Biochemical Engineering Journal 91 (2014) 16–22

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Biochemical Engineering Journal

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Improved capsular polysaccharide production by Streptococcus

pneumoniae serotype 14 using continuous cultivation
Verônica Maria Rodege Gogola-Kolling, Rafaela Taís Zanardo, Talita Souza Carmo, Natália
Dalfré Zampoli, Douglas Borges Figueiredo, Viviane Maimoni Gonc¸ alves ∗
Laboratório de Bioprocessos, Centro de Biotecnologia, Instituto Butantan, Av. Vital Brasil 1500, 05503-900 São Paulo, SP, Brazil

article info abstract

Article history: The capsular polysaccharide (PS) is the most important pneumococcal virulence factor and is currently used as antigen in all
Received 28 January 2014 pneumococcal vaccines. Despite its physiological and epidemiological importance, meager studies have been devoted to
Received in revised form 27 June 2014 improve PS production and understand its relationship with pneu-mococcal central metabolism. In this study, kinetics of
Accepted 9 July 2014 Available online 17 growth and production of PS by Streptococcus pneumoniae serotype 14 (PS14) in batch and continuous cultivation were
July 2014 investigated. Strong cell lysis was observed in batch cultivation, while accumulation of organic acids and autolysis was
avoided in continuous cultivation. In the continuous cultivation was possible to achieve higher concentration of biomass and
Keywords: PS14. Calculation of kinetic parameters demonstrated that PS14 is a cell-associated prod-uct. The coefficients for growth-
Pneumococcus associated stoichiometric true yield and maintenance were determined as 0.25 gglucose gbiomass −1 and 1.24 gglucose (gbiomass
Continuous culture h)−1 , respectively. The maximum productivity of PS14 released in the supernatant (PS14 R ) and cell-bound PS14 (PS14C )
Batch processing
Production kinetics were obtained at a dilution rate of 0.8 h−1 , respectively, 85 and 122 mg (gbiomass h)−1 . Compared to batch fermentation,
Microbial growth both PS14R and PS14C productivities were increased by about 300% in the continuous process. These findings demon-strate
Kinetic parameters that continuous cultivation is a promising strategy for PS production to be used in pneumococcal vaccines.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction America and worldwide and presents high level of antibiotic resis-tance [2].

The Streptococcus pneumoniae is a Gram-positive microorgan-ism that
colonizes the human respiratory tract. This pathogen can cause several
diseases with high incidence on children mortal-ity, as pneumonia,
bacteremia, meningitis and sepsis. The capsular polysaccharide (PS) is the
major virulence factor in pneumococ-cal diseases and is used as antigen in all
available pneumococcal vaccines.
The PS consists of a high molecular weight polymer of repeated
oligosaccharides and chemically differences in its structure differ-entiate the
pneumococci in 93 immunologically distinct serotypes [1]. The most frequent
serotypes in invasive diseases are selected for commercial vaccine
formulations. In Brazil, the serotype 14 is responsible for 39.8% of
pneumococcal disease in 0–6-year-old children [2]. This serotype is also the
most prevalent in Latin
In order to be effective in children immunization, polysaccha-rides have to
be covalently linked to carrier proteins, a process that results in conjugate
vaccines. Existing pneumococcal conju-gate vaccines are being produced with
proteins not related to the pneumococcal disease and their high price makes
them inaccessi-ble to the majority of the world population. In addition,
serotype replacement has been observed in all countries that introduced these
vaccines in their immunization programs [3]. Therefore, our research center is
developing a novel pneumococcal conjugate vaccine that includes PS of the
three most prevalent serotypes in Brazil conjugated to pneumococcal proteins.
This strategy has several advantages as reduced price due to lower number of
com-pounds, enlarged coverage and diminished serotype replacement
attributable to the protection given by the pneumococcal protein moiety [4–7].

