This is a solution of Unit 3 Treatment On Solid Pineapple Waste that describes about Developing business
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Collection and Storage of Biomass
The solid pineapple wastes were collected from Lee Pineapple Company at Jalan Skudai, Skudai, Johor Bahru. The collected pineapple waste were packed in a clean sealable plastic back and stored in freezer of below -20°C.
The pineapple wastes were dried in the incubator oven for 3 to 4 days under 50°C to 60°C. This range was obtained from monitoring the weight loss of the waste till it reaches a constant weight. After drying, the sample was stored in a dry air tight container to prevent fungus growth (Hames et al., 2008).
The solid pineapple wastes were grind and sieved at the range of 1mm to 0.5mm sizing. The size range is the suitable size for a pre-treatment process (Hames et al., 2008).
Preparation of Inoculum
In this study, Phanerochaete chrysoporium was used. The fungus culture was grown on a PDA agar and was allowed to grow. The fungus was incubated at 37°C for 7 to 15 days (Bak et al., 2009).
Harvesting of chrysoporium
The fungus was harvested by adding 1% (v/v) sterile Tween-80 by scraping the surface of the agar slowly with a glass hockey stick. All the spores will be diluted and then were collected into a centrifuge tube. The diluted spore then is centrifuged at 4000 rpm for 30 minutes under 4°C. After centrifuge, the supernatant was disposed (Shi et al., 2008). The left over pellet was mixed with 10 ml of sterile deionized water and vortex. The diluted pellet was then brought to spore count using Haemacytometer. The reading was done under 40X magnification (Shi et al., 2008). See more : Consumers Buying Behavior And Related Theories
The fungal pre-treatment of solid pineapple waste is done by inoculating P. chrysoporium spore into a 30 g of solid pineapple waste which was filled in 250 ml Erlenmeyer flasks.
Prior to the inoculation the solid pineapple waste was wet with 120 ml of basal media. The amount was applied to reach the 80% moisture content (Shi et al., 2008). The spore concentration of 5 × 106 spore/ml was inoculated into the substrate (Bak et al., 2009). The free cell technique fermentation was done for 14 days under the temperature of 29°C. Sampling of the fermentation was done every 2 days (Hodgson, 1998).
Preparation of Basal Media
The media was prepared according to the composition of Mendel Basal Medium (Mendel and Webber, 1969). The batch fermentation medium was prepared from the composition as in Table 3.1. The pH of the Basal Media was brought to pH 5.5 by adding distilled water. The composition of the substances was mixed and brought to the volume of 1L by adding distilled water. Prior to the usage of this media, autoclaving was done to achieve sterilization. The composition of Mendel Basal Medium is presented in Table 3.1.
Table 3.1: Composition of Mendel Basal Media (Mendel and Webber, 1969)
|FeSO4||5 x 10-3|
|MnSO4.H2O||1.6 x 10-3|
|ZnSO4.7H2O||1.4 x 10-3|
|CoCl2||2.0 x 10-3|
Preparation of Potato Dextrose Agar ( PDA )
The preparation of 1L agar is made by mixing 39 g of PDA powder into 1L of distilled water. The mixed solution was then autoclaved before pouring into the petri dishes. The petri dish was left to cool till the agar solidifies. The dishes were then sealed with parafilm tape before storing into the fridge.
Preparation of Chemicals
The chemical that was prepared for alkaline pre-treatment was sodium hydroxide with the concentration of 1% (v/v) and 3% (v/v). Sulphuric acid was prepared for the acid pre-treatment with the concentrations of 1% (v/v) and 3% (v/v) (Alicia et al., 2012).
The acidic and alkaline pre-treatment was done in a 250 ml Erlenmeyer ﬂasks. The flask which contains 30 g of substrate was mixed with 30ml of each concentrated acid to reach a 20% (w/v) of solid loading. The samples were then autoclaved (Alicia et al., 2012). The autoclave provides the temperature of 102°C for approximately 1 hour. After the pre-treatment the samples were washed with 1L of deionized water until it reaches pH 7 (Keshwani and Cheng, 2009). (Keshwani and Cheng, 2009).
Preparation of Samples
The concentrations of sodium hydroxide were prepared as mentioned in 3.3.1. The microwave pre-treatment involves time intervals of 5 minutes for 30 minutes. Thus 5 sets of pineapple solid waste are prepared. The amount of solution and biomass used was at the ratio of 1:10 (solid: liquid) (Keshwani and Cheng, 2009).
The pre-treatment was conducted in the microwave model NN-5626. The rated power was set at 240v-50Hz. This microwave was selected due to its availability of stirring device. The pre-treatment was carried out together with the stirring at 150 rpm (Hamisan et al., 2009).
The pre-treatment was carried out in a round bottom glass vessel. The samples were treated with different time intervals separately. The samples were pre-treated at 5 minutes time intervals up to 30 minutes. After the pre-treatment, the samples were washed with 1L of deionized water till it reaches pH 7.
