Title: A novel process of cultivating bacteria for yield
improvement of capsular polyoses.
Brief Description :
Capsular polyoses are important immunogens found on the surface
of bacteria involved in various bacterial diseases. This
feature has led to them being an important component in the
design of vaccines. They have proved useful in eliciting immune
responses especially when linked to carrier proteins.
Typically, capsular polyoses are produced using batch culture
in complex medium [Refer Shen et al. 2001 Vaccine
19: 850-61; Palazzi et al. 2004 J . Infect. Dis. 190:558-64;
Merritt et al. 2000 J . Biotech. 81:189-97 ;Dassy & Fournier 1996
Infect. Immunol. 64:2408-14 ;Suarez et al. (2001) Appl . Env.
Microbiol. 67:969-71 ;Wicken et al. (1983) J . Bact. 153:84-92]
The traditional DO-stat control of nutrient feeding is simply
based on the concept of DO rises/spikes (due to a reduction of
carbon substrate or cessation of oxygen consumption or
respiration) upon nutrient limitation or depletion. The DO-stat
control tries to maintain the culture at a constant DO level
within a limit (the DO setpoint) by increasing the nutrient feed
rate when DO rises above the setpoint and reducing the nutrient
feed rate when DO drops below the setpoint. The DO-stat strategy
typically works well in defined media where nutrient depletion
results in rapid DO rise. However, the DO-stat method often
fails in media supplemented with rich complex nutrients such
as yeast extract, tryptone, peptone, casamino acid, or Hy-Soy.
Rich complex nutrients are capable of supporting cellular
maintenance and respiration through amino acid catabolism such
that the DO level remains low (i.e. no apparent DO spikes) even
under carbon source limitation or depletion.
When a complex medium is used for culture growth, a pH-stat
strategy may be more suitable than DO-stat since the culture
pH tends to increase once the carbon source is depleted. In a
manner similar to DO-stat control, the pH-stat method maintains
a constant culture pH at about the setpoint by increasing the
nutrient feed rate as pH rises above the setpoint and reducing
the nutrient feed rate when pH drops below the setpoint. However,
since the change in culture pH upon nutrient depletion is less
responsive than that of DO, feeding control by pH-stat can be
relatively sluggish when compared to DO-stat.
Most studies used batch culture systems in which the growth rate,
nutrient levels and metabolic concentrations change during
cultivation. In such systems, alteration of one factor results
in changes in other factors associated with growth that can
affect yields unpredictably. Continuous cultures allow the
researcher to separate and define parameters that are
interdependent during batch culture growth, such as growth rate,
nutrient and product concentrations and cell density. During
continuous culture, fresh medium is added to a culture at a fixed
rate and cells and medium are removed at a rate that maintains
a constant culture volume.
In perfusion culture, fresh medium is added to a culture at a
fixed rate and cell-free spent medium are removed at a rate that
maintains a constant culture volume. However, continuous and
perfusion culture is prone to strain stability problems and
contamination, and is somewhat expensive due to the continuous
feed of medium and nutrients. Therefore, there is a need to find
alternatives to continuous culture for the high yield
production of capsular polysaccharides in order to overcome the
problems with continuous culture that are cited above.
One approach to overcome the drawbacks of continuous culture
is exemplified in WO 2007/052168. A complex fed batch
fermentation process has been developed to maintain a
nutritional environment and a growth rate favorable to cps
production. This process combines the advantages of batch and
continuous techniques, producing high cell densities due to
extension of the exponential growth phase and to conditions that
control substrate addition during fermentation. However, the
complex fed batch technique uses software with a complex
algorithm to manage the fermentation.
Another approach is disclosed in WO 20100272755 .A method for
cultivating the bacteria has been developed, wherein the
cultivation comprises two instantaneous additions of yeast
extract, followed by a linear addition of a glucose. Each
addition is initiated at a designated OD level. Thus here, the
linear addition of a carbon source without an algorithm is an
improvement over the previous complex fed batch
fermentation. However, this is a very tedious method requiring
continuous measurement of O.D. Further it fails to consider the
constant changing requirements (microenvironment ) of
organism.
