The instrument used in this study is a test tube, test tube racks, waterbath shaker, spectrophotometer , laminar air flow, microscope, a digital camera Sony Cybershot D33021)incubator (Barnstead), oven, electric autoclave, centrifuge (Hettich), refrigerator, digital scales (Mettler Toledo), vortex, hot plate, electric stove, desicator, plate-forming gel (Biorad), Eppendorf tubes, polypropylene tubes, micropipette, blue tip, yellow tip, thermometer, magnetic stirer, suction bulb, Bunsen, the needle loop, haemocytometer, glass objects, glass cover, handtally counter, bottle film, aluminum foil, petridishes, and glassware.
3.2 Preparation Samples
Samples of microorganisms to be used in this study were taken of microorganisms at oil palm plantation and palm oil mill in Pechaburi Province, Thailand.
3.3 Preparation of Acid Hydrolysis (Residues)
Oil palm empty fruit bunches are made into fibers by using a cruiser machine to destroy so that a small-sized fibers and fibers are added to a concentration of 2% sulfuric acid with a ratio of 1: 10 for 1 gram fiber and 10 ml of sulfuric acid. fibers and sulfuric acid put into 250 ml erlenmeyer sized and inserted into the autoclave with temperature 121oC for 75 minutes. after completion, the blend of fibers and sulfuric acid is filtered using Whatman filter paper No.1. liquid separated from solids. solids or residue that is taken to be a substrate for subsequent testing (Rahman et al., 2006).
3.4 Isolation Microorganisms
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Prior to the isolation of microorganisms do the preparation of the composition of liquid medium is 1 g KH2PO4, 0.5 g Nacl, 0.5 g MgS04.7H2O, 0.01 g FeSO4.7H20, MnSO4.H2O 0:01, 0:03 g NH4NO3 in 1 liter of distilled water, and added with the substrate 0.5% CMC as carbon source. liquid medium is inserted next sample suspected of containing microorganisms. for each sample made â€‹â€‹of liquid medium containing CMC. for substrate OPEFB fiber and residues were prepared as in preparation of a liquid medium using CMC substrate. Samples that have been incorporated into the liquid medium then carried shaker 200 rpm and incubated for 10 days at a temperature of 30oC.
After incubation, the samples suspected of containing microorganisms taken using a loop and then performed Streaked plate method to be grown in agar medium containing 5% CMC substrate and incubated for 7 days at room temperature. after seven days of incubation, the growth of microorganisms was observed subsequently calculated the number of microorganisms are grown for each sample and was recorded types of microorganisms and the number of microorganisms that grow on each medium in 0.5 % CMC medium agar. after recording, the microorganisms are transferred to the new CMC media to be grown and incubated in order to obtain a single colony of each microbe. Single colonies of microbes in part transferred to stored in the slant media or in 20% glycerol and partly transferred to be grown again in 0.5% CMC media to detect cellulase activity using congo red solution of 1% and 1% NaCl. Cellulase activity of microbial was detected if the clear zone is formed. Microbial yield clear zone and then proceed to characterize the morphology for use in subsequent testing.
3.5 Characterization of cellulolytic microorganisms
Characterization of cellulolytic microorganisms which do include morphological studies observation colony and Gram stain.
3.5.1 Morphological studies observation of microorganisms colonies
Morphological observation of colonies done by pure cultures of microbials grown on CMC medium slant taken with a loop and then streaked in quadrants on CMC agar. Cultures were incubated for 7-10 days at room temperature and then observed a single colony the shape, color, surface, internal structure and the structure of the edge of the colony.
18.104.22.168 Slide Culture Preparation for Fungi
In order to accurately identify many fungi it is essential to observe the precise arrangement of the conidiophores and the way in which spores are produced (conidial ontogeny). According to Riddel's (1950), simple method of slide culturing permits fungi to be studied virtually in situ with as little disturbance as possible. A simple modification of this method using a single agar plate is described below.
One plate of nutrient agar; potato dextrose is recommended, however, some fastidious fungi may require harsher media to induce sporulation like cornmeal agar or Czapek dox agar.
Using a sterile blade cut out an agar block (7 x 7 mm) small enough to fit under a coverslip.
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Marked to Standard
Flip the block up onto the surface of the agar plate.
Inoculate the four sides of the agar block with spores or mycelial fragments of the fungus to be grown.
Place a flamed coverslip centrally upon the agar block.
Incubate the plate at 26C until growth and sporulation have occurred.
Remove the cover slip from the agar block.
Apply a drop of 95% alcohol as a wetting agent.
Gently lower the coverslip onto a small drop of Lactophenol cotton blue on a clean glass slide.
