Direct Fermentation of Gelatinised Sago Starch to Solvent (Acetone Butanol-Ethanol) by Clostridium Acetobutylicum P262

Several approaches have been applied for the improvement of direct fermentation of sago starch to solvent by C. acetobutylicum. Optimization of medium based on gelatinised sago starch as a carbon source and kinetics of solvent-yielding batch fermentation of individual sugars and their mixture derived from enzymatic hydrolysis of sago starch were carried out using batch fermentation in Scoth bottle. Development of pH control strategy for improvement of solvent production was carried out in batch fermentation using 2 L stirred tank fermenter. The data gathered from batch fermentation were used for development of models to describe direct fermentation of sago starch to solvent. The 2 L and 0.5 L stirred tank fermenter were used to investigate the feasibility of using continuous culture (single stage and two stage) on the improvement of direct fermentation of sago starch to solvent. In all modes of fermentation investigated, the activities of enzymes relevant to solvent fermentation (crotonase, thiolase, phosphate butyryltransferase and β-hydroxybutyryl-CoA dehydrogenase) were determined in order to find their relationship with acid and solvent production. The use of 30 g/L gelatinised sago starch as the sole carbon source produced 11.2 gIL total solvent i.e. 1.5-2 times higher than fermentation using pure maltose or glucose. Enzymatic pretreatment of gelatinised sago starch yielding maltose and glucose hydrolysate prior to the fermentation did not improve solvent production as compared to direct fermentation of gelatinised sago starch. The inefficiency of the amylolytic enzymes secreted during the fermentation in hydrolyzing starch to maltose and glucose is the main problem in direct fermentation of sago starch to solvent. The a-amylase and glucoamylase of C. acetobutylicum have different pH and temperature optima. The pH optima for a-amylase and glucoamylase were found to be at 5.3 and 4.0-4.4, respectively. a-amylase showed a broad pH stability profile, retaining more than 80% of its maximum activity at pH 3.0-8.0 after 24 h incubation at 37°C. However, glucoamylase was only stable at pH 4.0-5.0, maintaining more than 90% of its maximum activity after 24b incubation at 37°C. Production of solvent (11.0 g/L) in fermentation using 30 g/L sago starch was comparable to fermentation using corn starch and about two times higher than fermentation using potato and cassava starches. At the range of sago starch concentration investigated (10 to 80 g/L), the highest solvent production (18.8 g/L) was obtained at 50 g/L. A mixture of yeast extract and ammoniwn nitrate produced total solvent (18.8 g/L) of about 6 times higher than that produced by fermentation using yeast extract alone. The individual concentration of nitrogen and carbon influenced solvent production to a greater extent than carbon to nitrogen (C/N) ratio. Simple unstructured models employing Logistic and Leudeking-Piret equations were found sufficient to describe the growth of C. acetobutybcum and the production of acid and solvent. In fermentation without pH controL initial culture pH 6 gave the highest solvent production (14.13 g/L) with the overall productivity of 0.5 g/L/h. Growth of C. acetoburylicum, rate of starch hydrolysis and solvent production were greatly reduced in fermentation where pH was controlled at 4.5 during acidogenic phase. Excessive acid production (32 g/L) was obtained in fermentation where pH was controlled at 6.0 during the acidogenic phase which reduced solvent production significantly (7.1 g/L). In order to reduce acid accumulation, the fermentation where pH was allowed to decrease naturally by self-acidification during acidogenic phase was suggested. Solvent production was also reduced when pH during the solventogenic phase was controlled at 6.5. Substantial improvement of solvent production was achieved in fermentation where pH was not controlled during acidogenic phase (initial culture pH was 6) and then the pH was controlled at 5.5 during solventogenic phase. Using this pH control strategy, the overall productivity (0.79 g/L.h) was improved by 1.6 times as compared to fermentation without pH control, though the final solvent concentration (16.82 g/L) was about the same. The highest crotonase and phosphate butyryltransferase activity was observed in fermentation where pH was controlled at 5 during acidogenic phase, which was corresponded to fairly high acid production but low solvent production. On the other hand, specific activity of β-hydroxybutyryl-CoA was the highest at pH 5.5, which was corresponded to high acid and solvent production. In fermentation with controlled pH during solventogenic phase, the highest thiolase specific activity was obtained at pH 5.25, which was corresponded to the highest production of acetone. On the other hand, the highest specific activity of crotonase, β-hydroxybutyryl-CoA dehydrogenase and phosphate butyryltransferase was observed at pH 5.5, and this was corresponded to the highest production of ethanol and butanol

The Isolation Of Kojic Acid Producing Fungi And The Optimization Of Kojic Acid Production

