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Sensitivity to Hydrogen Peroxide of Growth and Hyaluronic Acid Production by Streptococcus zooepidemicus ATCC 39920.

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Dev. Chem. Eng. Mineral Process. 13(5/6), pp. 531-540, 2005.
Sensitivity to Hydrogen Peroxide of Growth
and Hyaluronic Acid Production by
Streptococcus zooepidemicus ATCC 39920
M.D. Mashitah, K.B. Ramachandran' and H. Masitah*
Department of Chemical Engineering, Faculty of Engineering,
University Malaya, 50603 Kuala Lumpur, Malaysia
#
Dept of Biotechnology, Indian Institute of Technology, Chennai, India
The sensitivity to hydrogen peroxide (ff209 ofgrowth and hyaluronic acid (HA)
production by Streptococcus zooepidemicus A TCC 39920 was studied under various
conditions. In sheep blood agar-plates, no detectable zone was observed even when
the concentration of H202 was increased to 0.15 mM. With brain heart infision-agar
and chemically defined medium-agar plates, a profound zone was detected at 0.015
mM concentration of H202. To determine the minimal inhibitory concentration (MIC)
of the strain in culture broth, various concentrations of H 2 0 2 (0-200 mM) were
maintained in the medium prior to fermentation. The result showed that for higher
concentrations of H20z
in the medium, the greater was the inhibition. Streptococcus is
catalase-negative and known to produce H202 which may affect growth, HA
production and glucose utilization. In order to determine at which growth phase H202
had the maximum inhibitory activity, a batch fermentation of S.zooepidemicus was
conducted in shakeflask culture. it was found that H202
production took place during
the growth phase, and HA production started afier the growth had reach late
exponential phase when H202in the culture media was depleted. This indicates that
H202produced by the cells did not affect cell growth but influenced HA production.
Keywords: hyaluronic acid; hydrogenperoxide; Streptococcus zooepidemicus
Introduction
McLeod and Gordon [ 11 first reported on the production of hydrogen peroxide (H202)
by pneumococcus bacteria, others have since demonstrated that Hz02 is also
generated by many other microorganisms [2-4]. However, among the numerically
most prominent bacteria, peroxide production largely appears to be restricted to the
streptococci and occurs when a fermentable sugar such as glucose is catalyzed in the
presence of oxygen [5-61. According to Hardie and Whiley [7], in catalase-negative
* Authorfor correspondence (masitahhasan@um.edu.my).
53I
M D.Mashitah, K.B. Ramachandran and H. Masitah
streptococci, reactions with molecular oxygen mediated by flavoproteins can produce
toxic levels of HzOz and also intermediates such as highly reactive superoxides. These
reactive species are known to induce mitochondrial membrane permeability and the
drop in the mitochondrial membrane potential [8]. Such events can cause widespread
damage to biological macromolecules leading to lipid peroxidation, protein oxidation,
enzyme inactivation, DNA base modification and DNA strand breaks [9].
Recent investigations demonstrated that the oxygen-derived reactive species have
a profound effect on the production of hyaluronic acid [lo-111. These include the
superoxide anions (-Oi), hydroxyl (-OH')and singlet oxygen (-03
free radicals. The
lower cell yield, as well as the specific growth rate observed in aerated batch cultures,
was hypothesized due to the toxicity of H202,particularly since the microorganism is
catalase-negative [l I]. However, the degradation of HA by these oxygen species has
not been previously reported. Indeed, little is known concerning its mode of action on
HA production. This investigation aims at identifjmg and quantifjmg the sensitivity
of cell growth and hyaluronic acid production by Streptococcus zooepidemicus.ATCC
39920 to H202 in the medium.
Materials Used and Experimental Details
(i) Strain
Streptococcus equi subs-species zooepidemicus ATCC 39920 was obtained from
American Type Culture Collection (Rockville, Maryland, USA). It was maintained on
sheep blood agar (SBA) slants and kept at 4°C.