The S. pneumoniae is an anaerobic aerotolerant and nutritionally

fastidious microorganism. This bacterium obtains energy strictly via
∗ fermentation and is incapable of respiratory metabolism. The nutrients from
Corresponding author. Tel.: +55 11 2627 9821.
E-mail address: viviane.goncalves@butantan.gov.br (V.M. Gonc¸ alves). which the pneumococci normally obtain energy

http://dx.doi.org/10.1016/j.bej.2014.07.007
1369-703X/© 2014 Elsevier B.V. All rights reserved.
 V.M.R. Gogola-Kolling et al. / Biochemical Engineering Journal 91 (2014) 16–22
are carbohydrates which are oxidized to pyruvate via glycol-ysis [8]. The
main end-product of glycolysis is lactate, which inhibits the microorganism
+ Chemical
productivity by catabolic repres-sion. Depending on the ratio NAD /NADH,
pneumococcus, as other lactic acid bacteria, can also metabolize
carbohydrates to mixed-acid fermentation, producing acetate and formate [9].
The pneumococcal capsule synthesis requires the produc-tion of
nucleotide sugar precursors that will compose the oligosaccharide repeating
units. The capsular polysaccharide from serotype 14 (PS14) is made of
tetrasaccharide repeating units of →6)- -d-GlcpNAc-(1 → 3)- -d-Galp-(1 →
4)- -d-Glcp-(1→, with monosaccharide side-chains of a -d-Galp(1→ linked to
C4 of each N-acetylglucosamine residue [10]. Once synthesized, each
nucleotide activated monosaccharide is sequentially linked to a lipid carrier to
form the repeat unit. Except for serotypes 3 and 37, which are synthesized
without a lipid carrier, the repeat unit is transferred to the outer face of the
cytoplasmic membrane by a flippase, polymerized to form the mature PS, and
then attached to the peptidoglycan. The regulation of PS synthesis depends on
sev-eral factors, as activity of specific regulatory genes, production of
precursors and availability of lipid carrier [11].
Besides the capsule, pneumococci have a variety of factors that contribute
to virulence, including surface proteins and hydrolytic enzymes. These
enzymes could act directly on host cells or generate products from the
pneumococcal cell wall degrada-tion that mediate inflammation and toxicity.
LytA, the major pneumococcal autolysin, is an amidase that hydrolyses the
pneu-mococcal cell wall. It can be triggered to cause lysis of the bacteria in
stationary growth phase or upon antibiotic treatment [12].
Little attention has been given to the culture conditions of S. pneumoniae.
Although the PS is a critical cell surface virulence fac-tor, to our knowledge,
there are no data on kinetics of PS production, which will delineate the
strategy to improve its production for vaccine formulation. In addition, it is
very rare to found articles reporting yield and maintenance coefficients in
pneumococci cul-tivation. Few reports can be found in the literature related to
the medium study and culture conditions to investigate and optimize the
capsular polysaccharide production. Most of them have been conducted in
flasks, which do not reproduce large-scale conditions. Cultivation conditions
of bacterial growth in flasks have been inves-tigated for polysaccharide
production of serotype 1 [13], 4 [14] and 14 [15]. A study of the influence of
pH control and glucose addition in reactor batch cultures has been conducted
for serotype 14 [16] and a medium improvement for capsular polysaccharide
produc-tion in reactor has been reported only for serotype 23F [17], and this
medium was applied also for PS production by serotype 6B [18].
In our laboratory, we have experienced the influence of inocu-lum
conditions and the strong activity of autolysins in pneumococci cultivation
that could determine the success or failure of batch and fed-batch cultures.
These cultivation modes have dynamic physico-chemical conditions, resulting
in changes of metabolic and growth parameters that affect the composition of
the cell, producing complex patterns of data that are often difficult to
