Journal of Chemical Technology and Biotechnology
J Chem Technol Biotechnol 81:182–188 (2006) DOI: 10.1002/jctb.1377
Improved cultivation conditions for polysaccharide production by H. influenzae type b Mickie Takagi,1∗ Joaquin Cabrera-Crespo,1 Teresa Cristina Zangirolami,2 Isaias Raw1 and Martha Massako Tanizaki1 1 Instituto
´ ˜ Av. Vital Brasil, 1500 CEP 05503-900 Butanta, ˜ Sao ˜ Paulo, Brazil Butantan, Centro de Biotecnologia, Laboratorio de Fermentac¸ ao, ˜ Carlos, UFSCar Rodovia Washington Luis, Km 235 CEP 13565-905, de Engenharia Qu´ımica, Universidade Federal de Sao ˜ Carlos, Sao ˜ Paulo, Brazil Sao 2 Departamento
Abstract: Haemophilus influenzae b (Hib), an encapsulated Gram-negative cocco-bacillus, is one of the most common agents of meningitis worldwide. The capsular polysaccharide conjugated to a carrier protein is the antigen of the vaccine against Hib. An optimized cultivation process that could lead to an increase in the polysaccharide production would be of great interest for mass vaccination programs. The aim of this work was to evaluate different culture conditions in attempt to improve the capsular polysaccharide yield. Hib was cultivated in a bioreactor with modified soy-peptone and yeast-extract (MP) medium and optimal hemin and nicotinamide adenine dinucleotide (NAD) concentration in the culture medium was established at 30 mg L−1 and 15 mg L−1 , respectively. The batch experiments were carried out as follows: (a) overlay aeration without pH control; (b) air-sparged with dissolved oxygen tension (DOT) controlled at 10 and 30% air saturation, with and without pH control. The cultures with air-sparged aeration, without pH control, showed values for the specific production (SPp/x ) of 180–190 mg PRP g−1 dry cell weight (DCW) and overall polysaccharide productivity of 22–29 mg L−1 h−1 , accounting for an increase of ca 47% over the polysaccharide production with overlay aeration. Batch cultivations with air sparged aeration led to an improvement in the poly(ribosylribitol phosphate) (PRP) production for both conditions (DOT at 10 and 30% air saturation) investigated upon pH control, achieving up to 980 PRP mg L−1 . The SPp/x and overall polysaccharide productivity were 280–300 mg PRP g−1 DCW and 45–41 mg L−1 h−1 , respectively. The best production of capsular polysaccharide was obtained in the modified MP-medium, with 30 mg L−1 hemin and 15 mg L−1 NAD, upon sparged aeration and pH control. 2005 Society of Chemical Industry
Keywords: Haemophilus influenzae b; overlay aeration; air-sparged aeration; batch cultivation; polysaccharide production ABBREVIATIONS BHI brain heart infusion DCW dry cell weight DOT dissolved oxygen tension MP soy peptone plus yeast extract medium MMP modified MP medium NAD nicotinamide adenine dinucleotide PRP poly(ribosylribitol phosphate) VVM volume of air per unit volume of medium per minute WHO World Health Organization
INTRODUCTION Haemophilus influenzae b (Hib), an encapsulated Gram-negative cocco-bacillus, is one of the most
common agents of meningitis worldwide. Of the six known H. influenzae serotypes (a–f), type b is responsible for almost all of the invasive diseases.1 The capsular polysaccharide type b consists of repeated units of the polymer of ribosylribitol phosphate (PRP) and plays an important role in the virulence of this microorganism.2 In many developing countries, where vaccination against H. influenzae type b (Hib) disease has not been widely adopted, this microorganism is responsible for a substantial amount of meningitis cases in children under the age of five. In the USA, before the introduction of Hib vaccines, more than 70% of bacterial meningitis among children in this age group was attributed to Hib.3 However, after the introduction of the PRP–protein conjugate vaccines in
∗
´ ˜ Av. Vital Brasil, 1500 CEP Correspondence to: Mickie Takagi, Instituto Butantan, Centro de Biotecnologia, Laboratorio de Fermentac¸ ao, ˜ Sao ˜ Paulo, Brazil 05503-900 Butanta, E-mail:
[email protected] ˜ Paulo Research Foundation (FAPESP); contract/grant number: 00/08464-6 Contract/grant sponsor: The State of Sao ´ Contract/grant sponsor: Programa de Apoio ao Desenvolvimento Cient´ıfico e Tecnologico (PADCT)/Financiadora de Estudos e Projetos (FINEP) (Received 12 May 2005; revised version received 22 June 2005; accepted 22 June 2005) Published online 23 September 2005
