Optimization of medium and cultivation conditions for capsular polysaccharide production by Streptococcus pneumoniae serotype 23F

The influence of medium composition and culture conditions on Streptococcus pneumoniae serotype 23F cultivation was investigated in order to develop an industrial method for polysaccharide (PS) production. Acid-hydrolyzed casein (AHC) and dialyzed enzymatically hydrolyzed soybean meal (EHS) were investigated as nitrogen sources, and the vitamin solution of Hoeprich's medium and dialyzed yeast extract as vitamin sources. The influence of initial glucose concentration was also evaluated. In flask experiments, the best nitrogen source for PS production was AHC; EHS yielded small amounts of PS without interfering with bacterial growth. Dialyzed yeast extract provided an approximately 2-fold increase in PS production when compared to Hoeprich's vitamin solution. In a 5-l bioreactor, it was observed that the pneumococcus did not grow under aerobic conditions, CO(2) did not increase PS yield, glucose was inhibitory above 30 g l(-1), and the main glucose catabolism product was lactate, which had an inhibitory effect on cell growth. When anaerobic cultivation was performed under N(2) flow using the optimized medium, 240 mg l(-1) of soluble PS was obtained, which represents a 3-fold increase in yield as compared to that described in the published patent [Yavordios and Cousin (1983) European Patent 0 071515 A1]. Application of these results would considerably simplify upstream and downstream processes for PS production.     

Physiological factors affecting streptomycin production by Streptomyces griseus ATCC 12475 in batch and continuous culture

Abstract Conditions of growth are described for the production of streptomycin by Streptomyces griseus ATCC 12475 using chemically defined minimal medium and complex medium. It was found using batch cultures that early synthesis of the antibiotic occurred during growth in minimal medium but was delayed until the onset of stationary phase in complex medium. This effect was independent of whether spores or vegetative cells were used as inoculum. Stability of streptomycin biosynthesis in continuous culture was dependent on dilution rate and medium employed. Cultures were highly unstable when grown on complex medium but could be maintained in steady states in continuous culture using minimal medium when the dilution rate was increased in a stepwise manner, starting at a dilution rate of 0.02 h⁻¹ (15% of μ _max). The effect of changing dilution rate on growth, streptomycin production and the level of streptomycin phosphotransferase was examined using this technique.     

Introduction of air in the anaerobic culture of Streptococcus pneumoniae serotype 23F induces the release of capsular polysaccharide from bacterial surface into the cultivation medium

AIM: An approach to increase Streptococcus pneumoniae capsular polysaccharide (CPS) in the culture medium during fed-batch cultivation in bioreactor. METHODS AND RESULTS: Streptococcus pneumoniae serotype 23F was cultivated in a 5-l bioreactor with nitrogen-sparging and followed by addition of air in the stationary phase. The amount of CPS released in the supernatant progressively increased under air sparging. The profile of cellular viability and optical density was similar in both cultures. Immunoelectron microscopy showed that the amount of tightly cell-bound CPS was higher in bacteria cultivated under nitrogen than under air. CONCLUSIONS: The stress caused by the addition of air at the stationary phase promoted a large increase of free CPS into the medium, as a consequence of the morphologic change in the capsule. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of air in the stationary phase of the culture would greatly simplify the subsequent downstream process, allowing CPS purification from the supernatant. The direct consequence of this process improvement is the reduction of vaccine production costs.     