The slide can be left overnight to dry and later sealed with fingernail polish.
When sealing with nail polish use a coat of clear polish followed by one coat of red coloured polish.
3.5.2 Gram Staining for Bacteria
The Gram stain is the most important and universally used staining technique in the bacteriology laboratory. It is used to distinguish between gram-positive and gram-negative bacteria, which have distinct and consistent differences in their cell walls. Gram-positive cells may become gram negative through mechanical damage, conversion to protoplasts, or aging, in which autolytic enzymes attack the walls.
Transfer a loopful of the liquid culture to the surface of a clean glass slide, and spread over a small area. Two to four cultures may be stained on the same slide, which can be divided into 2-4 sections with vertical red wax pencil lines. To stain material from a culture growing solid media, place a loopful of tap water on a slide; using a sterile cool loop transfer a small sample of the colony to the drop, and emulsify. Allow the film to air dry. Fix the dried film by passing it briefly through the Bunsen flame two or three times without exposing the dried film directly to the flame. The slide should not be so hot as to be uncomfortable to the touch.
Flood the slide with crystal violet solution for up to one minute. Wash off briefly with tap water (not over 5 seconds). Drain.
Flood slide with Gram's Iodine solution, and allow to act (as a mordant) for about one minute. Wash off with tap water. Drain.
Remove excess water from slide and blot, so that alcohol used for decolorization is not diluted. Flood slide with 95% alcohol for 10 seconds and wash off with tap water. (Smears that are excessively thick may require longer decolorization. This is the most sensitive and variable step of the procedure, and requires experience to know just how much to decolorize). Drain the slide.
Flood slide with safranin solution and allow to counterstain for 30 seconds. Wash off with tap water. Drain and blot dry with bibulous paper. Do not rub.
All slides of bacteria must be examined under the oil immersion lens.
3.6 Detection of Cellulase Activity
Extracellular cellulases were tested using agar plate containing 1% (w/v) carboxymethylcellulose (CMC) (pH 7.0). The pure culture on agar plates was flooded with aqueous solution of congo red (1% w/v) for 15 min to detect cellulase production. The congo red solution was then poured off and the plates were destained with 1 M NaCl for 15 min. The formation of a clear zone of hydrolysis indicated cellulose degradation by microorganism (Ariffin et al., 2008).
3.7 Preparation of Microbial Growth Curve
Preparation of growth curve was carried out by each of the purified microorganisms isolates in liquid CMC media. Preparation of growth curves are intended to determine the phases of growth of cellulolytic isolates. Up to one loop isolates inserted into 100 ml of liquid CMC media. The suspension is then homogenized and incubated on a shaker at 150 rpm at a temperature of 30oC for 7 days. Optical measurement is then performed Density (OD) using a spectrophotometer at a wavelength of 540 nm. A total of 10% suspension of microbial culture by the number of cells 107 cfu / ml into 300 ml of media inserted so that the CMC in the medium containing 107 cfu / ml microbial (bacteria). The suspension is then homogenized and incubated on a shaker at a temperature of 30oC with a speed of 200 rpm. Bacterial cell growth curve was made by means of OD measurements every 6 hours during the first 24 hours, then samples were taken every 12 hours for 5 days to obtain the phases of the growth expected.
3.8 Activity assay of Cellulolytic Microbial
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Logarithmic phase of growth curves of the cellulolytic microbial isolates can be used to determine the cellulolytic microbial stock. This phase can also be used to determine the stock inoculum to be used for this test. Testing stages of this activity includes the preparation of standard curves of glucose, making a starter for the production of enzymes and enzyme activity measurements.
3.8.1 Preparation of Standard Curve Glucose
Standard solution of glucose was made at an interval of concentrations 0-1000 μug/ml. Of each concentration of glucose solution was taken and added as much as 1 ml, 1.5 ml of DNS solution then heated for 5 minutes and then added to 0.5 ml solution of sodium tartrate-Pottasium, after it is cooled with running water for 15 minutes, then the term measured OD 540 nm (Jeffries, 1996).
3.8.2 Starter preparations for the production of cellulase enzymes
Making a starter for the production of an enzyme made â€‹â€‹according to the method Gorska et al., (2001), taking 10% of the stock and put in production with the substrate CMC media, oil palm empty fruit bunch fiber and acid hydrolysis residues
3.8.3 Cellulase activity assay
Measurement of cellulase enzyme activity was measured by reduction of the amount of sugar produced by 1 ml of the supernatant of media production, is then added 1 ml of the CMC substrate was dissolved in phosphate buffer (0.05 M) (pH 7.0). the mixture was incubated at 37oC for 30 minutes and then the reaction was stopped with adding 3 ml solution DNS (2-hydroxy-3, 5 dinitrocalicylate). The mixture was then heated in boiling water for 5 minutes, then added 10 ml destilated water whose role is to stabilize the color, then cooled with running water for 15 minutes and the absorbance was measured using a spectrophotometer at a wavelength of 540 nm. One unit of enzyme activity is the amount of enzyme that produced 1 mol of glucose in 1 minute at specific measurement conditions (Susilawati et al, 2003). For Oil palm empty fruit bunch (OPEFB) and residues of acid hydrolysis substrates is followed the way of CMC substrate to cellulase enzyme activity assay.
