- Research article
- Open Access
Solid fermentation of wheat bran for hydrolytic enzymes production and saccharification content by a local isolate Bacillus megatherium
© El-Shishtawy et al.; licensee BioMed Central Ltd. 2014
- Received: 24 February 2014
- Accepted: 16 April 2014
- Published: 24 April 2014
For enzyme production, the costs of solid state fermentation (SSF) techniques were lower and the production higher than submerged cultures. A large number of fungal species was known to grow well on moist substrates, whereas many bacteria were unable to grow under this condition. Therefore, the aim of this study was to isolate a highly efficient strain of Bacillus sp utilizing wheat bran in SSF and optimizing the enzyme production and soluble carbohydrates.
A local strain Bacillus megatherium was isolated from dung sheep. The maximum production of pectinase, xylanase and α-amylase, and saccharification content (total soluble carbohydrates and reducing sugars) were obtained by application of the B. megatherium in SSF using wheat bran as compared to grasses, palm leaves and date seeds. All enzymes and saccharification content exhibited their maximum production during 12–24 h, at the range of 40–80% moisture content of wheat bran, temperature 37-45°C and pH 5–8. An ascending repression of pectinase production was observed by carbon supplements of lactose, glucose, maltose, sucrose and starch, respectively. All carbon supplements improved the production of xylanase and α-amylase, except of lactose decreased α-amylase production. A little increase in the yield of total reducing sugars was detected for all carbon supplements. Among the nitrogen sources, yeast extract induced a significant repression to all enzyme productivity. Sodium nitrate, urea and ammonium chloride enhanced the production of xylanase, α-amylase and pectinase, respectively. Yeast extract, urea, ammonium sulphate and ammonium chloride enhanced the productivity of reducing sugars.
The optimization of enzyme production and sccharification content by B. megatherium in SSF required only adjustment of incubation period and temperature, moisture content and initial pH. Wheat bran supplied enough nutrients without any need for addition of supplements of carbon and nitrogen sources.
- Bacillus megatherium
- Solid fermentation
Agricultural residues have an enormous potential as renewable carbon and energy sources. The main potential applications of agricultural residues are in food, animal feed, biofuel and pharmaceutical industries. Saccharification of agricultural residues by microbial hydrolytic enzymes (cellulases, xylanases, amylases and pectinases) is the first step of bioconversion of organic material into reducing sugars, like glucose and xylose . In the saccharification of agricultural residues, a potential effect was detected in presence of two or more enzymes . Cellulases for cellulose hydrolysis , xylanases for hemicelluloses hydrolysis , amylase for amylose hydrolysis  and pectinase for pectin hydrolysis  are cooperatively needed in the saccharification of agricultural residues. The reducing sugars obtained from these hydrolyzing actions could be utilized as carbon and energy sources in the fermentation industry, such as lactic acid , hydrogen  and ethanol . In addition, microbial hydrolytic enzymes utilized in several applications, such as food, textile, paper, pulp and detergent industries [9–12].
Solid state fermentation (SSF) is the growth of organisms on moist substrates in the absence of free-flowing water. The use of SSF for production of enzymes and other products has many advantages over submerged fermentation . These advantages included: easier recovery of products, the absence of foam formation and smaller reactor volumes. Moreover, contamination risks are significantly reduced due to the low water contents and, consequently, the volume of effluents decreases. Another very important advantage is that, it permits the use of agricultural and agro-industrial residues as substrates which are converted into products with high commercial value like secondary metabolites [13, 14]. Furthermore, the utilization of these compounds helps in solving pollution problems, which otherwise cause their disposal . For enzyme production, the costs of these techniques are lower and the production is higher than submerged cultures [16, 17]. A large number of fungal species was known to grow well on moist substrates in the absence of free-flowing water, whereas many bacteria are unable to grow under this condition [18–21]. As a result, most studies involving SSF have been conducted by using fungi. However, there are little reports of bacterial strains being used successfully for the production of enzymes by using SSF [4, 5, 22, 23]. Therefore, the aim of this study is to isolate strain of Bacillus sp. capable of using wheat bran in SSF to produce α-amylase, xylanase and pectinase. The saccharification content, total soluble carbohydrates and reducing sugars, of wheat bran was studied. Studies on optimizing production of enzymes and saccharification content were also carried out.
Isolation, identification and efficiency of the cellulose decomposing bacilli
Five isolates of Bacillus spp. were isolated from different samples i.e., sheep dung, horses waste, manure compost and rhizosphere soil. The efficiency of the five strains in cellulose decomposion was estimated using caboxymethyl cellulose (CMC) agar medium containing g/l: CMC, 5; peptone, 5; NaCl, 5; beef extract, 3; agar, 18 and pH was adjusted to 7 . The most efficient strain in cellulose decomposion was identified according to Bergey’s Manual of Systematic Bacteriology . The highest efficient strain in cellulose decomposion was isolated from sheep dung and identified as B. megatherium.