2005 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2005/$30.00
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Improved conditions for polysaccharide production of H. influenzae type b
1988, the incidence of meningitis by Hib in toddlers up to five years old decreased from 25 cases per 100 000, in 1984, to less than 2.5 cases per 100 000, in 1993.4 In Brazil, the incidence under the age of four, between 1987 and 1991, was 4.8 cases per 100 000 children.5 The conjugation of PRP to an immunogenic carrier protein was shown to convert the immune response from T-cell independent to T-cell dependent and to promote long-term immunological memory.6 Although conjugated PRP vaccines have been produced by different manufacturers, their costs remain too high because of the several production steps required: namely, bacterial cultivation, PRP extraction and purification, PRP protein conjugation and further purification. Therefore, improvement in any of these steps would contribute to enhance the process cost-efficiency. Studies on Hib cultivation processes available in the literature are rather scarce. Carty and collaborators described a medium containing soy peptone and yeast extract (MP) to be used in the polysaccharide production.7 Batch cultures were carried out at 37 ◦ C and 0.25 volume of air per unit volume of medium per minute (VVM), with constant agitation at 200 rpm, without pH control in both overlay and sparged aeration conditions. The synthesis of polysaccharide was increased in overlay aeration, despite low cellular density.7 MP medium supplemented with hemin and NAD was described in American patents and cultivation was performed with constant agitation at 100 rpm, 37 ◦ C, without pH control.8,9 More recently, Merrit and collaborators described a Hib-cultivation process using casamino acids and yeast extract as nitrogen sources, and hemin (40 mg L−1 ) and nicotinamide adenine dinucleotide (NAD) (20 mg L−1 ), as growth factors.10 The pH was controlled at 7.5, and the dissolved oxygen tension (DOT) was maintained at 50% of saturation by varying the airflow rate (0.6–0.8 VVM) and agitation rate (400–900 rpm); the temperature was kept at 36 ◦ C. Such conditions led to a production of 490 mg L−1 polysaccharide and 2.1 g L−1 dry cell weight (DCW). In summary, so far the capsular polysaccharide production process has been investigated in experiments carried out without pH control, under high or uncontrolled DOT. In Takagi et al. (2000), we first addressed the subject by using the same MP medium for batch cultures with overlay aeration (0.25 VVM) as proposed by Carty et al., without pH control.11 Under these conditions, we observed that cell growth followed a linear pattern, which could indicate some limitation in the medium composition that, ultimately, led to low PRP production. In the present study an optimized culture medium was established and tested in overlay and air-sparged modes. Furthermore, cell growth and PRP formation in cultivations carried out under pH control and DOT maintained up to 30% were also investigated. J Chem Technol Biotechnol 81:182–188 (2006)
MATERIAL AND METHODS Organism Haemophilus influenzae type b GB 3291 strain was kindly provided by the Center for Disease Control and Prevention (Atlanta, GA, USA). The manufacturing master seed was prepared in the Brazilian National Center of Meningitis, Adolfo Lutz Institute, Department of Bacteriology, S˜ao Paulo, Brazil, and the working seed was prepared in our laboratory and stored at −70 ◦ C.11 Inoculum The frozen sample of working seed was thawed, transferred to two chocolate agar-slant tubes and incubated for 18 h at 37 ◦ C in a candle jar. The surfacegrown cells were harvested and resuspended in 2 mL of culture medium. The resulting cell suspension was then transferred to a 2-L Erlenmeyer flask containing 400 mL of fresh culture medium (or a 300-mL Erlenmeyer flask containing 50 mL of fresh culture medium) and incubated at 37 ◦ C in a shaker incubator at 150 rpm for 5–6 h. Shake flask experiments Studies on medium composition improvement were conducted in Erlenmeyer flasks (500 mL) with 100 mL of the original MP or modified MP medium, containing (per liter): 10.0 g soy peptone, 5.0 g dialyzed yeast extract, 13.1 g Na2 HPO4 , 3.3 g NaH2 PO4 , 2.5 g K2 