The instant invention is based on a surprising finding and
relates to a novel feeding method , wherein the rate of feed
medium addition during fed-batch fermentation is equivalent to
the rate of alkali mixture addition for maintaining a preset
pH resulting in a volumetric increase in CP yield as compared
to previously existing batch fermentation methods.
Summary of the Invention:
The instant invention provides a novel fed batch fermentation
strategy resulting in 3 to 5 times increase in productivity of
capsular polyoses as compared to batch mode fermentation.
Particularly, the instant method relates to a method of
cultivating capsulated bacteria for higher volumetric
production of capsular polyoses (CP) , wherein said method
comprises (a) providing an inoculum of a strain of bacteria
expressing the CP; (b) cultivating the strain by fermentation
at pH 7.2, wherein the rate of feed medium addition is equivalent
to the rate of alkali mixture addition for maintaining a preset
pH;c) fermenting the culture medium at 35-38°C under stirring
at 50-150 RPM with an air flow rate of 0.1-0.5vvm.
Brief Description of Drawings :
Figure 1:Pneumococcal serotype 1 polysaccharide (fed-batch
fermentation) for different feeding methods
Figure2 :Pneumococcal serotype 1 polysaccharide
yield (fed-batch) for different ratios of bases in alkali mixture
for maintaining pH.
Figure 3:Pneumococcal serotype 1 , 5 , 6A, 6B, 7F, 9V, 14, 19A, 19F and
23F polysaccharide yield by fed-batch as compared to batch
fermentation .
Detailed Description of Invention:
The disclosure provides a process of cultivating
bacteria, wherein the rate of feed addition during fed-batch
fermentation is equivalent to the rate of alkali mixture
addition for maintaining a preset pH.
Another aspect of the instant invention is that alkali mixture
consisting of NaOH and Na2C03 in a specific ratio can be used
for a ) maintaining preset pH & b ) obtaining higher capsular
polyose yield. Use of only Na2C03 can provide suitable medium
for growth as well as pH control, however the limiting condition
of Na2C03 can provide a stress condition to make capsular
polyoses and higher requirement of Na2C03 quantity. Hence when
the mixture of NaOH and Na2C03 is used for maintaining pH, the
growth can be less with higher specific polyose productivity
as well as with higher volumetric productivity due to less
quantity of alkali consumption.
As per the instant process ,when glucose gets converted to
lactic acid and pH begins to decrease , it can be maintained
by alkali mixture addition with simultaneous glucose addition
to supplement the depleted glucose in media.
In a preferred embodiment of the instant invention, the
fermentation feed components can comprise of atleast one carbon
source, atleast one nitrogen source, atleast one magnesium
source that can be fed to the batch fermentation at a particular
cell density at a feed rate equivalent to the rate of alkali
mixture addition.
One embodiment of the current invention is that, said alkali
mixture contains at least two bases selected from the group
consisting of sodium hydroxide, sodium carbonate , sodium
bicarbonate, potassium hydroxide, potassium carbonate and
calcium hydroxide .Also a designated ratio of sodium hydroxide
and sodium carbonate is selected to achieve a higher volumetric
production of CP.
The designated ratio of sodium hydroxide to sodium carbonate
can be between 1:1 and 4:1.
According to the instant invention, NaOH was further enhancing
the productivity of CP when it was used with Na2C03 as mixture
for maintaining the pH. Only Na2C03 was providing suitable
medium for growth when it was used for maintaining the pH.
However the limiting condition of Na2C03 was providing a stress
condition to make capsular polyoses. When the mixture of NaOH
and Na2C03 was used for maintaining pH , the growth was less
but CP productivity was increased.
Preferably the invention provides a method of culturing
Streptococcus in fed batch culture, wherein a high yield of CP
is produced. Preferably, the yield of cps from the culture medium
is between 900 and 2000 mg/L or more. Thus, this fed-batch method
allows the production of CP at a volumetric yield increase
between 150 and 350% compared with batch culture. In some cases,
the yield may be at least twice the quantity produced using batch
culture, more preferably 4 times the quantity produced using
batch culture.