A local strain of fungus, which was found to be a high kojic acid producer has been isolated from Morning glory flower (Bixa orellana) using glucose-peptone medium. The pure strain was obtained through several steps of monospore isolation procedures using spread plate technique and identified by International Mycological Institute as Aspergillus flavus Link.Optimization of medium composition and cultural conditions for kojic acid production by this fungus were carried out in shake flask. The development of dissolved oxygen tension (DOT) control strategies aimed at improving kojic acid production by this strain was carried out using 1.5 L stirred tank reactor with automatic pH and DOT control systems. Initial culture pH 3 was found to POLO be optimum for kojic acid production. This strain was able to grow and produce kojic acid in various carbon sources such as glucose, starch, sucrose and xylose. However, the highest production of kojic acid was obtained at 100 g/L (w/v) glucose. In addition, 5 g/L of yeast-extract was found to be the best nitrogen source for fungal growth and enhancement of kojic acid production. Addition of 4% (v/v) of methanol to glucose yeast-extract medium increased kojic acid production by two times. The optimized medium for kojic acid production for this strain was proposed and the medium consisted of 100 g/L glucose, 1.0 g/L KH2PO4,0.5 g/L MgSO4. 7H20, 5.0 g/L yeast-extract and 4% methanol. Using this optimal medium the maximum kojic acid production in batch fermentation using shake flask was 39 g/L. This fermentation gave yield and productivity of 0.04 g/g and 0.07 g/L/h, respectively and is comparable to that reported in the literature for industrial strain. The DOT control strategy for the improvement of the kojic acid production by this strain was also developed. By controlling DOT at 80% saturation during growth phase and then switched to 30% saturation during production phase, increased the production of kojic acid by about two times as compared to uncontrol fermentation in the stirred tank fermenter which only produced maximum concentration of kojic acid of 15 g/L.. The effect of the addition of yeast-extract during the fermentation was also investigated using a constant fed-batch culture. The addition of 15 g/L yeast-extract at constant flow rate of 3.2 ml/h during the production phase caused excessive mycelial growth and decreased kojic acid production

Characterization of crude chitinase produced by Trichoderma virens in solid state fermentation

The objective of this study to ensure the appearance of the enzymes produced by Trichoderma virens and to obtain information on the optimum conditions for the enzymes in its specified reaction. Crude chitinase produced by Trichoderma virens presented three types of chitinolytic enzymes: endochitinase, exochitinase and protease. The optimal temperature for crude chitinase at 600C and optimum pH at 3.0.  Crude chitinase stability decreases as incubation temperature increases, however, crude chitinase was found to be stable over a range at pH acidic.  T. virens growth in the solid substrate with shrimp waste as substrate produced crude chitinase with several chitinolytic enzymes based on its protein visualization on SDS-PAGE.  This crude chitinase has endochitinase (50 and 42 kDa) exochitinase (33 and 25 kDa) and protease (18 kDa).Keyword : Chitinase, Trichoderma virens, Solid state fermentatio

Profile of enzymes relevant to solvent production during direct fermentation of sago starch by C. saccharobutylicum at different pH control strategies.

The profile of enzymes relevant to solvent production during direct fermentation of sago starch by clostidium saccharobutylicum P262 in a 2 L stirred tank fermenter was determined utilizing different pH control strategies. During fermentation without pH control (initial pH of 6), the specific activity of crotonase, thiolase, and β-hydroxybutyryl-CoA dehydrogenase increased proportionally with solvent production. The highest crotonase (3,450.7 kat) and phosphotransbutyrylase activity(1,475.6 kat) was observed in fermentation where pH was maintained at 5 during the acidogenic phase and corresponded to a fairly high acid accumulation but low solvent production. During fermentation with a controlled pH of 5.25 during the solventogenic phase, the highest thiolase specific activity (255.7 kat) was obtained and corresponded to the highest production of acetone. On the other hand, the highest specific activities of crotonase, β-hydroxybutyryl-CoA dehydrogenase, and phosphotransbutyrylase were observed at pH 5.5 and corresponded to the highest production of ethanol and butanol. Butyryl-CoA dehydrogenase had no significance role in solvent fermentation. These results suggested that pH control strategies were important for improvement of solvent production during direct fermentation of sago starch by C.saccharobutylicum. © KSB

Cloning and expression of pullulanase gene from locally isolated bacillus SP

Bacterial pullulanase represents one of th e starch-degrading enzymes that are widely used in the starch processing indu stry along with amylases. Amylases hydrolyze a -(1,4 )-glycosidic linkage in starch to produce a mixture of glucose , maltooligo sacchari de and limited a-dextrin. All the remaining a -(1,6)-glycosid ic branches in the products are hydrolyzed by p ullulanase. This is an advantage t o improve glucose production by coupling pullulanase and amylase in the p rocess. As such, many pullulana e enzyme has been isolated and one has been showing optimum pH of 10-10.5 which is suitable for use in dishwasher detergent additive in removal of star ch stain. We have recently iso lated a few bacterias that have shown potentially pullulanase producers by the holo-zone in pullulan-plate assay. One of them, we named Bacillus –1 sho ws a bigger holo-zone among others, Bacillus- 1 is highly active in pH more than 7. The enzyme also shows a mo derate activity to wards starch that may be indicates be side hydrolyzes a -(1,6)-glycosidic linkage in starch, it also hydrolyzes a -(1,4)- glycosidi c simi lar to a -amylase. Unfortunately the enzyme from wild-type bacteria is in lower yield an d in this studies, we intend to clone and sequence the pullulanase gene and also expressed the gene in a high expression system to be able to produce in a high yield before characterizing expressed protein