Hyaluronic acid production and molecular weight improvement by redirection of carbon flux towards its biosynthesis pathway

Hyaluronic acid (HA) production in Streptococcus zooepidemicus competes for the carbon source along with biomass formation, lactate formation (via glycolysis) and pentose phosphate pathway (PPP). In our studies, increase in HA molecular weight was observed by redirecting the carbon flux towards HA biosynthesis pathway by partially inhibiting the glycolytic pathway. Batch bioreactor (1.2 L) studies showed that with the addition of 25 μM sodium iodoacetate, 5 g/L tryptophan and 10 g/L pyruvate, which are glycolytic inhibitors, HA molecular weight increased to 3.2, 3.2 and 3.1 MDa respectively compared to control run (2.4 MDa). Yield coefficients Y_HA/S and Y_LA/S showed inverse relationship, indicating competition for glucose between HA and lactic acid formation. Addition of 5 g/L glutamine along with 25 μM sodium iodoacetate also increased the HA concentration to 5.0 g/L from 2.0 g/L in control run. Metabolic flux analysis studies show that concentration and molecular weight of HA is increased by decreasing carbon flux towards glycolysis and PPP and increasing carbon flux towards HA precursor formation. It was observed that specific growth rate of the cells correlated positively to the specific HA production rate and negatively to the molecular weight of HA produced. Addition of antioxidant tannic acid also increased molecular weight to 3.0 MDa.     

DBT desulfurization by decorating Rhodococcus erythropolis IGTS8 using magnetic Fe3O4 nanoparticles in a bioreactor

Today, crude oil is an important source of energy and environmental contamination due to the continued use of petroleum products is a matter or urgent concern. In this work, two technological platforms, namely, the use of a robust desulfurizing bacteria and the use of nanotechnology to decorate the surface of the bacteria with nanoparticles (NP), were combined to enhance biodesulfurization (BDS). BDS is an ecologically friendly method for desulfurizing petroleum products while avoiding damage to the hydrocarbons due to the high temperatures normally associated with physical desulfurization methods. First, a bacterium known to be a good organism for desulfurization (Rhodococcus erythropolis IGTS8) was employed in cell culture to remove a recalcitrant sulfur molecule from a common sulfur‐containing compound found in crude petroleum products (dibenzothiophene). 2‐Hydroxybiphenyl (2‐HBP) produced as a consequence of the BDS of dibenzothiophene was determined using Gibbs’ assay. The synthesized NP were characterized by field emission scanning electron microscope, transmission electron microscopy, Fourier transform infrared spectroscopy, X‐ray diffraction spectroscopy, and vibrating sample magnetometer. The field emission scanning electron microscope and transmission electron microscopy images showed the size of the NP is 7–8 nm. The decorated cells had a long lag phase, but the growth continued until 148 h (at OD₆₀₀ = 3.408) while the noncoated bacteria grow until 96 h before entering the stationary phase at OD₆₀₀ = 2.547. Gibbs’ assay results showed that production of 2‐HBP by decorated cells was 0.210 mM at t = 148 h, while 2‐HBP production by nondecorated cells was 0.182 mM at t = 96 h. Finally, the experiments were repeated in a fermenter.     

Stress-induced morphological and physiological changes in γ-linolenic acid production by Mucor fragilis in batch and continuous cultures

Batch and continuous cultures conditions were studied in order to increase γ-linolenic acid production by Mucor fragilis CCMI 142, in response to the presence of ethanol. Continuous cultures were used to add ethanol pulses to steady state pellet cultures. It was demonstrated that pellet size, which allowed homogeneous fungal cultures, can be obtained by means of pH adjustment, thus enabling steady state continuous growth at 2.90±0.05.

The 5 and 2% (v/v) ethanol pulses induced hyphal morphological changes with arthrospore formation. A 1% (v/v) pulse of ethanol did not immediately affect growth, but induced morphological changes, which led to autolysis at the pellet core. A 0.5% (v/v) pulse of ethanol did not affect neither the morphology nor the physiology of the microorganism to any significant extent. The 0.5 and 1% (v/v) ethanol pulses resulted in an increase in the proportion of γ-linolenic acid production up to 11%. Data from batch cultures showed a higher enhancement of ethanol, attaining 30% of γ-linolenic acid.

The increase of γ-linolenic acid content observed in batch and continuous conditions appears to be a response associated with stress induced by the ethanol which seems to be of value as an industrial medium component.