Production of Pullulanase by Raoultella Planticola Dsmz 4617 Using Sago Starch as Carbon Source

Production of pullulanase by Raoultella planticola DSMZ 46 17 was studied in batch fermentation using sago starch as a carbon source. The fermentations were carried out, firstly, in 500-mL Erlenmeyer flasks to search for a suitable cultivation medium for pullulanase production by this gram negative bacterium. For comparison, production of pullulanase by a locally isolated strain was also carried out. Secondly, further improvement of pullulanase production by R. planticola DSMZ 46 17 was done by using response surface methodology (RSM). The factors studied were sago starch, peptone and initial culture pH. The precise values of carbon-to-nitrogen (C/N) ratio that affect the pullulanase secretion by this gram-negative bacterium were also studied. Then, batch cultivations in 2-L stirred tank fermenter were carried out in an attempt to further improve pullulanase production by R. planticola DSMZ 461 7. Two important hydrodynamic parameters, i.e., aeration and agitation, were studied in detail. Unstructured model based on logistic and Luedeking-Piret equations were used to describe growth and pullulanase production by R. planticola DSMZ 4617 in both 500-mL shake flask and 2-L stirred tank ferrnenter. Cultivation of R. planticola DSMZ 4617 in modified mineral Czapek medium was found able to produce substantially high activity of pullulanase (320 times higher) as compared to 'Ohba-Ueda' medium. Among various carbon and nitrogen sources tested, sago starch and peptone were the best substrates for enzyme production, and under these conditions, R. planticola DSMZ 4617 produced 0.95 U/mL of pullulanase at initial culture pH around 7 and incubation temperature of 30°C. The partially purified pullulanase from R. planticola DSMZ 461 7 was optimally active at pH 6 to 7, and 50°C with stability ranges from pH 5 to 10. As compared with R. planticola DSMZ 4617, the local isolate B. cereus H1.5 was found to produce substantially high activity of protease during growth. This was the main reason that much of the pullulanase activity was lost during cultivation and partial purification processes and thus this local isolate is not appropriate for industrial applications. Therefore, the research work was focused on R. planticola DSMZ 4617. The RSM experiments based on central composite design (CCD) were found practical to derive a statisticai model for enhancement of pullulanase production by R. planticola DSMZ 4617. From this study, about 1.8-times of increment in pullulanase activity (1.70 U/mL) was achieved at 6.12 g/L sago starch, 15.34 g/L peptone and initial pH 7.23. Studies on C/N ratio hrther confirmed that the highest pullulanase production was obtained at ratio of 0.97 which correspondes to approximately 6.1 g/L of starch and 15.3 g/L peptone.The pullulanase productivity and yield were greatly influenced by the aeration and agitation conditions within the fennenter. High pullulanase activities in the fermenter were observed at aeration rate of 0.5 vvm and agitation speed of 250 rpm. Under this condition, the pullulanase production results were: pullulanase activity, 2.22 UImL; pullulanase productivity, 0.015 U/mLih and pullulanase yield, 369 U pullulanase g starch-'. Themodels proposed in this study fit significantly well to the experimental data with more than 95% confidence. This means that the proposed model can be used to explain growth and enzyme production at different chemical and physical conditions in a concise form which is comprehensible to those who wish to make use of the results. From the study, pullulanase production by R . - + + - .- _._ _ -+- . - -- - planticola DSMZ 4617 was found to be a non-growth associated process (a = O), where accumulation of pullulanase in the culture fluid occurred only during the nongrowth phase. An improved pullulanase fermentation process by R. planticola DSMZ 4617 has been successfully developed and it showed approximately 7-times increment of pullulanase production (2.22 UImL) in 2-L stirred tank fernenter with optimized medium composition and culture conditions as compared to cultivation employing original medium without optimized formulation in 500-mL shake flask (0.32 U/mL)

Anoxic-Aerobic Stabilization of Settled Activated Sludge from A Sew Age Treatment Plant