Degradation pathway of phenol through ortho- cleavage by Candida tropicalis RETL-Cr1

Phenols and its derivatives are environmental pollutant commonly found in many industrial effluents. Despite being toxic, phenol can be utilized by microbes as carbon and energy sources. The strain utilized up to 3mM phenol as a sole source of carbon and energy. Phenol catabolism was confirmed through the detection of the intermediary products namely catechol and cis,cis-muconic acid. Catechol was formed at the earlier stage of the reaction mixture while cis,cis-muconic acid was formed at the later stage of the biodegradation process. The maximum concentration of catechol was 20.4 mg L after 7 h incubation. The HPLC chromatography detected muconic acid and enzymatic assays performed were found to be negative for catechol 2,3 dioxygenase activity. Hence, these results showed that this indigenous phenol-degrading yeast, Candida. tropicalis RETL-Cr1 (AY725426) seemed to metabolize phenol via ortho-cleavage pathwa

Parameter estimation of stochastic differential equation

Non-parametric modeling is a method which relies heavily on data and motivated by the smoothness properties in estimating a function which involves spline and non-spline approaches. Spline approach consists of regression spline and smoothing spline. Regression spline with Bayesian approach is considered in the first step of a two-step method in estimating the structural parameters for stochastic differential equation (SDE). The selection of knot and order of spline can be done heuristically based on the scatter plot. To overcome the subjective and tedious process of selecting the optimal knot and order of spline, an algorithm was proposed. A single optimal knot is selected out of all the points with exception of the first and the last data which gives the least value of Generalized Cross Validation (GCV) for each order of spline. The use is illustrated using observed data of opening share prices of Petronas Gas Bhd. The results showed that the Mean Square Errors (MSE) for stochastic model with parameters estimated using optimal knot for 1,000, 5,000 and 10,000 runs of Brownian motions are smaller than the SDE models with estimated parameters using knot selected heuristically. This verified the viability of the two-step method in the estimation of the drift and diffusion parameters of SDE with an improvement of a single knot selection

Effect of agitation and aeration rates on chitinase production using Trichoderma virens UKM1 in 2 L stirred tank reactor

Shrimps have been a popular raw material for the burgeoning marine and food industry contributing to increasing marine waste. Shrimp waste, which is rich in organic compounds is an abundant source of chitin, a natural polymer of N-acetyl-D-glucosamine (GluNac), a reducing sugar. For this respect, chitinase-producing fungi have been extensively studied as biocontrol agents. Locally isolated Trichoderma virens UKM1 was used in this study. The effect of agitation and aeration rates using colloidal chitin as control substrate in a 2-l stirred tank reactor gave the best agitation and aeration rates at 200 rpm and 0.33 vvm with 4.1 U/l per hour and 5.97 U/l per hour of maximum volumetric chitinase activity obtained, respectively. Microscopic observations showed shear sensitivity at higher agitation rate of the above system. The oxygen uptake rate during the highest chitinase productivity obtained using sun-dried ground shrimp waste of 1.74 mg of dissolved oxygen per gram of fungal biomass per hour at the kappaL a of 8.34 per hour

Performance of Eular-Maruyama, 2-stage SRK and 4-stage SRK in approximating the strong solution of stochastic model

Stochastic differential equations play a prominent role in many application areas including finance, biology and epidemiology. By incorporating random elements to ordinary differential equation system, a system of stochastic differential equations (SDEs) arises. This leads to a more complex insight of the physical phenomena than their deterministic counterpart. However, most of the SDEs do not have an analytical solution where numerical method is the best way to resolve this problem. Recently, much work had been done in applying numerical methods for solving SDEs. A very general class of Stochastic Runge-Kutta, (SRK) had been studied and 2-stage SRK with order convergence of 1.0 and 4-stage SRK with order convergence of 1.5 were discussed. In this study, we compared the performance of Euler-Maruyama, 2-stage SRK and 4-stage SRK in approximating the strong solutions of stochastic logistic model which describe the cell growth of C. acetobutylicum P262. The MS-stability functions of these schemes were calculated and regions of MS-stability are given. We also perform the comparison for the performance of these methods based on their global errors