Another embodiment of the current invention is that the said
feed medium can comprise of atleast one carbon source, atleast
one nitrogen source ,atleast one salt and atleast one amino
acid.
Preferably the said carbon source is glucose. he nitrogen
source can be selected from yeast autolysates, yeast nitrogen
base, peptones, tryptone, casamino acids, soybean meal,
Hy-Soy, yeast extract and tryptic soy broth. The salt can be
selected from potassium sulphate, calcium chloride, magnesium
chloride ,magnesium sulphate and mixtures thereof.
A s per the instant invention the feed medium can comprise of
ingredients (gm/1) ,glucose within a range of 100-500 ,magnesium
sulphate within a range of 1-7 .5,Hy-soya within a range of
40-150, yeast extract within a range of 5-50 ,Thiamine
hydrochloride within a range of 0 .002-0 .005, cysteine within a
range of 0 .2-0 .5,calcium chloride within a range of 0.2-0.5.
Also the feed medium can comprise of ingredients (gm/1 ),glucose
within a range of 100-500, yeast extract within a range of 5-50,
Thiamine hydrochloride within a range of 0.002-0.005, cysteine
within a range of 0.2-0.5, calcium chloride within a range of
0.2-0.5.
Alternatively said feed medium can comprise of
ingredients (gm/1) ,glucose within a range of 100-500 , magnesium
sulphate within a range of 0.5-7.5, Hy-soya within a range of
40-150 and yeast extract within a range of 5-50.
A preferred embodiment of the instant invention is that the
feed medium can comprise of ingredients (gm/1) ,glucose within
a range of 100-200 ,magnesium sulphate within a range of
1-3, Hy-soya within a range of 50-150 and yeast extract within
a range of 15-25
According to the instant invention the said novel fed batch
process can be utilized for preparing capsular polyoses
selected from the group consisting of Escherichia coli,
Francisella tularensis, Haemophilus influenzae, Klebsiella,
Moraxella catarrhalis, Neisseria meningitidis ,groups A ,C , 3
Y and X , Porphyromonas gingivalis, Pseudomonas aeruginosa,
Burkholderia cepacia, Salmonella typhi, Salmonella typhimurium,
Salmonella paratyphi, Shigella dysenteriae, Shigella flexneri,
Shigella sonnei, Vibrio cholera, Enterococcus faecalis,
Enterococcus faecium, Group A Streptococcus, Group B
Streptococcus, Mycobacterium tuberculosis, Staphylococcus
aureus , Staphylococcus epidermidis and Streptococcus
pneumoniae.
EXAMPLES
Example 1
Evaluation of Feeding method for optimization of Fed batch
development for S . Pneumoniae Serotype 1 :
Different feeding methods were evaluated for feeding. DO- stat
method was not applicable for S . Pneumoniae fermentation. As
S . Pneumoniae is micro-aerof ilic bacteria only surface aeration
was required during fermentation process. After partial growth
of S . Pneumoniae in fermenter during batch mode fermentation,
dissolved oxygen was around zero.
Five different feeding methods were tried for fed batch
fermentation.
1 . pH stat method: The pH stat feed was controlled by a set-pH
point wherein when the pH of the culture rises above the
set-pH, the carbon source was added to the medium and when
the pH of the culture falls below the set-pH, the carbon
feed was stopped.
2 . Constant rate feeding method: In this method
concentrated feed was given at 4.2ml/m for 4 hrs in 1.6
1 of broth culture.
3 . Exponential feeding method: The feed rate was started with
lml/m and increased with OD. The feed rate was increased
by 0.5ml/m for lunit increment in OD.
4 . Constant glucose concentration: Residual glucose
concentration was tried to maintain around 0.5 g/1 during
fed batch fermentation.
5 . pH dependant feeding method: In this method pH was
maintained with Na2C03. The glucose was added for
depleted glucose in media at rate which Na2C03 was fed in
media for maintaining the pH.