interpret. In contrast, continuous cultivation allows the cell pop-ulation to
grow in a defined, ideally constant, and with controllable physico-chemical
conditions, generating meaningful information [19]. Using continuous
cultivation, a novel strategy to study pneu-mococcal metabolism, we are able
to minimize the influences of inoculum and autolysins, producing therefore
consistent results. By this system we attempted to determine kinetic
parameters that influence growth and PS production of S. pneumoniae
serotype 14, besides to improve the production of pneumococcal capsular PS.
17
Table 1
Medium composition for cultivation of S. pneumoniae serotype 14 [17].
Amount per liter
Acid-hydrolyzed casein (Bacto, BD) 30 g
Glucose 20 g
Ultrafiltered yeast extracta (Difco, BD) 20 g
K2 HPO4 5g
NaHCO3 1g
l-glutamine 0.625 g
Asparagine 0.1 g
Choline 0.01 g
Salts solutionb 2 mL
10% Thioglycolic acid 1 mL
a 5000 Da cut-off.
b
Salts solution (per liter): 250 g MgSO4 ·7H2 O; 2.5 g FeSO4 ·7H2 O; 0.4 g ZnSO4 ·7H2 O;
0.18 g MnSO4 ·H2 O; 10 mL HCl.
2. Materials and methods
2.1. Bacterial strain and maintenance
Streptococcus pneumoniae serotype 14 strain 5287 isolated by SIREVA
[20], identified by the National Centre for Streptococcus (Edmonton, Alberta,
Canada) and deposited at Nucleo de Colecao de Microrganismos, Instituto
Adolfo Lutz, was used in all experiments. The lyophilized strain was
suspended in 0.85% NaCl, transferred to blood-agar tubes and incubated at 37
◦ C in ∼3% CO atmosphere. After 48 h, the culture was suspended,
2
transferred to 50 mL of THY medium – Todd-Hewitt medium (Bacto, BD)
supplemented with 0.5% yeast extract (Difco, BD) – and incubated, until an
optical den-
sity at 600 nm (OD600 ) of 1.0 was reached. Finally, the culture was
centrifuged and the pellet was suspended in 1/10 of the original
volume with fresh THY medium containing 30% glycerol. The stock was
maintained in liquid N2 .
2.2. Growth systems and culture media
The stock culture was subcultured in 250 mL flasks containing complex
medium (Table 1), sterilized by filtration through a 0.22-m-pore-size
membrane filter. The incubation was conducted at 37 ◦ C in ∼3% CO2
atmosphere and under static cultivation, in order to obtain an OD600 of
approximately 1.0. In all cultivations, the
inoculum was used as seed to get an initial OD600 of 0.1 in the reactor
medium.
Batch and continuous fermentations were carried out in a 2-L bioreactor
®
(BioFlo3000 , New Brunswick, Edison, New Jersey, USA) with a working
volume of 1.5 L. Reactor cultivations were conducted under nitrogen
atmosphere (0.1 vvm), at 36 ◦ C and the pH was controlled at 7.0 by addition
of 5 M NaOH. The mixing time was maintained fewer than 5 s using a
mechanically driven impeller (100 rpm). At various intervals of time during
cultivation, 2-mL samples were removed and the cells were centrifuged at
20,000 × g, 15 min, 4 ◦ C. Both pellets and supernatants were frozen at −20 ◦
C. The pellets were submitted to extraction of cell-bound PS14 (PS14C ) and
the supernatants were analyzed for glucose, organic acids and released PS14
(PS14R ).
In continuous cultivation, the cells were grown in batch culture
up to an OD600 of 2.0 before the start of continuous operation. The volume
was maintained constant in 1 L by a weir. Medium inflow
was performed by using a peristaltic pump, with flux controlled by LabView
(National Instruments, Austin, Texas, USA), which pro-moted the adjustment
of the required dilution rate (D). After at least four residence times, three or
more independent samples were harvested at different times and the steady
state condition was confirmed by determination of standard deviation of
OD600 values, which should be inferior to 5%. Culture purity was con-firmed
by characteristic colony morphology and alpha-hemolysis
18 V.M.R. Gogola-Kolling et al. / Biochemical Engineering Journal 91 (2014) 16–22

