Improved cultivation conditions for polysaccharide production by H. influenzae type b

Abstract

AbstractHaemophilus 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−1h−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. Copyright © 2005 Society of Chemical Industry