(ii) Culture medium
The composition of the medium used in all experiments comprised of (in gA): glucose
30; yeast extract 10; KH2P040.5; Na2HP04.12H20 1.5; and MgS04.7H200.5. The
medium was prepared and autoclaved at 121°C for 20 minutes. Glucose solution was
autoclaved separately and mixed aseptically with the other components on cooling.
(iii) Agar-plate culture
Three types of agar plates were used to determine the sensitivity of hydrogen peroxide
towards the tested strain of S. zooepidemicus. These include sheep blood agar (SBA),
brain heart infbsion-agar (BHI-agar), and chemically defined media-agar (CDMagar). Sheep blood agar as recommended by ATCC comprised of tryptic soy broth
powder 3.0% (w/v), agar 1.5% (w/v) and 5% (v/v) of fresh sterile sheep blood from
the research farm of the University of Malaya. For the CDM-agar, media composition
was similar as above (ii), except that 1.5% (w/v) agar was included.
(iv) Shake flask culture
Cell suspension for the shake flask culture was prepared by inoculating aseptically a
stock culture of S. zooepidemicus onto SBA-plates and incubating overnight at 37°C.
The colonies that were formed were punched with a sterile cork borer to produce a
round disk (0.85 cm diameter), and 10 disks were then put into a sampling bottle
532
Growth and Hyaluronic Acid Production by S.zooepidemicus A TCC 39920
containing 50 ml of sterile distilled water. The sampling bottle was vortexed for 3
minutes so that the cells were evenly distributed in the liquid.
Two conditions of incubation were used. First, 15 ml of the cell suspension was
inoculated in a 500 ml Erlenmeyer flask containing 135 ml of culture media, and
incubated in a rotary shaker at 250 rpm, and 37°C for 12 h. Second, the suspension
was inoculated similarly except the flasks were incubated under static conditions at
37°C. Samples were taken every hour and kept at 4OC for later analysis.
(v) Inhibition test
The inhibition of HzO2 on S. zooepidemicus was detected using the plate-hole
diffiion technique as described by LeBien and Bromel [121. Surfaces of all the agar
plates (40 ml per plate) were uniformly streaked with 0.3 ml cell suspension of the
tested strain. A sterilized cork borer (0.85 cm diameter) was then used to punch a hole
to the bottom of each plate. A 100 pl of H202
at various concentrations (0-0.4 mM)
were then added to each well and left on the laminar flow cabinet for 4 hours to allow
the Hz02to difhse evenly into the agar. The plates were then incubated at 37°C for
24 hours. Plates were scored positive if the zone of inhibition of at least 1 mm wide
was observed around the disk.
To determine the sensitivity of growth and product formation to H2O2in culture
broth, 0-200 mM of HzOZsolution (at 20% v/v of the working volume) was added to
110 ml of culture media in a 500 ml Erlenmeyer flask. Cell suspension (10 ml) was
then added and incubated at 37"C, 250 rpm for 24 hours; growth rate was measured
by changes in optical density at 600 nm. The highest dilution of HzOz that showed no
visible growth (turbidity) indicated the minimum inhibitory concentration (MIC).
(vi) Analytical methods
Samples were withdrawn fiom all the flasks every hour and analyzed for cell, glucose,
product and by-product concentrations. Cell concentration was determined by
measuring optical density (OD) at 600 nm by Jenway Spectrophotometer and dry cell
method. A correlation between dry cell weight and OD600was established. Both
hyaluronic acid and glucose concentrations were determined using the method as
described by Mashitah et al. [13] and hexokinase (Sigma), respectively. The
concentration of HzOz was analyzed by the spectrophotometric method as suggested
by Emiliani and Riera [ 141, with slight modification.
Results and Discussion
In view of the toxicity of hydrogen peroxide (H202) during growth of S.
zooepidemicus ATCC 39920, several investigations were carried out in an attempt to
elucidate its effect on hyaluronic acid (HA) production. Hence, plate and shake flask
cultures at various conditions were studied.