HPO4 , 5.0 g glucose. Modifications of MP medium were made in the NAD and hemin concentrations: (a) original medium MP (control) containing 5.0 mg L−1 NAD and 10 mg L−1 hemin; (b) 20 mg L−1 hemin and 10 mg L−1 NAD; (c) 30 mg L−1 hemin and 15 mg L−1 NAD; (d) 40 mg L−1 hemin and 20 mg L−1 NAD; (e) 80 mg L−1 hemin and 40 mg L−1 NAD. For all experiments, the medium pH was adjusted to 7.5 with 5 mol L−1 NaOH. The medium was sterilized by filtration in a Millipore system with an A20 pre-filter and a 0.22-µm sterile membrane filter (GVWP 293 A20). The Erlenmeyer flasks containing the different culture media were inoculated with 10 mL of bacteria suspension (inoculum) and incubated in a shaker at 200 rpm at 37 ◦ C for 15 h. Cultivations Studies on the influence of the aeration mode and dissolved oxygen tension on PRP productions were carried out as batch cultivations in a 13-L fermentor [Bioflo 2000 (New Brunswick Scientific Co.)] containing 7.6 L of modified MP medium (MMP), which was sterilized as described previously, under agitation (100–700 rpm) at 37 ◦ C. The air supply was maintained at 0.25 VVM, and batch experiments were conducted as follows: (a) overlay aeration without pH control; (b) airsparged aeration and dissolved oxygen tension (DOT) controlled at 10% of air saturation: (1b) without pH control and (2b) with pH controlled at 7.2 and (c) air-sparged aeration and dissolved oxygen 183
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tension (DOT) controlled at 30% air saturation: (1c) without pH control and (2c) with pH controlled at 7.2. During the fermentation process, the pH was controlled by automatic addition of 5 mol L−1 NaOH. The dissolved oxygen tension was measured using a DO meter probe (Mettler Toledo) and adjusted to the desired value by changing the stirring speed. To restore the initial concentration (5.0 g L−1 ) a calculated volume of 50% (w/v) glucose solution was added twice during the batch cultivations when glucose was near depletion. Foaming was controlled by the addition of antifoam UCON LB 652. Analyses Biomass concentration Samples of 10 mL were withdrawn from bioreactor and distributed in pre-weighed centrifuge tubes (in triplicate). After centrifugation at 6000 × g at 4 ◦ C for 60 min, the pellet was resuspended in 10 mL of 0.15 mol L−1 NaCl and submitted to a new centrifugation in the same conditions described above. The supernatants were discarded and the washed cells were dried at 60 ◦ C to constant weight.11 Glucose concentration Glucose concentration was determined by the glucose oxidase method.12 The catabolized glucose was calculated considering the difference between total input and residual glucose present in the medium in every sample-collecting time. PRP concentration Samples of 10 mL were withdrawn from bioreactor and centrifuged at 4000 × g, 4 ◦ C for 60 min. The supernatant were submitted to dialysis (membrane cut-off 12 000–14 000) against distilled water for 24 h in order to eliminate low molecular weight components from the medium. The polysaccharide concentration was measured by the modified Bial method, using ribose as standard.13 The PRP concentration was determined by multiplying the ribose concentration by 2.55, according to the PRP structural formula reported by Crisel et al.2
Organic acids concentrations The metabolic acids produced by Haemophilus were measured by high-performance liquid chromatography (HPLC; Shimadzu Co.) using an automatic sampler, an aminex HPX-87H (300 mm × 7.8 mm; BioRad) type column, an UV detector (210 nm) and an integrator (class VP, version 6.2; Shimadzu Co.). An aliquot of 20 µl of cell-free supernatant, collected during cultivation, was diluted with 0.1 mol L−1 H2 SO4 (1:5), filtered in Millex 0.22 µm and injected into the column at 35 ◦ C. A 0.005 mol L−1 H2 SO4 solution was used as mobile phase with a flow rate of 0.6 ml. min−1 . Kinetics parameters The fermentation parameters were estimated on the basis of the results obtained for biomass formation, glucose consumption, PRP and organic acids production in shake flask experiments and batch cultivations. All parameters were estimated at the end of the growth, when the maximum PRP concentration had been reached. The data collected from the two independent fermentation batches were statistically processed at the 95% confidence interval.