The cellulase enzyme activity assay using treatment parameters long incubation, pH, temperature and rpm. for the long incubation period is 6, 12, 24, 36, 48, 60 and 72 hours for bacteria and fungi and actinomyces are for 1, 2, 3, 4, 5, 6 and 7 days. treatment of the effect of pH is 3, 4, 5, 6, 7, and 8. For cellulase treatment temperature effect was tested by using the temperature is 25 oC, 30oC, 35oC, 40oC, 45oC and 50oC. Treatment of the effect of rpm is 100, 125.150, 175, and 200.
3.9 Preparation for Production Etanol Using Saccharomyces cerevisiae
Oil palm empty fruit bunches are made into fibers by using a cruiser machine to destroy so that a small-sized fibers and fibers are added to a concentration of 2% sulfuric acid with a ratio of 1: 10 for 1 gram fiber and 10 ml of sulfuric acid. fibers and sulfuric acid put into 250 ml erlenmeyer sized and inserted into the autoclave with temperature 121oC for 75 minutes. after completion, the blend of fibers and sulfuric acid is filtered using Whatman filter paper No.1. liquid separated from solids. solids or residue that is taken to be a substrate for subsequent testing but before solids have to neutralization by using NaOH until obtained pH 6 - 7.
For enzymatic saccharification OPEFB from the acid hydrolysis process was soaked a 100 ml of acetate buffer solution (pH 4.8) and mixed with cellulase (70 FPU mL-1 from microorganism at temperature of 48oC and rate of agitation 150 rpm for 48 hour. The OPEFB hydrolysate obtained was used for fermentation.
3.9.1 Inoculums Preparation
Saccharomyces cerevisiae was initially grow on yeast-peptone-glucose (YPG) and incubated at a temperature of 35oC and a rate of agitation 150 rpm for 18 to 24 hour. In this study, YPG medium consisted of (gL-1); 10 g yeast extract, 20 g peptone and 20 g glucose. After incubation period, the cells were then harvested by centrifugation at 3000 rpm at 4oC for 15 minute. The pellet was then rinsed twice with sterilized saline solution before being re-suspended in sterilized saline solution to yield an Optical Density (OD) of 1.0 at 600nm. The standardized S. cerevisiae was used for subsequent study.
3.9.2 Ethanol Production
Fermentation of Residues of acid hydrolysis pretreatment process was carried out by using S. cerevisiae. A total of 150 mL of residues from enzymatic saccharification was prepared in a 250 mL conical flask. A total of 10% (v/v) of standardized active S. cerevisiae was added to the hydrolysate prior to incubation into a shaker at temperature 30oC, pH of 4 and agitation rate of 150 rpm. OPEFB hydrolysate was harvested every 12 to 24 hour interval. The harvested samples were filtered using a 0.45 µm membrane filter and then the filtered samples were put in 2.5 mL vials prior to being analyzed (Mohd. Kassim et al., 2011).
Oil palm empty fruit bunch (OPEFB)
Input on Cruise Machine
Mix OPEFB with H2SO4 (1g OPEFB : 10ml H2SO4)
Add H2SO4 , 2%
Input on Autoclave, 121oC for 75 minutes
Filtrated by Whatman paper No.1
Figure 9. Flow Chart Preparation Acid Hydrolysis Residue from OPEFB
Plantation Palm Oil Field
Factory of Palm Oil
Incubation on OPEFB substrate + Mineral Medium, 30oC for 7 days
Incubation on CMC substrate + mineral medium,
30oC for 10 days
Incubation on Acid Hydrolysis Residues substrate + Mineral Medium, 30oC for 7 days
Single colony of isolates microorganisms
Put on CMC Media,
Checking clear zone by congo red
The best isolates of microorganisms
Preparation growth microorganism curve
Preparation Glucose standard curve
Activity enzym assay
The Best Condition test :
pH, temp, time, rpm
Production Ethanol using S. cerevisiae
Figure 10. Flow Chart of Hydrolytic Microorganisms Associated with Palm Oil Industry
Incubation on CMC substrate + mineral medium,
30oC for 7 days