I Plate culture experiments
Table 1 depicts the experimental data taken fiom plate-cultures incubated for 24 hours
at 37°C. With SBA-plates, no detectable zone was observed even when the
533
M.D. Mashitah, K.B. Ramachandran and H.Masitah
concentration of H202 was increased to 0.15 mM. When the tested strain was grown
on BHI-agar and CDM-agar plates, a profound inhibition zone was detected. The
minimum inhibitory concentration (MIC) of H202 for both types of plates was
0.015 mM. A higher MIC concentration for cells grown on SBA is probably due to
the enzyme catalase present in heme which tends to break down the peroxide to water
and oxygen [15]. These results indicate that the cell growth is inhibited by the
additional H202.
11 Shake flask experiments
A study was also conducted to determine the minimal concentration of H202that
inhibits the growth of the tested strain in the culture broth. The results showed that the
higher the concentration of H202,then the greater is the inhibition (see Figure la).
However, when treated with a low concentration of HzOz,the response curve tends to
be biphasic. Between 0 to 5 mM of H202the OD6oodecreased, but then rose again
above 5 to 10 mM (see Figure lb).
Further increase in the level of H202above 10 mM resulted in further decrease in
cell growth. Earlier investigations suggested that two modes of killing were proposed
to explain the curve [ 16-17]. First, cells were killed primarily by DNA damage caused
by OH- radicals produced during the Fenton reactions. Second, higher levels of H202
are thought to scavenge OH-, thus suppressing first mode killing. Furthermore, as the
concentration of H202 rises still M e r , cell growth is decreased by second mode of
killing, although the cellular targets in this instance are not known. According to
Thibessard et al. [ 181, above a certain concentration, a protective response to Hz02
was induced by the cells. However, as the H202 concentration increases further, the
cell growth again decreased due to the saturation of this protective system.
Figure 2 shows the profiles of cell biomass, HA, glucose and H202production
with time for the shake flask culture. It can be seen that the HA (HAtota!and HAso~u~~e)
concentration continued to decrease with an increase in amount of HzOz inoculated.
Table 1. Inhibitory zones produced during growth of S. zooepidemicus at various
BHI
0
0.003
0.015
0.03
0.10
0.15
0.30
0.35
0.40
534
NIL
NIL
NIL
NIL
NIL
1.0 f 0.15
1.0 k 0.22
2.5 f 0.31
2.5 f 0.05
of inhibition
NIL
NIL
0.5 f 0.12
1.0 f 0.17
3.0 f 0.23
4.0 f 0.14
5.0 It 0.05
7.0 f 0.33
8.0f0.11
CDM
0.45 f 0.15
2.0 f 0.22
2.5 f 0.005
5.0 f 0.12
6.0 f 0.36
6.5 f 0.25
1
Growth and Hyaluronic Acid Production by S.zooepidemicus ATCC 39920
0.6
0.5
0.4
% 0.3
A
0
v
x
0.2
0
0.1
0"
ji
J 0.0
- 4
0.0
0
40
80
120 160 200
15
0
30
45
YO, concentration
YO, concentration
(W
(W
60
Figure I . Sensitivity of S. zooepidemicus to hydrogen peroxide, growth was
determined by measuring the optical density at 600 nm; (a) high concentrations of
Hz02;
(b) low concentrationsof H202.
0.35
,
r 2.5
12
,
.-0
I=
2
2
m
>r
I
---.---Wsol
1
-Wlola
--r-CCdlblOrress
I
I
I
+HZOZ
+glucose
I
I
Figure 2. Fermentation profi(es ofgrowth, hyaluronic acid, glucose and extracellular
H202 production by S. zooepidemicus after exposure to appropriate H202
concentrations.