Analytic Methods of Pre-treatments
High Performance Liquid Chromatography (HPLC) Analysis
The HPLC was utilised in order to determine the presence of the six different sugar such as the glucose, galactose, arabinose, mannose, xylose, and cellobiose. Since this research project of focuses on the saccharification for lactic acid, the glucose, fructose and sucrose content were measured by HPLC. The HPLC analysis was conducted by using a 300 mm ´ 7.8 mm, Rezex RCM-Monosaccharide column (Phenomenex) with RI detector. The mobile phase used was 100% nanopure water at a flow rate of 0.6 ml per minute, 20 µl of injection volume and ambient temperature and the carbohydrate will be identified relative to the standard carbohydrates (Association of Official Analytical Chemists (AOAC), 1984). Prior to the HPLC analysis, the supernatant was filtered through 0.3µm Milipore membrane filters into HPLC vials.
The Determination of Lignin, Cellulose and Hemicellulose Content
This analysis was made possible by applying various solvents to separate the insoluble part of the plant cell wall and also the result of lignification can be determined. The cellular components were also suitable to determine the amount of sugars, starch, lipids, protein, organic acids and soluble ash (Van Soest and Wine, 1967).
The Determination of Neutral Detergent Fiber (NDF)
As mentioned in Chapter 2, the solid pineapple waste consists of non-structural carbohydrates, structural carbohydrates, fat, protein and ash. The structural carbohydrate consists of lignin, cellulose and hemicellulose. The non- structural carbohydrate consists of pectin, sugar and starch.
To separate the insoluble cell wall fraction from the soluble once, the detergent with a neutral pH was applied by boiling it together with the sample. This method permits the measurement of non-structural carbohydrate (NSC) (Van Soest and Wine, 1967).
The Determination of Acid Detergent Fibre (ADF)
The ADF is carried out to drawback the content of hemicellulose. The procedure for this analysis is followed according to the procedure done in (NDF). The neutral substance is replaced with an acid detergent, instead of boiling it with a neutral detergent (Van Soest and Wine, 1967).
The Determination of Lignin Composition
The lignin composition was determined from applying the samples obtained from ADF method. The samples from ADF consists of lignin, cellulose and ash, by mixing the sample with 72% H2SO4, the ash and cellulose will be diluted leaving the lignin structure. The lignin was then washed, filtered, dried and weighed (Van Soest and Wine, 1967).
The Determination of Cellulose and Hemicellulose Content
The analysis of cellulose requires the data from the determination of ADF and NDF. The analysis for the determination of cellulose and hemicellulose the filter membrane which consists of the sample after the determination of ADF was reused.
Hemicellulose = NDF – ADF
Cellulose and Lignin = Obtained from formula
Ash = NDF – Lignin – Cellulose – Hemicellulose
The Determination of Reducing Sugar – DNS Method
The DNS method was carried out to determine the presence of reducing sugar in the pre-treated samples. The DNS method was focused on the characteristic of glucose as the reducing sugar. This glucose in the sample if present decreases the oxidized form of DNS reagent to form (3-amino, 5-nitrosalicylic acid) from (3, 5-dinitrosalicylic acid). The reading is obtained when the reduced form of DNS reagent absorbs light at the wavelength of 535 nm. The wavelength and the calorimetric change are compared with the glucose amount from samples (Miller, 1959).
The Determination of Enzyme Activity
The determination of enzyme activity is done by conducting few analyses such as, Carboxymethylcellulase (CMCase) activity and FPase. These enzymes were analysed to distinguish their function in the pre-treatment of solid pineapple waste.
The Determination of Carboxymethylcellulase (CMCase) activity
The CMCase activity was determined by using CMC of 2% mixed with sodium acetate buffer of pH 5. This solution acts as the substrate for the enzyme present, the glucose emitted was analysed at the wavelength of 540 nm. See more : Unit 1 Business Assignment Help
The CMCase activity was carried out to determine the activity of endoglucanases enzyme. The Carboxymethylcellulase (CMCase) activity was carried out according to the Standard IUPAC procedures. The CMCase defines one mole activity as the release of 1 µmole of glucose per mL enzyme per minute. The reducing sugar content was then calculated by using the glucose standard curve as a reference. This analysis defines the activity of the enzyme in the hydrolysis of cellulose.
The Determination of FPase
The IUPAC defines one unit of FPase activity as the release of 1µmole of glucose from the Whatman No. 1 filter paper per minute. The glucose content that was produced was determined by using dinitrosalicylic acid (DNS) reducing sugar assay (Wood and Bhat, 1988).
- Chemical and Physical Pre-treatment
- The Determination of Reducing Sugar – DNS Method
The analysis done for these two pre-treatments were carried out by following the steps in section 18.104.22.168.
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