A Study on Production of Poly-β-Hydroxybutyrate Bioplastic from Sago Starch by Indigenous Amylolytic Bacteria

Bacillus sp. PSA10 and Bacillus sp. PPK5 were two indigenous strain amylolytic bacteria from SoutheastSulawesi that have ability to produce bioplastic poly-β-hydroxybutyrate (PHB) from sago starch. The study wasattempted to determine the mechanism of PHB production by bacteria amylolytic was grown on sago starchcontainingmedia. Two amylolytic bacteria i.e. Bacillus sp. PSA10 and Bacillus sp. PPK5 was grown for 168 hin a mineral salts medium with sago starch as carbon source. Growth of amylolytic bacteria was monitoredby cell dry weight. Extraction of PHB was done by N-hexane acetone-diethyl ether method and PHB contentwas quantifi ed with UV spectrophotometer at 235 nm. Glucose level was determined by using kit of glucoseGOD 10” and was quantifi ed with spectrophotometer at 500 nm. Sago starch concentration was determinedby phenol method using specthrophotometer at 490 nm. The result of the study showed that Bacillus sp.PSA10 was produced PHB up to 66,81 % (g PHB/g cell dry weight) at 48 h and Bacillus sp. PPK5 up to 24,83% (g PHB/g cell dry weight) at 84 h. Bacillus sp. PSA10 has ability to converse sago starch to be PHB directlywithout glucose accumulation in the media, whereas Bacillus sp. PPK5 have to accumulate glucose as productof sago starch hydrolysis to produce of PHB. PHB synthesis by Bacillus sp. PHB production on sago starchof the Bacillus sp. PSA10 was found to be growth-associated whereas Bacillus sp. PPK5 was found to be nongrowth-associated. Therefore, two indigenous amylolytic bacteria were having of difference in biosynthesismechanism of PHB in sago starch medium and their characteristics of PHB synthesis should be consideredin developing cultivation methods for the effi cient production of PHB. Keywords : Production, PHB, Amylolytic bacteria, Sago starch.

Optimizing Sporulation of Clostridium perfringens

Many sporulation media have been developed for Clostridium perfringens, but none stimulates sporulation for all strains. The aim of our experiments was to develop a sporulation method using Duncan and Strong (DS) medium, which supports sporulation of a wide variety of strains. Different inoculation levels were tested, and the effects of sporulation-promoting substances and acid shock were evaluated. Furthermore, DS medium was compared with other sporulation media. Highest spore numbers in DS medium were obtained with a 10% 24-h fluid thioglycollate broth inoculum (5.0 × 105/ml). Addition of theophylline and replacement of starch by raffinose increased spore yields for some strains, but most strains were not affected (average increases in log N/ml of 0.2 and 0.3, respectively). One strain was enhanced by the addition of bile, but other strains were strongly inhibited (average decrease in log N/ml of 2.5); agar did not influence sporulation. Neither short-time acid exposure nor addition of culture supernatant fluids of well-sporulating strains resulted in higher spore numbers in DS medium. None of the tested methods enhanced sporulation in general; only strain-dependent effects were obtained. Peptone bile theophylline medium was the most promising sporulation medium tested; peptone bile theophylline starch medium yielded highest spore numbers (2.5 × 105/ml), but some strains failed to sporulate. In conclusion, adding theophylline to DS medium may optimize sporulation of C. perfringens, but peptone bile theophylline medium with or without starch is most suitable.

Differential glucoamylase expression in Schwanniomyces castellii induced by maltose

Different levels of glucoamylase expression occurred when Schwanniomyces castellii strain 1402 was shifted during growth on glucose to either glucose, maltose, or soluble starch medium. Extracellular glucoamylase activity was greatest from cells grown on maltose (~22×), slightly less on soluble starch (~16×), and least on glucose (1×). Glucoamylase biosynthesis was further studied by labelling of total proteins in vivo with [35S]methionine and immunoprobing with a polyclonal anti-glucoamylase antibody. Mature active glucoamylase is 146 kDa. Maltose cultures expressed four cellular (75, 78,138, and 146 kDa) and two extracellular (78 and 146 kDa) polypeptides. Neither the 138- nor the 146-kDa products were detected in cells in the presence of glucose; the 78-kDa product is expressed at approximately 5% the level obtained from cells in maltose. The 146-kDa glucoamylase is expressed within 30 min after transfer of cells from glucose- to maltose-containing medium. This expression appears to be cell growth and concentration independent and is therefore similar to galactose-inducible enzyme expression in Saccharomyces cerevisiae.

The production of constitutive invertase and inulinase by the mushroom Panaeolus papillonaceus in submerged culture

Five different mushrooms were screened for extracellular inulinase and invertase activities. The enzymes were found to be produced constitutively by the mushrooms, but the carbon source, glucose, sucrose, and starch, in the medium influenced the production of individual enzymes. The highest producers of inulinase and invertase were Panaeolus papillonaceus growing in starch medium and Agaricus bisporus growing in glucose medium, respectively. A medium for the optimum production of invertase and inulinase by P. papillonaceus was developed by determining the optimum concentrations of suitable carbon and nitrogen sources. Some of the physicochemical properties of the enzymes present in the culture filtrate were studied. Both of the activities have pH optima at 6.5 and temperature optima at 60 °C. Km values for both enzyme activities were found to be identical at 2.5 mg of substrate/mL, whereas the νm for the inulinase activity was more than two times greater than that for invertase activity.