Conventional aerobic sludge stabilization is a power-intensive process. Another major upset is the resulting drop in mixed liquor pH (MLpH). Furthermore, the high concentration of ammonical nitrogen CN"H4-N) , nitrate nitrogen (N03-N), nitrite nitrogen (N02-N) and ortho-phosphate (P04-P) in the filtrate, could be a strong water-polluting agent. In this study, the anoxic-aerobic stabilization system incorporates, at regular intervals, non-aerated periods during aerobic stabilization. Four different stabilization modes (anoxic-aerobic, anoxic-aerobic with methanol, aerobic with lime control and aerobic) were applied to remove both the mixed liquor volatile suspended solids (MLVSS) and soluble nutrients simultaneously. Both the anoxic-aerobic systems were found to have higher solids decay rate, i.e., 0.064 dai1 and 0.057 dai1, respectively, as compared with lime-controlled (0.049 day-I) and aerobic systems (0.033 day-I). Anoxic-aerobic stabilization also maintained neutral MLpH levels at 'no cost', which is superior to daily lime dosing. The unfavorable environment within the aerobic digester (pH 5.51 - pH 6.59) is believed to retard the endogenous metabolisms of the sludge. The aerobic digester showed a significantly high build-up of NH4-N (95% higher) but lower concentrations in both anoxic-aerobic and lime-controlled systems. Since both the anoxic-aerobic digesters experienced anoxic conditions on a regular basis, the peak nitrate were much lower (2.6 mg/L in anoxic-aerobic digester and 1.4 mg/L in anoxic-aerobic digester with methanol). The addition of methanol did accelerate the denitrification process. In contrast, nitrate levels in the lime-controlled digester and aerobic digester were greater than 30.0 mg/L, which do not comply with the World Health Organization standards. Since P04-P is not lost from the digester as gas, the concentration in the four digesters increased with digestion time. The lowest P04-P levels in the lime-controlled digester could be a function of calcium phosphate fonnation. In contrast, the highest level of P04-P in the fully aerobic digester was due to the low MLpH. Alkalinity was consumed and produced during the anoxic-aerobic stabilization process, thus, there was no net change in the alkalinity level. The experimental results indicate that the anoxic-aerobic digestion system is definitely a suitable method for the stabilization of sludge, in terms of solids reduction and soluble nutrients removal

Pullulanase type II from a local Bacillus isolate H1.5 : isolation, production and characterization of the enzyme

Enzymes that specifically break down the branch points – debranching enzymes – have been known for more than a decade. Pullulanase (EC 3.2.1.41, pullulan 6- glucanohydrolase), which was first reported in Aerobacter aerogenes splits the α-1,6- glucosidic linkages in pullulan and give rise finally to maltotriose (Yuzuru and Masaharu, 1983). In most cases, pullulanase can also cleave the α-1,6 link of other branched polysaccharides, such as starch, glycogen and maltodextri

Nutritional composition of unripe and ripe freeze-dried Terung Asam from Sarawak

This study aimed to assess and compare the nutritional composition between unripe and ripe freeze-dried Terung Asam from Sarawak. Terung Asam was checked for its maturity upon collection. Whole fruits were freeze-dried and used for proximate analyses. The redness/greeness (a*) of unripe and ripe fresh Terung Asam used in this study were -14.20 ± 3.11 and 12.57 ± 1.60 respectively. The moisture content of unripe fresh Terung Asam was significantly higher than ripe fresh Terung Asam (p < 0.05). There were statistically significant differences in crude protein (p < 0.05), crude fat (p < 0.05), ash (p < 0.05) and crude fibre (p < 0.001) between unripe and ripe freeze-dried Terung Asam (UFTA and RFTA respectively). RFTA had higher crude fibre (19.87 ± 0.68g/100g) and ash (6.20 ± 0.18g/100g) than UFTA (13.86 ± 0.58g/100g crude fibre, 5.43 ± 0.12g/100g ash). In contrast, UFTA was higher in protein (10.77 ± 0.30g/100g) and fat (0.74 ± 0.05g/100g) than RFTA (9.43 ± 0.40g/100g crude protein, 0.48 ± 0.08g/100g fat). The results of this study suggested that whole fruits of UFTA and RFTA were good source of crude protein, crude fibre and mineral. The data gathered from this research can contribute to the body of knowledge of this underutilized indigenous fruit as well as becoming useful for future product development using Terung Asam