of cells grown overnight at 37 ◦ C on plates of blood agar media Table 2

Calculation of kinetic parameters.
supplemented with brain heart infusion (Difco, BD).
Parameter Batcha Continuousb
2.3. Analytical procedures Growth yield coefficient based (X − X0 ) / (S0 − S) X/ Sf − S
on glucose consumption
Bacterial growth was monitored by OD600 and calibrated against dry cell (YX/S ) (g g−1 )
weight measurements. A volume of 200 mL culture in exponential growth PS14R yield based on glucose (PSR − PSR0 ) / (S0 − S) PSR / Sf − S
was inactivated with 0.74% formaldehyde overnight. The cells were consumption (YPSR /S )
(mg g−1 )
centrifuged at 12,857 × g, 4 ◦ C, 30 min and washed with 0.9% NaCl. The
PS14C yield based on glucose (PSC − PSC0 ) / (S0 − S) PSC / Sf − S
pellet was washed and resuspended consumption (YPSC /S )
with 0.9% NaCl in order cover an OD600 range of 0.1 to 10. The tubes (10 (mg g−1 )
mL) with different values of OD600 were centrifuged at PS14R yield based on cell (PSR − PSR0 ) / (X − X0 ) PSR /X
growth (YPSR /X ) (mg g−1 )
20,000 × g, 15 min, 4 ◦ C and dried at 60 ◦ C to constant weight to measure (PSC − PSC0 ) / (X − X0 )
PS14C yield based on cell PSC /X
biomass concentration. By linearization, a change of 1.0 U
growth (YPSC /X ) (mg g−1 )
of OD600 was shown to be equivalent to 0.366 g of dry mass per liter. Total PS concentration (PSt ) PSC + PSR
(mg L−1 )
For organic acid and residual glucose analyses in sample super-natants, an Overall PS14 yield based on (PSt − PSt0 ) / (S0 − S) PSt / Sf − S
Aminex HPX-87H column (Bio-Rad, Hercules, California, USA) in HPLC glucose consumption (YPS/S )
(mg g−1 )
system was used. The column was maintained at 60 ◦ C, and the compounds
PS14R productivity (QPSR ) (PSR − PSR0 ) / (t − t0 ) PSR × D
were eluted with 5 mM H2 SO4 at a flow rate of 0.6 mL min− . Glucose was
1
(mg L−1 h−1 )
detected by refractive index and organic acids by UV absorbance at 210 nm. PS14C productivity (QPSC ) (PSC − PSC0 ) / (t − t0 ) PSC × D
Peaks were identified and quantified by comparison to the retention times of (mg L −1 −1
h )
authentic standards. a
Subscribed 0 indicates the beginning of the fermentation.
b Subscribed f indicates the concentration in the feeding medium and all other are the
The protocol proposed by Bender et al. [21] was used to extract cell- concentration in the steady-state. X is the biomass concentration (g L−1 ); S is the glucose
bound PS14. The protocol consists in the incubation of cell pel-lets with concentration (g L−1 ); PSR is the PS14 concentration released in the supernatant (mg L−1 ) and
lysozyme in an adequate buffer. The lysozyme hydrolyzes the peptidoglycan PSC is the cell-bound PS14 concentration (mg L−1 ); PSt is the total polysaccharide
releasing the PS cell-bound to the buffer phase, separated from cells by concentration (mg L−1 ) and D is the dilution rate (h−1 ).

centrifugation. The concentration of both PS14 C and PS14R was determined

by Sandwich ELISA [22]. culture, assuming that 1 mol of ATP is produced per mol of lactate formed
and 2 mol of ATP per mol of acetate:
2.4. Determination of kinetic parameters ATP

In batch cultivation, the maximum specific growth rate ( max ) was Y =

ATP/S Sf − S (3)
determined by the slope in a regression analysis of the linear portion of the The equations used to determine other kinetic parameters in batch and
logarithmic growth curve. In continuous cultivation, the max was calculated continuous cultivation are displayed on Table 2.
from a washout experiment, when the
dilution rate was increased to 1.5 h− and the OD600 was followed for 2 h.
1
2.5. Statistics
The slope of the logarithmic curve of biomass decrease is
equivalent to max minus D. One-way analysis of variance (ANOVA) for independent samples and the
The model proposed by Luedeking and Piret [23] was applied to
Tukey Test were used to compare parameters and values obtained at different
determine the product kinetics (Eq. (1)) in continuous cultivation, where the
D in continuous cultivation. The Student t-test was applied to compare batch
rate of product synthesis ( p ) is a function of growth rate alone ( ), or and continuous cultivation.
bacterial density alone or a function of both bacterial density and growth rate
together.
3. Results

