspergillus
niger were corrected because the original units for maintenance coefficient were
g oxygen/g biomasslh. Taking into account that for each mole of glucose (180g)
consumed in an aerobic fermentation 6 moles of O2 (l92g) are necessary, the
correction factor employed was 0.94.
In Table 3 the values obtained in the case of Candida utilis and Aspergillus niger
are reported. A two time interval was considered to determine whether the
maintenance coefficient could be considered constant during the whole process. In
the case of Aspergillus niger such an assumption is not valid. This was attributed to
the fact that inducible enzymes (pectinases and cellulases) were synthesized during
consumption of the initial sugar content. When comparing the value obtained for
Lactobacillus rhamnosus with the highest values reported for Aspergillus niger and
Yields and Maintenance Coefficient on Fermentation 171

for Candida utilis, an increase of 350% was observed, indicating, according to the
procedure here analyzed, that production of metabolites was important, an element
that was not taken into consideration when the data was evaluated.

Table 3. Comparison of maintenance coefficient (g substrate/g of Biomass/h) values obtained for

different fermentations.
Microorganism Fermentation type Substrate Maintenance
coefficient (m)
Lactobacillus rhamnosus Submerge molasses 0.0945
Candida utitis Solid citric residues 0.0202*
0.0245*
Aspergillus niger Solid citric residues 0.0064*
0.0181 *
*values corrected and estimated at two different time interval

6,00
:..:
5,00
4,00
3,00
2,00
1,00
0,00
0 5 10 15
t(h)

Figure 1. Comparison between the biomass measured during the fermentation of sugar cane molasses at
20 g!l with astrain of Lactobacillus rhamnosus and the biomass predicted by Equation (E6) with the
values of Y,J, =0.239 and m =0.0945 g substrate/g biomass/h.

Conclusions
In the case of sugar cane molasses fermentation with Lactobacillus rhamnosus, the
value obtained for the maintenance coefficient indicated that around of 10% of the
sugar consumed during the process was employed in cell maintenance and
metabolites synthesis.
The procedure hereby employed confirmed that the fermentation with Lactobacillus
rhamnosus was not as efficient in relation to biomass synthesis. In the latter case
there was a five-fold increase of the value for the maintenance coefficient, as
compared to the other processes considered.
172 J .A. Rodriguez-Leon et af.

The value obtained for m when considering the production of lactic acid was still
high indicating that other metabolites must be considered, referring to the
possibility that the fermentation was heterolactic.
The procedure here reported allowed the prediction of the maintenance coefficient
and gave more accurate calculations for the yield based on sugar consumption and
specific metabolite production.

Acknowledgements
The present work was partially financed by the National Technical and Scientific
Program from CITMA, Agriculture Branch. Projects NO.0.03.00.004 and 0.08.0070

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