Effect of cultivation conditions on spore production from Bacillus amyloliquefaciens B128 and its antagonism to Botrytis elliptica.

AIMS: To maximize spore production by Bacillus amyloliquefaciens B128, and its antagonism to the fungal pathogen Botrytis elliptica B061. METHODS AND RESULTS: In the 5-l stirred-tank bioreactor (STR), with the 0.5 vvm aeration rate, an agitation rate of 200 rev min(-1) significantly enhanced the spore yield compared to the same in 300 rev min(-1) cultivations. In a 20-l airlift bioreactor (ALR) the maximal spore production was further increased with a controlled aeration rate of 2.5 vvm operated in a 24-mesh net-draft tube mode, and no pH control cultivation. This spore yield in the 20-l ALR was five- and eightfold higher; in addition the cultivation period was 19 h shorter, compared to that obtained from shaker flask and in the 5-l STR cultivations respectively. CONCLUSIONS: Although culture conditions are still to be optimized, by using an ALR with net-draft tube, a scaling up from shaker flasks and STR to ALR of spore production by the strain B128 is technically feasible. SIGNIFICANCE AND IMPACT OF THE STUDY: The spore yields obtained using bioreactors were much higher than those previously reported. The freshly produced spore preparations from the B128 strain significantly antagonized the grey mould pathogen B. elliptica.

Percutaneous transhepatic gall bladder drainage: a better initial therapeutic choice for patients with gall bladder perforation in the emergency department.

OBJECTIVES: To investigate clinical features and outcomes in patients with acute cholecystitis with gall bladder perforation receiving open cholecystectomy or percutaneous transhepatic gall bladder drainage in the emergency department. METHODS: From 1996 through 2005, 33 patients with non-traumatic gall bladder perforation, among 585 patients with acute cholecystitis, were enrolled. Patients were divided into two groups: open cholecystectomy in 16 patients and percutaneous transhepatic gall bladder drainage in 17 patients. Medical records, including demographic data, past history of systemic diseases or gallbladder stones, initial clinical presentations, laboratory data, physical status, therapeutic interventions, and outcomes, were analysed. RESULTS: Mean patient age was 72.6 years (range 54-92 years). 28 patients (84.8%) were male. Median time of symptom onset before emergency department diagnosis was 5 days (range 0.5-30 days). Estimated incidence of gall bladder perforation was 5.6% (33/585). 27 patients (81.8%) had gallstones operatively or in image studies. All patients had either right upper quadrant pain/tenderness or epigastric pain/tenderness. Only 9 (27.3%) patients had positive Murphy's sign. Six patients in the percutaneous transhepatic gall bladder drainage group received further open cholecystectomy. Overall mortality was 24.2% (8/33). The direct cause of death was disease related sepsis in all patients. Patients receiving percutaneous transhepatic gall bladder drainage had a higher survival rate than those receiving open cholecystectomy (100% vs 50%, p<0.001). No differences in complications and length of hospital stay of survivors were observed between groups. CONCLUSIONS: In this study, we delineated clinical features of patients with gall bladder perforation. Better clinical outcome is observed for percutaneous transhepatic gall bladder drainage, and this is suggested as an initial therapeutic choice, especially in high risk patients who are likely to need surgery.

Exchange bias dependence on interface spin alignment in a Ni80Fe20/(Ni,Fe)O thin film.

A Ni80Fe20/(Ni,Fe)O thin film exhibits a positive exchange bias when cooled in a zero field and a negative exchange bias when field cooled. With transmission electron microscopy and electron energy loss spectrometry, the composition and magnetic structure has been ascertained and a distribution of magnetization easy axes about the interface extrapolated. The results indicate that the positive exchange bias is from antiferromagnetic interface moments perpendicular to their ferromagnetic counterparts. With field cooling the alignment is put into a parallel configuration resulting in a negative exchange bias.

Effect of rice--glycerol complex medium on the production of Lovastatin by Monascus ruber.

Response surface methodology (RSM) was employed to study the effect of the composition of the rice-glycerol complex medium on the production of lovastatin (Lvs) by the ascomycete Monascus ruber in mixed solid-liquid (or submerged) cultures at 25 degrees C. Four components (rice powder, peptone, glycerol, glucose) were studied to evaluate the approximate polynomial for all dependent variables, explaining their effects on the production of Lvs. The best composition derived from RSM regression was (in g/L) rice powder 34.4, peptone 10.8,, glucose 129, KNO3 8.0, MgSO4.7H2O 4.0 and glycerol 36.4 mL/L. With this composition, the Lvs production was 157 mg/L after 10 d of cultivation. In comparison with glycerol and glucose, the rice powder becomes a more suitable carbon source and represents a great potential for the production of Lvs.

Production of cyclodepsipeptides destruxin A and B from Metarhizium anisopliae.