p = ˛ ×+ ˇ (1)
3.1. Batch fermentation
The constants ˛ and ˇ are determined from the plots of p
against , equivalent to D in steady state of continuous cultures, The time course of batch fermentation of S. pneumoniae serotype
where ˛ is equal to the slope of the straight line and ˇ is equal to 14 in complex media is shown in Fig. 1. The exponential growth
the p intercept. If ˛ = 0, the product kinetics will be non-growth phase was between 0 and 2.5 h of cultivation, giving a max of
1.20 h− . The maximum cell concentration (1.61 0.08 g L− ) was
=
1 1 ±

associated; if ˇ = 0, it will be growth associated and if ˛, ˇ / 0,

partially growth associated.
In the maintenance model proposed by Pirt [24], the specific
substrate uptake rate ( s ) is expressed as linear function of the
specific growth rate, equivalent to D in steady state of continuous
cultures, by attributing the substrate consumption to a growth-
associated coefficient (YG ) and a non-growth-associated parameter
(ms ), which represents the substrate uptake for maintenance (Eq.
(2)).
1 period. On the other hand, the production of PS14 R seems to be
s = YG ×+ ms
The ATP yield based on glucose consumption was calculated
according to the following equation in steady state of continuous
reached in 3.5 h (Fig. 1). After this point, the cell lysis began and the
cell concentration was reduced more than 67% in the next 2 h. More
than 80% of initial glucose was consumed at the end of cultivation,
mostly directed to lactic, formic and acetic acid production, which
reached, respectively, 16.7, 1.1 and 3.0 g L− after 6 h of cultivation.
1
The profile of PS14C production was clearly growth-associated.
At the point where autolysis began to the next 2 h, the PS14 C con-
centration decreased in about 74%, close to cell decay in the same
(2) partially growth associated, since the PS14R concentration rapidly
increased when the cell growth ceased. However, this increase
should be represented mostly by the release of PS14 C from the
cell. Once the enzyme autolysin hydrolyses the peptidoglycan, it
 V.M.R. Gogola-Kolling et al. / Biochemical Engineering Journal 91 (2014) 16–22 19

Fig. 1. Biomass ( ), PS14R ( ), PS14C ( ), glucose ( ) concentration profiles from batch

cultivation of S. pneumoniae serotype 14 strain 5287. Average and standard deviation of three
independent experiments.

is expected that once this activity is triggered the cell-bound PS14 is released
to medium cultivation.

3.2. Continuous culture

A continuous steady-state culture with dilution rate ranging from D = 0.4

to 0.9 h− was used to determine the growth param-eters of S. pneumoniae
1
serotype 14 strain 5287 (Figs. 2 and 3 and Table 3).

Fig. 3. Rates of (A) PS14R and PS14C formation, (B) lactate formation and (C) glucose
consumption during continuous culture of S. pneumoniae serotype 14 strain 5287. The error bars
represent the standard deviation of at least 3 samples per point taken at different times after
reaching the steady state.

Among the D tested, the highest productivity was obtained at D = 0.8 h−

1

for all products and biomass (Fig. 2B). At dilution rate of 0.9 h− , the
1
consumption of glucose dimin-ished considerably (Table 3) together with a
decrease in the biomass concentration, clearly showing that growth at this
dilution rate was close to the washout condition (Fig. 2A). At D = 1.5 h− ,
1

max was determined by the dynamic method. The max obtained in continuous
cultivation (1.23 h− ) was in conformity with that found in batch cultivation
1

(1.20 h− ). The statistical analysis showed that the biomass and PS14 R
1

production at D = 0.8 h− was significantly higher (p < 0.01) than the other
1
Fig. 2. Concentration (A) and productivity (B) profiles of biomass (no lines), PS14R (horizontal
two D, while the biomass at D = 0.4 h − was not dif-ferent from that at D =
lines), PS14C (diagonal lines) as a function of the dilution rate in contin-uous culture of S.
1

0.9 h− . The same was observed for PS14C production (p < 0.05). The
pneumoniae serotype 14 strain 5287. The error bars represent the standard deviation of at least 3 1
samples taken at different times after reaching the steady state. * p < 0.05 and ** p < 0.01
against the other D (Tukey Test).
biomass productivity was significantly
20 V.M.R. Gogola-Kolling et al. / Biochemical Engineering Journal 91 (2014) 16–22