Application of a high density adsorbent in expanded bed adsorption of lipase from Burkholderia pseudomallei

The application of STREAMLINE Direct HST adsorbent in expanded bed adsorption of lipase from Burkholderia pseudomallei was explored in this study. Scouting of optimum binding and elution condition was performed in batch binding mode. Theaddition of 0.2 M salt in acetate buffer (pH 5) during adsorption has increased thespecificity and quantity of lipase binding onto the adsorbent. The addition of 0.4 M salt in phosphate buffer (pH 7) achieved the highest purification fold (2.5) in elution. The high density of the adsorbent allowed the EBA to be operated at linear velocityas high as 657 cm/h with feedstock containing 4.5% (w/v) wet biomass. The Richardson-Zaki correlation obtained for this EBA system at the presence of 4.5% (w/v) wet biomass is 5.14, a value closed to the laminar flow regime of 4.8, demonstrated that a stable bed is achieved under this operating condition.Meanwhile, a flow velocity of 343 cm/h with bed expansion of 3.2 gave highest dynamic binding capacity (4979.28 U/ml) and productivity (61.52 U/ml.min) for this EBA operation. It also demonstrated that biomass concentration up to 4.5% (w/v) wet weight showed slightly drop of sorption efficiency (0.82) compared to lower biomass concentration (0.94). Further increase of biomass concentration above 4.5% (w/v) wet weight has greatly decreased the equilibrium and dynamic capacity. Application of high density adsorbent tolerated to high density and biomass has reduced the processing time and increased the productivity

Chemical analysis on the honey of Heterotrigona itama and Tetrigona binghami from Sarawak, Malaysia

This study aims to compare the chemical composition of honey samples produced by Heterotrigona itama and Tetrigona binghami which originated from Sarawak, Malaysia. One hundred and six (106) honey samples were collected from local bee farms and analysed in terms of their chemical profiles. The chemical analysis conducted includes physicochemical composition such as moisture, total phenolic content, sugar, 5-hydroxymethylfurfural (5-HMF), pH and organic acids and proximate analysis which included ash, protein, carbohydrates and energy. Independent T-test was used as a statistical tool to investigate the significant difference between the composition of both honey samples. The results showed that honey samples of Heterotrigona itama and Tetrigona binghami possessed significant difference (p<0.05) in moisture, total phenolic content, fructose, glucose, pH, protein, gluconic acid, acetic acid, ash, carbohydrates and energy. The honey samples of Heterotrigona itama exhibited significantly higher fructose and glucose at the average of 22.00 ± 3.48 g/ 100 g and 23.45 ± 3.23 g/100 g, respectively. Besides, the honey samples also possessed higher pH value, gluconic acid, ash, carbohydrates and energy. Meanwhile, Tetrigona binghami honey samples possessed significantly (p< 0.05) higher moisture content, total phenolic content, protein and acetic acid compared to the Heterotrigona itama’s honey samples. To conclude, the geographical and floral origins of honey are the two important quality parameters which fundamentally affect the physical-chemical properties as well as biological activities of honey samples

Primary recovery of lipase derived from Burkholderia sp. ST8 with aqueous micellar two-phase system.

The partitioning and recovery of lipase derived from Burkholderia sp. ST8 strain was explored using temperature-induced aqueous micellar two-phase system (AMTPS) composed of single nonionic surfactant. Nonionic surfactant Triton X-114 and Pluronic series (triblock copolymer) were evaluated in terms of their clouding phenomenon (cloud-point temperature) and the performance of the lipase partitioning in these AMTPSs. Pluronic L81 showed the most optimum partition efficiency for the recovery of lipase to the micellar phase of the AMTPS. Based on the AMTPS which consisted of 24% (w/w) Pluronic L81 and 0.5% (w/w) potassium chloride (KCl), the selectivity of lipase partitioned to bottom phase has been enhanced to 0.035 and the lipase was purified 7.2 fold. Furthermore, the lipase from the micellar phase was consecutively extracted to a new aqueous solution, with an aim of removing the surfactant from the purified lipase. It was attained by replacing the aqueous top phase from the primary recovery of AMTPS with a new potassium thiocyanate (KSCN) solution. The lipase was then recovered in the newly formed bottom aqueous phase which culminated in the yield of 89% and partition coefficients of 0.34 and 4.50 for lipase and surfactant, respectively. AMTPS offers a convenient and efficient method for the primary recovery of lipase with low cost, large loading capacity and the potential of linear scale up

