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The instrument used in this survey is a trial tubing, trial tubing racks, waterbath shaker, spectrophotometer, laminal air flow, microscope, a digital camera Sony Cybershot D33021 ) brooder ( Barnstead ) , oven, electric sterilizer, extractor ( Hettich ) , icebox, digital graduated tables ( Mettler Toledo ) , vortex, hot home base, electric range, desicator, plate-forming gel ( Biorad ) , Eppendorf tubings, polypropene tubings, micropipette, bluish tip, xanthous tip, thermometer, magnetic stirer, suction bulb, Bunsen, the needle cringle, haemocytometer, glass objects, glass screen, handtally counter, bottle movie, aluminium foil, petridishes, and glasswork.

3.2 Preparation Samples

Samples of micro-organisms to be used in this survey were taken of micro-organisms at oil thenar plantation and palm oil factory in Pechaburi Province, Thailand.

3.3 Preparation of Acid Hydrolysis ( Residues )

Oil thenar empty fruit Bunches are made into fibres by utilizing a patrol car machine to destruct so that a small-sized fibres and fibres are added to a concentration of 2 % sulphuric acid with a ratio of 1: 10 for 1 gm fibre and 10 milliliter of sulphuric acid. fibres and sulphuric acid put into 250 milliliters erlenmeyer sized and inserted into the sterilizer with temperature 121oC for 75 proceedingss. after completion, the blend of fibres and sulphuric acid is filtered utilizing 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

Prior to the isolation of micro-organisms do the readying of the composing 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 litre of distilled H2O, and added with the substrate 0.5 % CMC as C beginning. liquid medium is inserted following sample suspected of incorporating micro-organisms. for each sample made aˆ‹aˆ‹of liquid medium incorporating CMC. for substrate OPEFB fibre and residues were prepared as in readying of a liquid medium utilizing CMC substrate. Samples that have been incorporated into the liquid medium so carried shaker 200 revolutions per minute and incubated for 10 yearss at a temperature of 30oC.

After incubation, the samples suspected of incorporating micro-organisms taken utilizing a cringle and so performed Streaked home base method to be grown in agar medium incorporating 5 % CMC substrate and incubated for 7 yearss at room temperature. after seven yearss of incubation, the growing of micro-organism was observed later calculated the figure of micro-organisms are grown for each sample and was recorded types of micro-organisms and the figure of micro-organisms that grow on each medium in 0.5 % CMC medium agar. after entering, the micro-organisms are transferred to the new CMC media to be grown and incubated in order to obtain a individual settlement of each bug. Single settlements of bugs in portion transferred to stored in the slant media or in 20 % glycerin and partially transferred to be grown once more in 0.5 % CMC media to observe cellulase activity utilizing Congo ruddy solution of 1 % and 1 % NaCl. Cellulase activity of microbial was detected if the clear zone is formed. Microbial output clear zone and so continue to qualify the morphology for usage in subsequent testing.

3.5 Word picture of cellulolytic micro-organisms

Word picture of cellulolytic micro-organisms which do include morphological surveies observation settlement and Gram discoloration.

3.5.1 Morphologic surveies observation of micro-organism settlements

Morphologic observation of settlements done by pure civilizations of microbials grown on CMC medium angle taken with a cringle and so streaked in quarter-circles on CMC agar. Cultures were incubated for 7-10 yearss at room temperature and so observed a individual settlement the form, colour, surface, internal construction and the construction of the border of the settlement.

3.5.1.1 Slide Culture Preparation for Fungi

In order to accurately place many Fungis it is indispensable to detect the precise agreement of the conidiophores and the manner in which spores are produced ( conidial growth ) . Harmonizing to Riddel ‘s ( 1950 ) , simple method of slide culturing licenses fungi to be studied virtually in situ with every bit small perturbation as possible. A simple alteration of this method utilizing a individual agar home base is described below.

One home base of alimentary agar ; potato dextrose is recommended, nevertheless, some fastidious Fungis may necessitate harsher media to bring on monogenesis like Indian meal agar or Czapek dox agar.

Using a unfertile blade cut out an agar block ( 7 x 7 millimeters ) little plenty to suit under a coverslip.

Flip the block up onto the surface of the agar home base.

Inoculate the four sides of the agar block with spores or mycelial fragments of the fungus to be grown.

Topographic point a flamed coverslip centrally upon the agar block.

Incubate the home base at 26C until growing and monogenesis have occurred.

Remove the screen faux pas from the agar block.

Use a bead of 95 % intoxicant as a wetting agent.

Gently lower the coverslip onto a little bead of Lactophenol cotton blue on a clean glass slide.

The slide can be left nightlong to dry and subsequently sealed with fingernail gloss.

When sealing with nail Polish usage a coat of clear Polish followed by one coat of ruddy colored gloss.

3.5.2 Gram Staining for Bacteria

The Gram discoloration is the most of import and universally used staining technique in the bacteriology research lab. It is used to separate between Gram-positive and Gram-negative bacteriums, which have distinct and consistent differences in their cell walls. Gram-positive cells may go gram negative through mechanical harm, transition to energids, or aging, in which autolytic enzymes attack the walls.