Table 3 Table 4
Steady-state values and calculated parameters from continuous culture of S. pneu-moniae Comparison of the maximum PS14C and PS14R concentration and kinetic parameters for batch
serotype 14 strain 5287. Average and standard deviation of at least 3 samples of the steady- and continuous cultivation of S. pneumoniae serotype 14 strain 5287.
state.
Parameter Batcha Continuousb
Parameter Dilution rate (h−1 ) Biomass (g L −1
) 1.61± 0.08 2.77 ± 0.09*
Y (g g −1 )
0.4 0.8 0.9 x/s biomass glucose 0.19± 0.02 0.21 ± 0.01
YPS14R /s (mgPS14R gglucose −1 ) 7.1± 1.1 8.2 ± 0.7
Residual glucose (g L−1 ) 6.40 ± 0.68 7.02 ± 0.73 11.68 ± 1.20a
YPS14C /s (mgPS14C gglucose −1 ) 12 ± 1 12 ± 2
Lactate (g L−1 ) 13.03 ± 0.85 15.42 ± 0.91 11.08 ± 0.98b
Overall PS14 yield (mg gglucose −1 ) 19 ± 1 21 ± 2
Acetate (g L−1 ) 0.00 ± 0.00 1.31 ± 0.64a 0.12 ± 0.02
YPS14R /x (mgPS14R gbiomass −1 ) 38 ± 7 38 ± 3
Formate (g L−1 ) 3.01 ± 0.12a 1.17 ± 0.35c 0.45 ± 0.04 −1 65 ± 4 59 ± 8
Y (g g −1 )
Y
0.17 ± 0.01 0.21 ± 0.01 0.28 ± 0.04d,e
PS14R (mg L−1 ) 106 ± 7*
x/s biomass glucose
61± 12
YPS14R /s (mgPS14R gglucose −1 ) 5.76 ± 0.37a 8.21 ± 0.67a 10.9 ± 1.22a PS14C (mg L−1 ) 105 ± 1 163± 22*
YPS14C /s (mgPS14C gglucose −1 ) 8.45 ± 1.24c 12.6 ± 1.76 13.9 ± 3.55 85± 5*
PS14R productivity (mg L−1 h−1 ) 17 ± 3
YATP/s (molATP molglucose −1 ) 1.54 ± 0.06f 2.40 ± 0.28 2.35 ± 0.42
68.0 ± 3.4 64.9 ± 3.6 39.6 ± 5.5a PS14C productivity (mg L−1 h−1 ) 29 ± 0 130± 18
*
% Glucose consumption
YPS14R /x (mgPS14R gbiomass −1 ) 33.7 ± 1.02 38.4 ± 2.56 39.0 ± 3.42 a
Period considered: 0 to 3.5 h of cultivation.
YPS14C /x (mgPS14C gbiomass −1 ) 49.3 ± 5.19 58.8 ± 7.93 49.1 ± 5.23 b D = 0.8 h−1 ; average of 5 samples taken at different times after reaching the steady-state.

Statistical significance (Tukey Test).

a p < 0.01 compared to the other two samples. b p < Statistical significance (t-test): * p
< 0.01.
0.01 compared to D = 0.8 h−1 .
c
p < 0.05 compared to D = 0.9 h−1 . d
p < 0.01 compared to D = 0.4 h−1 . e p
3.3. Comparison between batch and continuous cultivation
< 0.05 compared to D = 0.8 h−1 .
f
p < 0.05 compared to the other two samples.

A comparison of PS14R and PS14C productivities and yields between

batch and continuous cultures of S. pneumoniae serotype 14 strain 5287 in
different for all three D evaluated with p < 0.01. The PS14 C produc-tivity was complex medium is illustrated on Table 4. Although 1.7-fold higher biomass
significantly lower at D = 0.4 h− (p < 0.01) than the other two D, and at D =
1
and PS14R concentration and 1.5-fold higher PS14C concentration were
0.8 h− it was significantly higher when compared to D = 0.9 h − (p < 0.05).
1 1
reached in continuous cultivation at D = 0.8 h − (p < 0.01, t-test), the yield
1