The NBS Bioflo 115 and Bioengineering AG fermenter was used
for all these experiments. The present study was carried out
at 3 L NBS Bioflo 115 fermenter model and it was scaled-up to
30 L Bioengineering AG Fermenter. The further scale up could
be carried out at 450L Bioengineering fermenter. The 200 g/1
glucose and 5 g/1 gS04 were used as feeding media for all fed
batch fermentation. The fermentation condition were same for
all fed batch experiments. The pH was maintained with 20% Na2C03
solution during fermentation process.
The following parameters were maintained during the fed batch
fermentation. Temperature (36 .5+0.5), pH(7.1 +0.2), RPM-100,
Airflow (surface aeration) -0 .5vvm.
Table 1:CP yield of serotype 1 for different feeding methods
Feeding method CP yield (mg/1)
No fee 210
pH stat method 170
Constant rate feeding method 198
Exponential feeding method 180
Constant glucose concentration 160
pH dependant feeding method 260
Example 2
Feed media optimization for Fed batch fermentation
Pneumoniae Serotype 1 CP
The following different media were used for feeding during
fermentation:
1 . Glucose- 200g/l
2 . Glucose (200g/l) , gS0 (2 .5g/l)
3 . Glucose-200g/l, MgS04-2.5 g/1, Yeast extract- 25g/l.
4 . Glucose-lOOg/l, MgS04-5g/l, Yeast extract- 15g/l,
Hy-soya- 50 g/1
5 . Glucose-200g/l, MgS04-2 .5g/l, Yeast extract- 25g/l,
Hy-soya- lOOg/1
6 . Glucose-200g/l, MgS04-2 .5g/l, Yeast extract- 25g/l,
Hy-soya- lOOg/1, Thiamine Hydrochloride-0 .02g/l ,
Cacl2 .2H2O-0 .0.02g/l, Cysteine-0 .2g/l .
7 . Glucose-lOOg/l, MgS04-lg/l, Yeast extract- lOg/1,
Hy-soya- 40g/l, Thiamine Hydrochloride-0 .04g/l ,
Cacl2.2H2O-0. 04g/l, Cysteine-0 .4g/l
All the fermentation conditions were maintained as mentioned
above. The pH dependent feeding method was used for fed batch
fermentation feeding for all batches. The 4th Feed medium was
utilized for further fed batch experiments.
Table 2 : CP yield of serotype 1 for different feeding media
composition
Example 3
The mixture of alkali used for maintaining the pH
The NaOH was further enhancing the productivity of CP when it
was used with Na2C03 as mixture for maintaining the pH. Only
Na2C03 was providing suitable medium for growth when it was used
for maintaining the pH. The limiting condition of Na2C03 was
providing a stress condition to make CP. When the mixture of
NaOH and Na2C03 was used for maintaining pH , the growth was
less but CP productivity was increased. The 8% NaOH and 20%
Na2C03 was used in ratio of 1:1, 2:1, 3:1 and 4:1. Feed media
composition 3 in example 2 was used as feed for the following
fed-batches .
Table 3 : The mixture of alkali used for maintaining the pH for
fed batch fermentation of S . Pneumoniae Serotype 1
Example 4
Different serotype of S . Pnevanoniae were tried with optimized
fed batch condition .
For the serotype 1 fed batch conditions were used for other
serotype also. The following condition were taken into
consideration running the fed batch fermentation.
1 . The pH dependant feeding method
2 . Mixture of alkali used
3 . Optimized feeding media
4 . Other fermentation were similar as mentioned above
The serotypes 1 , 5 , 6A, 6B, IF, 9V, 14 ,19A, 19F and 23F were used
using new optimized conditions of fed-batch process.
yield in fed batch fermentation for different
Serotype 19F 510 1530 3
Serotype 23F 550 1410 2.56
Serotype 7F 517 1800 3.48
Serotype 6A 508 1200 2.36
Thus fed batch method of the current invention results in
volumetric increase in CP yield ranging from 150 to 350%, wherein
final CP yield ranges between 900 and 2000 mg/1.