RESULTS Shake flask experiments The results (Table 1) showed that medium 5, containing 80 mg L−1 hemin and 40 mg L−1 NAD, presented the highest biomass concentration (3.6 g L−1 ). However, medium 3, containing 30 mg L−1 hemin and 15 mg L−1 NAD, presented the highest specific production (SPp/x , 103 mg PRP g−1 DCW) and polysaccharide concentration (253 mg L−1 ). Hence, medium 3 [referred to as modified medium MP (MMP)] was the best choice for H. influenzae cultivations. All subsequent experiments in bioreactors were conducted in MMP medium and resulted in exponential growth profiles (Figs 1 and 2). Batch cultivations The effect of dissolved oxygen tension (DOT) on PRP production was studied at two different levels (10 and 30%) in experiments carried out under airsparged mode. For comparison, the results of our reference experiment carried out under air overlay aeration mode, are also presented. The time course
Table 1. Influence of growth factors on polysaccharide and biomass concentrations as well as yield coefficient and productivitya
Experiments 1 (control) 2 3 (MMP) 4 5
Hemin (mg L−1 )
NAD (mg L−1 )
PRP (mg L−1 )
X (g DCW L−1 )
Yp/s (mg PRP g−1 G)
SPp/x (mg PRP g−1 DCW)
10 20 30 40 80
5 10 15 20 40
144.0 ± 5.0 219.5 ± 9.5 253.5 ± 8.5 240.0 ± 8.5 195.5 ± 4.5
1.9 ± 0.1 2.7 ± 0.2 2.4 ± 0.3 2.5 ± 0.1 3.6 ± 0.1
25.1 ± 1.5 28.3 ± 1.1 37.3 ± 1.8 37.6 ± 1.2 21.5 ± 1.1
76.1 ± 4.0 80.4 ± 5.0 103.7 ± 3.5 81.0 ± 4.0 55.2 ± 3.0
a X = biomass; DCW = dry cell; PRP = polysaccharide; G = glucose. Experiments carried out in shake flasks.
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J Chem Technol Biotechnol 81:182–188 (2006)
Improved conditions for polysaccharide production of H. influenzae type b (c) 18
(b)
6
600
4
2
16 glucose consumed (g L-1)
PRP concentration (mg L-1)
Biomass concentration (DCW g L-1)
(a)
500 400 300 200 100
0 5
10 15 20 25 Time (h)
12 10 8 6 4 2 0
0 0
14
0
5
0
10 15 20 25 Time (h)
5
10 15 20 25 Time (h)
Figure 1. Growth, PRP production and glucose consumption in batch cultivations of Haemophilus influenzae carried out without pH control using: overlay aeration (square, ); air-sparged aeration with DOT controlled at 10% (circle, ) and at 30% air saturation. (triangle, ). The arrows indicate glucose addition time. (A) biomass concentration; (B) PRP concentration; (C) consumed glucose.
ž
(a)
(c)
(b) 1100
6
4
2
800 700 600 500 400 300 200
0 0
5
12 10 8 6 4
0 0
10 15 20 25 Time (h)
14
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100 0
18 16
900 Consumed glucose (g L-1)
PRP concentration (mg L-1)
Biomass concentration (DCW g L-1)
1000
5 10 15 20 25 Time (h)
0
5
10 15 20 25 Time (h)
Figure 2. Growth, PRP production and glucose consumption in batch cultivations of Haemophilus influenzae carried out with pH control, using air-sparged aeration with DOT controlled at 10% (circle, ); and at 30% air saturation (triangle, ); and pH control. The arrows indicate glucose addition time. (A) biomass concentration; (B) PRP concentration; (C) consumed glucose.