Finally, when comparing the PS14R pro-ductivity, it was observed that it was coefficients did not differ, as they were calculated before the beginning of cell
also significantly lower at D = 0.4 h−
1 lysis in batch. Differently, the maximum productivity for both PS14R and
(p < 0.01) and no statistically
significant difference was found between D = 0.8 h− and 0.9 h− .
1 1
PS14C was increased by approximately 300% in continuous com-pared to
The residual glucose was significantly higher at D = 0.9 h− than at the
1 batch culture, which could be a consequence of the highest concentration of
biomass reached in the continuous cultivation, as the PS14 is a growth-
other D, as a consequence the glucose consumption was lower (p < 0.01 in
associated product, together with the effect of the steady state conditions on
both, Table 3). The lactate production was lower at D = 0.9 h − when
1
the impairment of the autolysis.
compared to =0.8 h− (p < 0.01), but it not differ between the other D. The
1

highest acetate production was obtained at D = 0.8 h− (p < 0.01), which may
1
4. Discussion
explain the highest production of biomass and PS14, i.e., since 1 mol of
acetate produces 3 mol of ATP, at D = 0.8 h− more energy should be
1
Several studies documented the capsular polysaccharide as an important
available for growth and synthesis of PS14. The conversion of glucose into virulence factor; however until now there is little knowl-edge about the
cell was signifi-cantly increased with D and the conversion of glucose into mechanisms and kinetics of PS production. The understanding of the
products, as well as into ATP was lower at D = 0.4 h− (Table 3), indicating
1
association between biomass and capsu-lar polysaccharide production is
that the efficiency in the use of glucose to obtain energy and to generate extremely important to improve, optimize and scale-up the production process
biomass and products increased with the growth rate. On the other hand, the of this antigen. In the present work, batch and continuous cultures were
yield of PS14 per biomass did not change significantly with the D tested
performed using a complex medium to produce capsular polysaccharide of S.
(Table 3).
pneumoniae serotype 14.
For PS14R , as well as for PS14C , the kinetics of product formation was
associated to cell growth (ˇ = 0) (Fig. 3A), which confirms that the sharp The most remarkable characteristic of pneumococcal batch cultures was
increase of PS14R in batch culture was due to the release from the cells rather the strong lytic activity after the growth phase, a phe-nomenon that has also
than a partially growth associated production. The growth-associated been observed by Kanclerski and Möllby
coefficients (˛) were determined accord- [25] and Leal et al. [16] in batch fermentation of S. pneumoniae serotype 14
ing to Eq. (1) as 39 mg gbiomass − for PS14R and 47 mg gbiomass − for
1 1
with controlled pH. It is already known that the start of autolysis process
PS14C . could be triggered by several factors: organic acid production [26,27], activity
The production of lactate, on the opposite, was partially growth associated of specific peptides [28] and LytA activ-ity [12]. Nonetheless, the exact
(Fig. 3B) (˛, ˇ =/ 0). The growth-associated coefficient (˛) and non-growth- conditions in which these factors are involved remain poorly understood.
associated coefficient (ˇ) were determined
according to Eq. (1) as 4.78 g gbiomass − and 0.40 g (gbiomass h)− ,
1 1
Despite of the relatively high PS14 and cell concentrations in terms of
respectively. This profile was likewise verified to other lactic acid pneumococci cultivation that were obtained in our batch cultures compared to
bacteria [32–34]. other works [15,16], the autolysis might be unfavorable for PS14 purification,
As illustrated in Fig. 3C, the specific consumption of glu-cose increased because when it occurs, the har-vest will contain high titers of proteins,
linearly with the dilution rate. Therefore, the growth-associated stoichiometric
nucleic acids and other contaminants, which reduce values of recovery and
true yield coefficient (YG ) and the maintenance coefficient (ms ) were purification factor. In addition, it is difficult to perform metabolic studies and
determined according to Eq. (2) optimize culture parameters in a situation of eminent autolysis. Since one of
as 0.25 gglucose gbiomass − and 1.24 gglucose (gbiomass h)− . The ms and YG
1 1
the most valuable characteristic of continuous culture lies in the removal of
values found for S. pneumoniae serotype 14 are comparable to metabolites and other effects that negatively