Optimizing the acceptability of jam from Baccaurea Angulata fruit peel

The purpose of this study was to utilize the fruit peel of Baccaurea angulata to produce a fruit jam. The B. angulata fruit was an underutilized fruit known as “Belimbing Hutan” or wild starfruit in Sabah and Sarawak. The peel of B. angulata fruit makes up more than 60% of the fruit and is a significant source of food waste having potential to be explored. Using conventional jam making technology, the peel of B. angulata was used as the principal ingredient to produce a jam besides sugar and pectin. To find the optimal formulation, the D-Optimal of Mixture Design of Design Expert Version 10 software was employed. The software generated sixteen formulations and from those sixteen formulations, sensory evaluation was conducted to determine the optimal formulation. The optimal acceptability of the B. angulata fruit peel jam was determined by using seven sensory responses namely sweetness, sourness, spreadability, colour, texture, aroma and overall acceptance. Of the sixteen formulations, formulation number 9 (49.5% puree, 49.5% sugar and 1.0% pectin) was selected by the sensory panellists with the highest mean score of overall acceptability (7.2). The contour plots generated using the software explained the various combinations of ingredients that affected the seven sensory responses of the fruit jam

Schizophyllum commune

Schizophyllum commune UTARA1 was used for lipase production under solid state fermentation (SSF) of sugarcane bagasse (SB) impregnated with used cooking oil medium. Pretreatments of steam, microwave, hydrochloric acid (HCl), sodium hydroxide (NaOH), and their combinations, such as steam-assisted HCl, steam-assisted NaOH, microwave-assisted HCl, and microwave-assisted NaOH, on the milled SB, were done prior to SSF to investigate their effects on lipase production via SSF. The highest lipase activity among the pretreated SB was 0.200 U/gSB, using steam-assisted HCl treated SB, which is lower than the lipase activity produced from the untreated SB, which was 0.413 U/gSB. Scanning Electron Microscope (SEM) imaging showed significant rupture of the SB structure after steam-assisted-HCl pretreatments where the thin walls of the SB pith were wrinkled and collapsed, with no distinctive cell wall structure. The HCl pretreated SB gave the highest crystallinity index (CrI), 91.43%, compared to the untreated, 61.90%. Conversely, microwave and NaOH pretreatments reduced the CrI, which were 46.15% and 43.36%, respectively. In this study, the results obtained indicated that pretreated SB did not improve the lipase production of Schizophyllum commune UTARA1 under SSF

A newly developed kelulut honey drink enriched with seaweed by using design of experiment approach

A novel functional drink, Kelulut honey enriched with seaweed (designated as KhS), was successfully developed by using design of experiment (DOE) approach in this study. A two-level-factor Full Factorial Design (FFD), 22, was employed to screen the interactions among the two major composition (Kelulut honey and seaweed) as well as to analyse the effect of these ingredients towards the sensory and proximate properties of the newly developed product. The design of experiment (DOE) and data analysis were executed by using Design- Expert Software. Two main approaches of hedonic tests, i.e., the measurement of preference and the measurement of acceptance were applied during sensory evaluation. Results indicated that amount of seaweed and honey used during the preparation of KhS did affect the moisture content and pH of the end product significantly. Sensory evaluation revealed that the overall acceptability score of the newly developed Kelulut honey enriched with seaweed drink was prefered moderately by most of the panelists and thus it could be a favourable product to be commercialised in the market