°
for biomass production (Fig. 1A), PRP production (Fig. 1B) and catabolized glucose (Fig. 1C), in the fermentations carried out without pH control, is shown, and overall kinetics parameters are given in Table 2. In all cases, the exponential growth phase lasted 10–12 h, and the exponential period for PRP production under air-sparged condition was extended by about 2 h. Consequently, the overall PRP production was higher in air-sparged conditions (529–574 mg L−1 ) than in the overlay one (approximately 420 mg L−1 ). In all experiments, the total concentration of glucose supplied was 15 g L−1 . However, the glucose consumption pattern changed according to the cultivation condition used. The glucose consumption at 10% air saturation was 14 g L−1 within 17 h, under which conditions the biomass concentration was significantly higher than at 30% air saturation, which, in turn, catabolized 10 g L−1 glucose in 20 h (Fig. 1C). The biomass yield J Chem Technol Biotechnol 81:182–188 (2006)
on consumed substrate (YX/S ) at 10% was higher (0.33 g DCW g−1 ) than at 30% (0.29 g DCW g−1 ), confirming that this microorganism grows better at low dissolved oxygen levels if pH is not controlled. In contrast, the product yield (YP/S ) at 10% was inferior (44.7 ± 7mg g−1 ) to that at 30% (55.0 ± 0.6mg g−1 ), showing that the oxygen level had the opposite influence on PRP production. The acetic acid concentration reached 7 g L−1 in all above conditions. Cultivations carried out in air-sparged conditions at 10 or 30% showed higher values for the specific production (SPp/x ), 179.9 ± 15 to 193.7 ± 22 mg PRP g−1 DCW, being at least 26–36% better than in overlay conditions (Table 2). In the experiments without pH control, values of pH declined from 7.5 to 6.8 in the end of cultivation. To verify the effect of pH in the PRP production, experiments with DOT maintained at 10 and 30% air saturation and pH controlled at 7.2 by the addition of 185
M Takagi et al. Table 2. Polysaccharide and cell yield coefficients, productivity and maximum specific growth rate in batch cultivations carried out in bioreactors, using modified MP medium, for different aeration modes and dissolved oxygen tension, with and without pH control
Without pH control DOT (% air saturation) Kinetics parameters −1
Maximum biomass concentration, Xmax (g DCW L ) Maximum PRP concentration, PRPmax (mg L−1 ) Maximum acetic acid concentration, HAmax (g L−1 ) Polysaccharide yield coefficient (Yp s−1 ) Cell yield coefficient (Yx s−1 ) Specific production (SPp/x ) Overall PRP productivity (mg PRP L−1 h−1 ) Maximum specific growth rate, µmax (h−1 )
pH control DOT (% air saturation)
Overlay
10
30
10
30
4.3 ± 0.5 420.8 ± 93 7.2 ± 0.6 48.8 ± 11 0.39 ± 0 126.0 ± 38 28.0 ± 1.1 0.38
4.9 ± 0.5 529.74 ± 25 7.7 ± 0.2 44.69 ± 7 0.33 ± 0 179.9 ± 15 22.1 ± 2.0 0.39
3.3 ± 0.1 574.3 ± 66 7.7 ± 0.1 55.1 ± 0.6 0.29 ± 0 193.7 ± 22 29.3 ± 7.0 0.35
3.6 ± 0 984.1 ± 73 9.4 ± 0.3 63.5 ± 3 0.22 ± 0 284.8 ± 14 45.0 ± 4.0 0.36
3.1 ± 0.4 943.3 ± 94 9.3 ± 0.1 68.5 ± 2 0.22 ± 0 312.3 ± 20 41.0 ± 4.0 0.34
The data are given with 95% confidence interval.