values of other lactic bacteria and streptococci species [35–37].
 V.M.R. Gogola-Kolling et al. / Biochemical Engineering Journal 91 (2014) 16–22
affect cell growth [19], we decided to study the PS14 production in
continuous cultures. Indeed, the continuous cultivation avoided the
accumulation of organic acids and autolysins. As a consequence, autolysis did
not occur and the process reached elevated concen-trations of PS14 and
biomass and consequently productivity values were improved (Table 4).
The model proposed by Luedeking and Piret [23] was applied to
demonstrate that the kinetics of product formation was asso-ciated to cell
growth for PS14R as well as for PS14C . This kinetics is in agreement with
PS14 biosynthetic pathway, given that the same nucleotide sugar precursors
form PS14 and also other cellu-lar structures (peptidoglycan, teichoic acid,
and lipoteichoic acid) and all these precursors are derived totally from
intracellular pools [11]. Consequently, the synthesis of precursors does not
require dedicate enzymes encoded within the capsule loci and is mostly
dependent of pneumococcal central metabolism. The mechanism of PS14
synthesis is also similar to the synthesis of S. agalactiae cap-sules and a direct
association between capsule production and cell growth has already been
suggested for this microorganism [29].
The translocation of PS14 unit also seems to be a process associ-ated to
cell growth. The production of PS14 comprises the synthesis of repetitive
subunits, polymerization and attachment to the cell wall. Although the
posterior release of PS14 to the medium is not a well-defined process, we
hypothesized that the growth associated production profile of PS14R could be
connected to cell-division. Once cell division requires the enzymatic activity
of autolysins, which hydrolyse the peptidoglycan, some amount of PS14 C is
also released to the medium, then becoming PS14R . Then, a faster cell
division (increase of D) implies in faster PS14R production, as veri-fied in
Fig. 2.
As expected, the production of lactate by S. pneumoniae was par-tially
growth associated. In their classical model, Luedeking and Piret [23]
proposed that, in lactic acid bacteria, the cell dissimi-lates glucose to lactic
acid in order to obtain the energy required to form new bacteria and at the
same time it does so as a normal metabolic activity independent of growth,
therefore justifying the partially growth associated production profile of
lactate (Fig. 3). It is worth to note that it was not possible found a linear
correlation between D and formation of the two other organic acids found in
pneumococcal culture, acetic and formic.
Maintenance energy represents the energy expenditures nec-essary to
maintain an energized membrane, to repair damaged cellular components, to
transfer some nutrients and products in and out of the cells, for motility and to
adjust the osmolarity and opti-mal internal pH of the cells [30]. Sinclair and
Kristiansen [31] have reported that the values of the maintenance coefficient
can range
from 0.02 to 4.0 gsubstrate (gbiomass h)− , depending on the environ-mental
1
conditions and on the rate of cell growth. Don and Shoparwe
[35] have also found that ms varied according to the concentration of initial
substrate in batch cultivations. The ms and YG values found for S.
pneumoniae serotype 14 were compared to values of other lac-tic bacteria and
streptococci species. The YG was similar to values found for other
streptococci [35–37], while the ms was in the same range of values for L.
lactis [32,34].
5. Conclusions
The continuous cultivation allowed us to demonstrate that the PS14
production is cell-associated and to compare some pneu-mococcal
physiologic activities to other lactic acid bacteria, as the partially growth-
associated production of lactic acid and values of true yield and maintenance
coefficients. These new data will help to delineate new strategies to optimize
medium and improve PS14 production. Compared to batch, the continuous
cultivation reached the highest values of biomass, PS14 concentration and
21
productivity, while avoided autolysis, a frequent event in batch cultivations of
pneumococcus. These findings make the continuous cultivation not only a
good tool to metabolic studies, but also a promising strategy for PS14
production.
Acknowledgements
We would like to thank Instituto Adolfo Lutz for gently pro-viding the
strain. This work was financially supported by Sao Paulo Research
Foundation (FAPESP grants 2008/10364-1 and 2008/05207-4) and
Coordenadoria de Aperfeicoamento de Pessoal de Nível Superior (CAPES,
Brasilia, Brazil).
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