Four dried agricultural residues, i.e. wheat bran, date seeds, grass and palm leaves were used as substrates for solid state fermentation (SSF).
Physicochemical parameters of SSF
Physicochemical parameters of SSF were studied for optimization production conditions of soluble carbohydrates, reducing sugars, α-amylase, pectinase and xylanase by B. megatherium. The agricultural residues were sperately sterilized in an autoclave for 20 min at 121°C. B. megatherium was grown in 50 ml Erlenmeyer flask included 5 g of the respective sterilized agricultural residue and appropriate amount of water needed to adjust the moisture of dried substrate, which contained 10% moisture after dring. Optimized physicochemical parameters including: incubation period, incubation temperature, and moisture content of the substrate and incubation pH. The pH was adjusted using 0.1 M NaOH or HCl. The influence of supplementation of carbon sources (glucose, maltose, starch, sucrose, and lactose at 1% w/v) and nitrogen sources (yeast extract, urea, sodium nitrate, ammonium sulphate, and ammonium chloride at 1% w/v) has been studied. Each experiment was done in triplicate.
Soluble carbohydrate and enzyme extraction
Soluble carbohydrate and enzyme were extracted by mixing the fermented substrate with 50 ml distilled water and shaked on a rotary shaker at 180 rpm overnight. The suspension was then centrifuged at 12000 rpm for 10 min and the supernatant was designated as a crude extract.
Determination of total reducing sugars
Total reducing sugars were determined by the method of Miller . The reaction mixture contained 0.5 ml of crude extract and 0.5 ml dinitrosalicylic acid reagent. The tubes were heated in a boiling water bath for 10 min. After cooling to room temperature, the absorbance was measured at 560 nm. Glucose served as the calibration standard for total reducing sugar determination.
Determination of total soluble carbohydrates
Total soluble carbohydrates were determined by the method of Dubois et al. . The reaction mixture contained 25 μl of a 4:1 mixture of phenol and water, 0.8 ml of crude extract and 2 ml of concentrated sulfuric acid. Then mixed well, and heated in a boiling water bath for 30 min. The absorbance was determined at 480 nm. Glucose served as the calibration standard for total carbohydrate determination.
α-Amylase, pectinase and xylanase activities were assayed by determining the liberated reducing end products using maltose, galacturonic acid and xylose as standards, respectively . Substrates used were starch, polygalacturonic acid and birchwood xylan for α-amylase, pectinase and xylanase, respectively. The reaction mixture (0.5 ml) contained 1% substrate, 0.05 M sodium acetate buffer pH 5.5 and 0.1 ml crude extract. Assays were carried out at 37°C for 1 h. Then 0.5 ml dinitrosalicylic acid reagent was added to each tube. Then the reaction mixture was mixed well, and heated in a boiling water bath for 10 min. After cooling to room temperature, the absorbance was measured at 560 nm. One unit of enzyme activity is defined as the amount of enzyme which liberated one μmol of reducing sugar per min under standard assay conditions.
All the experimental work was run in triplicates.
The obtained data were statistically analyzed as a randomized complete block design with three replicates by analysis of variance (ANOVA) using the statistical package software SAS (SAS Institute Inc., 2000, Cary, NC., USA). Comparisons between means were made by F-test and the least significant differences (LSD) at level P = 0.05. Correlations coefficient among the different parameters were also calculated by SAS.
The effect of agricultural residues
The effect of incubation period
The effect of initial moisture content
The effect of incubation temperature
The effect of pH
The effect of supplementation carbon and nitrogen sources
In conclusion, the production of pectinase, xylanase and amylase and saccharification content (total soluble carbohydrates and reducing sugars) by a newly local isolat B. megatherium using wheat bran in SSF will have several advantages. The optimization of enzyme production and sccharification content required only adjustment of incubation time and temperature, moisture content and initial pH. Wheat bran supplied enough nutrients without any need for addition of supplements of carbon and nitrogen sources. All these combined together could greatly reduce the overall cost of production of enzymes and saccharification content by B. megatherium. In the future, the reducing sugars will be used for hydrogen production.
This Project was funded by the King Abdulaziz City for Science and Technology (KACST) under grant number 11-ENE1527-03. The authors, therefore, acknowledge with thanks KACST for support for Scientific Research. Also, the authors are appreciating the kind cooperation provided by the Deanship of Scientific Research (DSR), King Abdulaziz University.
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