(a) 1000
Yp/s = 41.72 and R = 0.99264 Yp/s = 57.65 and R = 0.99585
900
(b) Yp/s = 67.36 and R = 0.98821 Yp/s = 47.02 and R = 0.99085
PRP production (mg L-1)
800 700 600 500 400 300 200 100 0 0
2
4
6
8 10 12 14 16 18
0
2
4
6
8
10 12 14 16 18
Glucose consumption (g L-1) Figure 3. Estimation of polysaccharide yield coefficient on glucose (YP/S ). Air-sparged aeration, with DOT controlled at 10% (A) and at 30% (B) air saturation; without pH control (filled circle, ; filled triangle, ) and with pH control (open circle, ; open triangle, ).
ž
NaOH were carried out. The PRP production values in the experiments with and without pH control were rather similar during the exponential growth phase at the first 10 h of cultivation. However, this similarity completely disappeared when the stationary growth phase was reached, after 15 h of cultivation (Figs 1B and 2B). The PRP production in the cultures performed without pH control followed cell growth, while, at constant pH condition, PRP production continued throughout the stationary phase. In other words, the release of polysaccharide to supernatant observed in the experiments with controlled pH took place in both exponential and stationary growth phases. As a consequence, the overall PRP production under pH control within 22 h of cultivation was about 980–940 mg L−1 (Fig. 2B), being significantly higher than the production without pH control (550–575 mg L−1 ). Although the specific growth rates (µmax ) were nearly the same irrespective of pH control, the PRP specific production (SPp/x ) and PRP yield per consumed glucose (Yp/s ) were significantly higher at the constant pH cultivations 186
°
than the values found for the pH-uncontrolled experiments, as illustrated in Table 2. YP/S calculated from experimental data (Fig. 3) increased from 42 mg PRP g−1 to 58 mg PRP g−1 under pH control, regardless the DOT variation of 10 or 30% air saturation. Furthermore, controlled pH allowed an improved YP/S (PRP synthesis/glucose) conversion ratio (Figs 3A and 3B) as well as extended PRP production for longer within the cultivation period (Figs 1B and 2B). Thus, it was reasonable to infer that capsular polysaccharide synthesis benefited from stabilization of the pH. The glucose consumption rates were almost similar in the experiments performed at 10 and 30% air saturation with (Fig. 2C) and without pH control (Fig. 1C). For both experiments carried out at 10% air saturation, the supplied glucose was almost completely consumed. With the implementation of the pH control, acetic acid concentration increased to about 9.0 g L−1 at the end of the cultivations. The specific production (SPp/x ) and volumetric polysaccharide productivities were 280–300 mg PRP g−1 DCW and 45–41 mg L−1 h−1 , J Chem Technol Biotechnol 81:182–188 (2006)
Improved conditions for polysaccharide production of H. influenzae type b
higher than the 180–194 mg PRP g−1 DCW and 22–29 mg L−1 h−1 without pH control.