Maltose and peptone were the best carbon and nitrogen sources for the production of destruxins from Metarhizium anisopliae. With the addition of 0.1% (w/v) beta-alanine to the basal medium, the yields of cyclodepsipeptides DA and DB were 7.2 and 279 mg/L, respectively, which was 2-fold higher than that of control experiment. Response surface methodology (RSM) was applied to optimize the compositions of maltose, peptone, beta-alanine, and glucose used in a shaker-flask cultivation of M. anisopliae for the production of DA and DB. Estimated optimal compositions for the DA production were maltose 2.58%, peptone 0.72%, beta-alanine 0.02%, and glucose 0.55%. The predicted DA yield was 18.5 mg/L. On the other hand, the optimal compositions for DB production were maltose 2.51%, peptone 0.75%, beta-alanine 0.02%, and glucose 0.43%. A maximum DB yield of 232 mg/L was predicted. These were confirmed by cultivation experiments conducted at the optimized conditions for maximum destruxins production in a shaker-flask. Furthermore, a modest high level of DA (49 mg/L) and DB (268 mg/L) yields were obtained by employing the response surface methodology optimized DB production medium in a no-baffle, stirred-tank fermentor.

Characterization study of the sporulation kinetics of Bacillus thuringiensis.

A wild-type and an rDNA strain of Bacillus thuringiensis were cultured in a net-draft-tube modified 20-L airlift bioreactor. A comparison of the sporulation patterns suggests that the early sporulation strain has a lower final spore count. Results from off-gas analysis suggests that the CO(2) profile could be an alternative indication to spore counts for the examination of fermentation performance or even the mortality in bioassay of the cultivation product. The difference in mortality tests exhibited by the microorganism was attributed to different patterns of sporulation as well as different levels of gene control inside the cell itself. The sporulation kinetics of B. thuringiensis was simulated by a simple modified Hill equation, where the initial glucose concentration could affect the timing of the onset of sporulation. The equation matches well with the experimental sporulation data for B. thuringiensis in both wild-type and rDNA strains.

Quantitative analysis of thuringiensin by high-performance liquid chromatography using adenosine monophosphate as an internal standard.

An analytical method for thuringiensin using adenosine monophosphate (AMP) as an internal standard is established. AMP, with high stability and availability, is an appropriate internal standard for thuringiensin quantitative analysis using high-performance liquid chromatography and ultraviolet absorbance detection at 260 nm. A good correlation between the concentration of thuringiensin and the peak-area ratios of thuringiensin to AMP is demonstrated. From this general equation of linear regression line, the concentration of thuringiensin can be assessed in fermentation broth or semi-purified product.

Production of thuringiensin from Bacillus thuringiensis using a net-draft-tube, modified airlift reactor.

A net-draft-tube, modified airlift reactor and a stirred-tank reactor were used for thuringiensin production by Bacillus thuringiensis subsp. darmstadiensis growing with various concentrations of molasses. The optimum concentration of molasses for thuringiensin production in both reactors was 15 g/l. There was a 6 h delay in sporulation in the modified airlift reactor compared with that in the stirred-tank reactor. Thuringiensin yield in the modified airlift reactor (2.2 g/l) was consequently higher than that in the stirred-tank reactor (1.1 g/l).

Fed-batch culture of Bacillus thuringiensis for thuringiensin production in a tower type bioreactor.

Fed-batch culture of Bacillus thuringiensis in a modified airlift reactor has been developed by using adaptive control of glucose concentration in the reactor. The glucose concentration was estimated via a correlation equation between carbon dioxide production rate and glucose consumption rate. The estimated glucose concentration as the output variable was fed back to computer for calculation of substrate addition. The modified reactor was an airlift reactor with a net draft tube. The airlift reactor had high oxygen transfer rate and low shear stress which were important factors for production of thuringiensin. Fed-batch culture of Bacillus thuringiensis in the modified airlift reactor provided significant improvement of thuringiensin production.

Penicillin-G enhanced production of thuringiensin by Bacillus thuringiensis sp. darmstadiensis.

The effect of penicillin-G on the production of the potential microbial insecticide thuringiensin by Bacillus thuringiensis sp. darmstadiensis was studied. Shake flask and 3-L jar fermentor studies showed that the addition of 360 units/mL penicillin-G at 9 h, when the fermentable sugar in the medium was about to be mostly consumed, improved thuringiensin production by more than 1-fold relative to the control. The dosage of 360 units/mL penicillin-G had only a modest effect on the growth of the microorganism. However, cell growth was inhibited at higher dosages of the antibiotic. Since penicillin-G could interfere with cell wall synthesis, which facilitated the release of thuringiensin, a high thuringiensin productivity of 2600 mg/L was attained in this study, which is about 2-10-fold higher than those values reported in the literature.