It will be evident to those skilled in the art that the invention
is not limited to the details of the foregoing illustrative
examples and that the present invention may be embodied in other
specific forms without departing from the essential attributes
thereof, and it is therefore desired that the present
embodiments and examples be considered in all respects as
illustrative and not restrictive, reference being made to the
appended claims, rather than to the foregoing description, and
all changes which come within the meaning and range of
equivalency of the claims are/therefore intended to be embraced
therein .
We claim,
1 . A method of cultivating capsulated bacteria for higher
volumetric production of capsular polyoses (CP) , wherein said
method comprises (a) providing an inoculum of a strain of
bacteria expressing the CP; (b) cultivating the strain by
fermentation at pH 7.2, wherein the rate of feed medium addition
is equivalent to the rate of alkali mixture addition for
maintaining a preset pH;c) fermenting the culture medium at
35-38°C under stirring at 50-150 RPM with an air flow rate of
0.1-0. 5vvm.
2 . The method according to claim 1 ,wherein the bacteria is grown
in fed-batch culture.
3 . The method according to claim 2 ,wherein the volumetric
increase in CP yield by fed-batch culture is between 150 to 400%
as compared to batch or continuous culture conditions.
4 . The method according to claim 3 ,wherein the yield of CP by
fed-batch is between 900 mg /l and 2000 mg/1.
5 . The method according to claim 1 ,wherein the feed medium can
comprise of atleast one carbon source, atleast one nitrogen
source ,atleast one salt and atleast one amino acid.
6 . The method according to claim 5 ,wherein the said carbon
source is glucose.
7 . The method according to claim 5 ,wherein the said nitrogen
source consists of three or fewer from the following list : yeast
autolysates, yeast nitrogen base, peptones, tryptone, casamino
acids, soybean meal, Hy-Soy, yeast extract, and tryptic soy
broth .
8 . The method according to claim 5 ,wherein the said salt can
be selected from potassium sulphate, calcium
chloride, magnesium chloride ,magnesium sulphate and mixtures
thereof .
9 . The method according to claim 5 ,wherein the feed medium can
comprise of ingredients (gm/1) ,glucose within a range of
100-500 ,magnesium sulphate within a range of 1-7 .5,Hy-soya
within a range of 40-150, yeast extract within a range of
5-50, Thiamine hydrochloride within a range of
0 .002-0 .005, cysteine within a range of 0 .2-0 .5 ,calcium
chloride within a range of 0.2-0.5.
10. The method according to claim 9 ,wherein the feed medium can
comprise of ingredients (gm/1) ,glucose within a range of
100-500, yeast extract within a range of 5-50 ,Thiamine
hydrochloride within a range of 0 .002-0 .005, cysteine within a
range of 0.2-0 .5,calcium chloride within a range of 0.2-0.5,
11. The method according to claim 9 ,wherein the feed medium can
comprise of ingredients (gm/1 ) ,glucose within a range of
100-200, magnesium sulphate within a range of l-3,Hy-soya within
a range of 50-150 and yeast extract within a range of 15-25.
12. The method according to claim 1 ,wherein said alkali mixture
contains at least two bases selected from the group consisting
of sodium hydroxide, sodium carbonate , sodium bicarbonate,
potassium hydroxide, potassium carbonate, and calcium
hydroxide .
13. The method according to claim 12, wherein a designated ratio
of sodium hydroxide and sodium carbonate is selected to achieve
a higher volumetric production of CP.
14. The method according to claim 13, wherein said ratio ranges
between 1: and 4:1.
15. The method according to claim 13, wherein the concentration
of sodium hydroxide is between 1.5 and 3.0 .
16. The method according to claim 13, wherein the concentration
of sodium carbonate is between 10 and 20% (w/v) .
17. The method according to claim 1 , wherein said bacteria is
selected from Streptococcus pneumoniae, Neisseria meningitidis
Staphylococcus aureus, Haemophilus influenzae , Group A
Streptococcus and Group B Streptococcus.