DISCUSSION Improved poly(ribosylribitol phosphate) (PRP) productivity was accomplished with three important modifications in the cultivation protocol described by Carty et al:7 medium composition, sparged aeration and pH controlled at 7.2. The MP medium described by Carty et al.7 was shown to be limited for growth of Haemophilus by Takagi et al.11 Moreover, the early study of Lwoff and Lwoff showed that a Haemophilus strain cultivated in a limiting hemin condition presented increased respiratory activity and limited cell growth, proportional to the amount of hemin in the medium.14 Taking all into account, we designed our study so as to test different concentrations of the growth factors, while maintaining both nitrogen sources (soy peptone and yeast extract) at the original values. The best condition turned out to be the one with threefold the original concentration of growth factors (15 mg L−1 NAD and 30 mg L−1 hemin), confirming our hypothesis that NAD and hemin were at limiting concentrations in the original MP medium.11 No significant difference was found in the PRP production at 10 and 30%, which means that capsular polysaccharide formation was not influenced by DOT in the range investigated. Yet, at 10% air saturation, glucose consumption and biomass concentration were higher than at 30% (Figs. 1A and 1C). These data suggest that this microorganism grows better at lower levels of dissolved oxygen. Stabilization of pH, in turn, has allowed PRP production for a longer period (20–24 h) and directed glucose consumption to PRP synthesis, resulting in overall increase from 550 mg L−1 to 950 mg L−1 . Consequently, PRP productivity has almost doubled. Another interesting phenomenon observed upon pH stabilization was that the production of H. influenzae capsular polysaccharide has changed from cell growth associated to cell growth non-associated.11,15 Merrit et al. reported for H. influenzae cultivated with controlled pH 7.2 an overall PRP production much lower (ca 400 mg L−1 ) than ours. Their PRP production was restricted to the cell growth phase, what could ultimately be associated with the high dissolved oxygen tension used in their study (50% air saturation).10 In addition to the high production achieved, the purification of the polysaccharide from the supernatant was performed, revealing that its structure maintained original chemical composition and molecular weight in accordance to the WHO requirement16 (data not shown). HPLC analysis of acid metabolites showed that the main by-product accumulated in the broth during fermentation was acetic acid; this was different from that reported by Tuyau et al.17 and Merrit et al.,10 who J Chem Technol Biotechnol 81:182–188 (2006)
instead had found succinic acid. Acetic acid formation was not influenced by the dissolved oxygen level in the range investigated. However, the acetic acid titer increased in the experiments carried out with pH control (Table 2). There are few studies in the literature addressing the influence of pH on polysaccharide and by-product formation. In general, the milieu pH may affect the cell membrane function, its morphology and structure, causing stress to the cell. The variation of pH usually affects the bacterial metabolism and can provoke changes in end products or in the polysaccharide composition, as described in many different bacteria: Propionibacterium microaerophilum,18 Azospilillum brasiliense19 or Lactobacillus helveticus.20 Furthermore, synthesis of EPS from L. helveticus was improved in low pH environment.20 Morse and Habeler have long demonstrated the importance of environmental pH on the glucose metabolism of Neisseria gonorrhoeae, in which the pH altered the major metabolic routes and, consequently, the relative concentration of end products.21 In cases of a simple polysaccharide, such as cellulose, where a single enzyme is involved in its synthesis, changes in pH affecting the enzyme function directly affect the product formation.15 However, for complex biosynthetic pathways, as for the capsule synthesis of H. influenzae, the metabolic effects of pH variation are not easy to identify. Thus, various genes have been shown to be involved in the regulation and synthesis of bacterialcapsular polysaccharides.22 In H. influenzae, a gene amplification mechanism was found responsible for increased capsule production.23 It is therefore possible that, in this bacterium, the pH and the level of oxygen could affect gene expression and regulation of enzymes involved in the polysaccharide synthesis. In conclusion, by increasing the concentration of growth factors and maintaining the pH of sparged cultures at 7.2, the production of PRP was significantly enhanced. As both modifications do not require additional reagents or equipment, the present data would provide an important contribution for large scale processes without elevating the costs.
ACKNOWLEDGMENTS We gratefully acknowledge financial support from The State of S˜ao Paulo Research Foundation (FAPESP), S˜ao Paulo, Brazil (grant 00/08464-6) and the Programa de Apoio ao Desenvolvimento Cient´ıfico ´ e Tecnologico (PADCT)/Financiadora de Estudos e Projetos (FINEP). We also thank Lourivaldo In´acio de Souza and Inˆes do Amaral Maurelli for technical assistance.