535
M.D.Mashitah. K.B. Ramachandran and H. Masitah
However, the glucose and H202concentration appeared to increase from 0.04 to
21.74 g/L and 0.004 to 9.890 mM, respectively. This could be attributed to the
toxicity of H202towards the bacteria, altering membrane permeability followed by
damage to the metabolic system. This would reduce the utilization of glucose inside
the media. According to Piard and Desmazeaud [19], H202 can damage bacterial
nucleic acids, leading to reversible or irreversible alterations. In fact, it causes breaks
in the carbon phosphate backbone of DNA, releasing nucleotides and preventing
chromosome replication. However, Condon [151 stated that the relative toxicity of this
oxidizing agent towards bacteria is somewhat controversial. These results show that
H202 inhibits both growth and HA production.
1 OD14
25,
OD12
*
OD
0
2
4
6
8
10
12
lncubtion t i m e (h]
A
Cell concentration
-e-
total hyaluronic acid
+hydrogen
percotide
Figure 3. Fermentation profile of batch culture of Streptococcus Zooepidemicus
ATCC 39920 in shaking condition.
In order to determine the effect of H202
produced by the cells itself on the growth
and HA production, additional experiments were carried out in shake flasks without
externally added H202.In the shake flask culture, growth, H202and HA production
were also influenced by the condition of incubation. Figure 3 shows the profiles of
H202,cell mass and HA concentration with time for a shaken culture. It can be seen
that H20zis produced in the early phase of the growth period. It remained constant for
about 4 hours and then started falling sharply, it then reached a very low value after
6 hours of fermentation. The HA production increased steadily with cell growth to a
maximal level at the onset of, or during, the stationary phase. This showed that even
as produced by the cells themselves, HA
though cells grew in the presence of H202
production significantly increased only after H202
concentration became very low in
the medium. In fact, the relationship between growth and HA production was not
linear and reflects the characteristics of mixed-growth-associated product formation.
According to Papagianni et al. [20]in exo-polysaccharides fermentation, the specific
rate of product may either be independent of growth rate or it may increase with it,
depending on the microorganism and the growth limiting nutrient.
536
Growth and Hyaluronic Acid Production by S. zooepidemicus ATCC 39920
In static conditions (see Figure 4), H202was produced by the cells in the early
growth phase but also began decreasing in this early period. This could be due to nonThe
availability of oxygen in static culture to maintain the production of HzO2.
concentration of H202in both types of fermentation was about the same in both
shaken and static cultures. Product formation again took place only after the produced
H202was completely degraded. HA production displayed a lag-phase of 7 hours
which may be attributed to the time required for the germination and growth of cells
or spores used for the production of HA.
In general, major differences were observed in the time of HA production during
incubation under both conditions. The reason could be the presence of Hz02which
was produced by S. zooepidemicus in small quantities, and presumably accounted for
the growth inhibition observed in cultures. Furthermore this compound is inherently
toxic and reactive, and the tested strain is also catalase-negative [7,21]. Moreover, all
streptococci posses superoxide dismutase (SOD),the enzyme responsible for
changing the O i to HzOz. As streptococci are catalase-negative and lack a cytochrome
system, the H202is not degraded but rather excreted and accumulated in the broth.
0.018
0.8
0.01 4
0.6
s
1
t
E0
Y
0.010
0.4
0.006
0.2
0.m2
0
0.o
-01302
0
2
4
6
8
10
12
Incubationtime (h)
-Gel concentration
Total hyduronic acid
VHyckogenperatide
Figure 4. Fermentation profile of batch culture of Streptococcus zooepidemicus
ATCC 39920 in static conditions.
The results of this study also showed that the release of H202 occurred at the
early stage of the exponential growth phase. This could be explained by the fact that
during fermentation, S. zooepidemicus may consume oxygen during growth.
Therefore, when the maximum biomass was reached, i.e. beginning of the stationary
phase, oxygen was limiting and H202inhibition was reduced, thus increasing the
production of HA. In general, growth inhibition in aerated batch cultures was due to
the accumulated concentration of oxygen-derived free radicals [22]. Whether such
low concentrations may inhibit growth is still uncertain.
