Sensitivity to Hydrogen Peroxide of Growth and Hyaluronic Acid Production by Streptococcus zooepidemicus ATCC 39920.код для вставкиСкачать
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 , in catalase-negative * Authorfor correspondence (firstname.lastname@example.org). 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 . Such events can cause widespread damage to biological macromolecules leading to lipid peroxidation, protein oxidation, enzyme inactivation, DNA base modification and DNA strand breaks . 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.  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 . 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 , 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. 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 . 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.  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. 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