REFERENCES 1 Norden CW, Haemophilus influenzae type b, in Bacterial infections of humans, ed by Evans AS and Feldman HA. Plenum, New York, pp 259–273 (1982). 187
M Takagi et al. 2 Crisel RM, Baker RS and Dorman DE, Capsular polymer of Haemophilus influenzae, type b. I–Structural characterization of the capsular polymer of strain Eagan. J Biol Chem 250:4926–4930 (1975). 3 Schuchat A, Robinson K, Wenger JD, Harrison LH, Farley M and Reingold AL, Bacterial meningitis in the United States in 1995. N Engl J Med 337:970–976 (1997). 4 Wenger JD, Epidemiology of Haemophilus influenzae type b disease and impact of Haemophilus influenzae type b conjugate vaccines in the United States and Canada. Pediactr Infect Dis J 17:S132–136 (1998). 5 Guia de Vigilancia Epidemiologica. Meningite por Haemophilus influenzae. Cap 5.21. www. Funasa.gov.br [accessed 1 May 2003]. 6 Breukels MA, Rijkers GT, Voorhorst-ogink MM and Zeger BJM, Regulatory T cells in the antibody response to Haemophilus influenzae type b polysaccharide. Infec Immun 67:789–793 (1999). 7 Carty CE, Mancinelli R, Hagopian A, Kovach FX, Rodriguez E, Burke P, Dunn NR, McAleer WL, Maigetter RZ and Kniskern P, Fermentation Studies with Haemophilus influenzae. Dev Indust Microbiol 26:763–767 (1985). 8 Hilleman MR, Tai JY and Tolamn RL, Coupled Haemophilus influenzae type b vacine. US Patent 4 459 286 (1984). 9 Marburg S, Kniskern PJ and Tolman RL, Covalently-modified bacterial polysaccharides, stable covalent conjugates of such polysaccharides and immunogenic proteins with bigeneric spacers and methods of preparing such polysaccharides and conjugates and of confirming covalency. US Patent 4 882 317 (1989). 10 Merrit J, Allard G, O’Toole L, Swartz R and Licari P, Development and scale-up of a fed-batch process for the production of capsular polysaccharide from Haemophilus influenzae. J Biotechnol 81:189–197 (2000). 11 Takagi M, Cabrera-Crespo J, Baruque-Ramos J, Raw I, Zangirolami TC and Tanizaki MM, Characterization of polysaccharide production of Haemophilus influenzae type b and its relationship to bacterial cell growth. Appl Biochem Biotech 110:91–100 (2003).
188
12 Dahlqvist A, Determination of maltose and isomaltose activities with a glucose-oxidase reagent. Biochem J 80:547–55 (1961). 13 Ashwell G, Colorimetric analysis of sugar, in Methods of Enzymology, Vol. III. Academic Press, New York, p 87–90 (1957). 14 Lwoff A and Lwoff M, Role physiologique de l’hemine pour H influenzae. Ann Inst Pasteur 59:129 (1937). 15 Anderson P, The synthesis and release of polyribophosphate by Haemophilus influenzae type b in vitro. Infect Immun 13:581–589 (1976). 16 World Health Organization, Requirements for Haemophilus influenzae type b conjugate, Technical Report Series, No 814, Annex 1 (1991). 17 Tuyau JE, Sims W and Williams RAD, The acid end products of glucose metabolism of oral and other Haemophili. J Gen Microbiol 130:1787–1793 (1984). 18 Kouss´emon M, Combet-Blanc Y and Ollivier B, Glucose fermentation by Propionibacterium microaerophilum: effect of pH on metabolism and bioenergetic Curr Microbiol. 46:141–145 (2003). 19 Konnova SA, Fedonenko YP, Makarov OE and Ignatov VV, Effect of cultivation conditions on the composition of extracellular polysaccharide-containing substances in the bacterium Azospirillum brasilense. Biol Bull 30:430–437 (2003). 20 Torino MI, Taranto MP, Sesma F and Font de Valdez G, Heterofermentative pattern and exopolysaccharide production by Lactobacillus helveticus ATCC 15 807 in response to environmental pH. J Appl Microbiol 91:846–852 (2001). 21 Morse SA and Hebeler BH, Effect of pH on the growth and glucose metabolism of Neisseria gonorrhoeae Infect Immun 21:87–95 (1978). 22 Dillard JP, Vandersea MW and Yother J, Characterization of the cassette containing genes for types 3 capsular polysaccharide biosynthesis in Streptococcus pneumoniae. J Exp Med 181:973–983 (1995). 23 Gilsdorf JR, Antigenic diversity and gene polymorphisms in Haemophilus influenzae. Infect Immun 66:5053–5059 (1998).
J Chem Technol Biotechnol 81:182–188 (2006)