Figure 5 shows the profiles of product yields (YtHm) plotted against time. It can
be seen that, with shaking cultures, product yields were higher than that produced
when grown statically. Therefore, cell growth was accompanied by HA production
537
M.D. Mashitah, K.B. Ramachandran and H. Masitah
regardless of H20z being released into the media. However in static conditions,
product yield displayed a lag-phase of 7 hours. On further fermentation, the yield
increased to a peak value of 0.027 g tHA per g cells, then decreased gradually until
the end of the fermentation period. During lag phase, the concentration of H202
was
higher (see Figure 4), thus inhibiting growth and production of HA. This indicates
that H202might suppress the metabolism of the streptococcal cells more in static
cultures than in aerated cultures.
0.30
I
+stitc
+shaking
0.20
0.1 5
0.05
0.00
0
2
4
6
8
1
0
1
2
IncBatimtime (h]
Figure 5. Fermentation profile of product yield during growth of S.zooepidemicus
ATCC 39920
0
J
0
:
:
:
1
2
3
:
4
:
5
:
8
:
7
:
8
:
0
:
:
:
I0
I 0 1 1 12
habairntime(h)
Figure 6. Glucose profile of batch culture Streptococcus zooepidemicus A TCC 39920.
Recent investigations demonstrated that the HA production was directly related
to the oxygen uptake rate, a linear relationship between the two factors which in turn
was linked to agitation conditions [11, 231. With agitation, more dissolved oxygen is
available and stimulates further uptake of glucose and the utilization of phosphate. If
the cell oxygen demand is not satisfied, the nutrient consumption will definitely be
538
Growth and Hyaluronic Acid Production by S. zooepidemicus ATCC 39920
affected. Sometimes the metabolites may act as inhibitors, e.g. sugar phosphate
(G-6-phosphate or F-6-phosphate) arising from the phospho-transferase.
Figure 6 illustrates the glucose profile of the tested bacteria for the 12 hour
incubation. For shaking cultures, glucose was slowly utilized in the beginning and
reduced gradually to 10.4 g/l at the end of the fermentation period. There is a distinct
increase in glucose utilization profile after 5 hours of incubation, when the H202
concentration was small. Similar observations was made under static conditions.
Therefore, the presence of this preferred carbon source, i.e. glucose, resulted in an
increase in H202production by the bacteria. However as the level of glucose declined,
the HA production began, indicating that glucose hydrolysis and uptake of hydrolysis
products occurred concurrently.
In biopolymer production, as well as the operating conditions, the culture pH also
plays an important role in an overall fermentation productivity. As shown in Figure 7,
the culture pH fell from 7.0 to 5.5 in both situations. This is probably due to the
formation of acidic metabolites and HA, which contain acidic functional groups.
However, this finding contradicts the findings of Kyriakides et al. [24] who stated that
the culture pH in xanthan production by X. campestris may either decrease or increase
throughout the fermentation, depending on the strain and medium composition.
;::-.:*-\
5
z
4-
-shslarrg
3 -.
2 -.
1
-*
+ddc
0
1
1
1
1
1
1
4
-. 3
-. 2
3-
1
i
1
1
1
.
1
-. 1
1
I
r O
Conclusions
From these preliminary studies, it is shown that in the presence of heme in SBA
plates, no detectable inhibition to growth was observed. This is probably due to the
enzyme catalase present in heme, which breaks down the Hz02 to water and oxygen.
In other culture plates, H202inhibited growth. In culture broth, the results showed
that the higher the concentration of added H202, the greater is the inhibition.
Hydrogen peroxide produced by the cells also affects growth and HA production.
Furthermore, the condition of incubation also affects the production of HA in a shake
flask culture. With agitation, H202does not inhibit cell growth but influenced HA
production. However in static conditions, growth and HA production started as soon
as H202 in the culture media was depleted.
539
M.D. Mashitah, K.B. Ramachandran and H. Masitah
Acknowledgements
The authors are gratell to the University of Malaya (Vote F: 0066/2001A,0148/2002
and 0158/2003A)for the financial support of this research.
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