Transfer a loopful of the liquid civilization to the surface of a clean glass slide, and spread over a little country. Two to four civilizations may be stained on the same slide, which can be divided into 2-4 subdivisions with perpendicular ruddy wax pencil lines. To stain stuff from a civilization turning solid media, topographic point a loopful of tap H2O on a slide ; utilizing a unfertile cool cringle reassign a little sample of the settlement to the bead, and emulsify. Let the movie to air dry. Fix the dried movie by go throughing it briefly through the Bunsen fire two or three times without exposing the dried movie straight to the fire. The slide should non 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 H2O ( non over 5 seconds ) . Drain.

Flood slide with Gram ‘s Iodine solution, and let to move ( as a mordant ) for about one minute. Wash off with tap H2O. Drain.

Remove extra H2O from slide and smudge, so that intoxicant used for decolorization is non diluted. Flood slide with 95 % intoxicant for 10 seconds and rinse off with tap H2O. ( Smears that are overly thick may necessitate longer decolorization. This is the most sensitive and variable measure of the process, and requires experience to cognize merely how much to bleach ) . Drain the slide.

Flood slide with safranin solution and let to counterstain for 30 seconds. Wash off with tap H2O. Drain and smudge prohibitionists with boozy paper. Do non rub.

All slides of bacteriums must be examined under the oil submergence lens.

3.6 Detection of Cellulase Activity

Extracellular cellulases were tested utilizing agar home base incorporating 1 % ( w/v ) carboxymethylcellulose ( CMC ) ( pH 7.0 ) . The pure civilization on agar home bases was flooded with aqueous solution of Congo ruddy ( 1 % w/v ) for 15 min to observe cellulase production. The Congo ruddy solution was so poured off and the home bases were destained with 1 M NaCl for 15 min. The formation of a clear zone of hydrolysis indicated cellulose debasement by micro-organism ( Ariffin et al. , 2008 ) .

3.7 Preparation of Microbial Growth Curve

Preparation of growing curve was carried out by each of the purified micro-organism isolates in liquid CMC media. Preparation of growing curves are intended to find the stages of growing of cellulolytic isolates. Up to one cringle isolates inserted into 100 milliliter of liquid CMC media. The suspension is so homogenized and incubated on a shaker at 150 revolutions per minute at a temperature of 30oC for 7 yearss. Optical measuring is so performed Density ( OD ) utilizing a spectrophotometer at a wavelength of 540 nanometers. A sum of 10 % suspension of microbic civilization by the figure of cells 107 cfu / milliliter into 300 milliliter of media inserted so that the CMC in the medium incorporating 107 cfu / milliliter microbial ( bacterium ) . The suspension is so homogenized and incubated on a shaker at a temperature of 30oC with a velocity of 200 revolutions per minute. Bacterial cell growing curve was made by agencies of OD measurings every 6 hours during the first 24 hours, so samples were taken every 12 hours for 5 yearss to obtain the stages of the growing expected.

3.8 Activity check of Cellulolytic Microbial

Logarithmic stage of growing curves of the cellulolytic microbic isolates can be used to find the cellulolytic microbic stock. This stage can besides be used to find the stock inoculant to be used for this trial. Testing phases of this activity includes the readying of standard curves of glucose, doing a starting motor for the production of enzymes and enzyme activity measurings.

3.8.1 Preparation of Standard Curve Glucose

Standard solution of glucose was made at an interval of concentrations 0-1000 ?ug/ml. Of each concentration of glucose solution was taken and added every bit much as 1 milliliter, 1.5 milliliter of DNS solution so heated for 5 proceedingss and so added to 0.5 ml solution of Na tartrate-Pottasium, after it is cooled with running H2O for 15 proceedingss, so the term measured OD 540 nanometer ( Jeffries, 1996 ) .

3.8.2 Starter readyings for the production of cellulase enzymes

Making a starting motor for the production of an enzyme made aˆ‹aˆ‹according to the method Gorska et al. , ( 2001 ) , taking 10 % of the stock and put in production with the substrate CMC media, oil thenar empty fruit clump fibre and acerb hydrolysis residues

3.8.3 Cellulase activity assay

Measurement of cellulase enzyme activity was measured by decrease of the sum of sugar produced by 1 milliliter of the supernatant of media production, is so added 1 milliliter of the CMC substrate was dissolved in phosphate buffer ( 0.05 M ) ( pH 7.0 ) . the mixture was incubated at 37oC for 30 proceedingss and so the reaction was stopped with adding 3 ml solution DNS ( 2-hydroxy-3, 5 dinitrocalicylate ) . The mixture was so heated in boiling H2O for 5 proceedingss, so added 10 milliliter destilated H2O whose function is to stabilise the colour, so cooled with running H2O for 15 proceedingss and the optical density was measured utilizing a spectrophotometer at a wavelength of 540 nanometer. One unit of enzyme activity is the sum of enzyme that produced 1 mol of glucose in 1 minute at specific measuring conditions ( Susilawati et al, 2003 ) . For Oil thenar empty fruit clump ( OPEFB ) and residues of acerb hydrolysis substrates is followed the manner of CMC substrate to cellulase enzyme activity check.

The cellulase enzyme activity assay utilizing intervention parametric quantities long incubation, pH, temperature and revolutions per minute. for the long incubation period is 6, 12, 24, 36, 48, 60 and 72 hours for bacteriums and Fungis and actinomyces are for 1, 2, 3, 4, 5, 6 and 7 yearss. intervention of the consequence of pH is 3, 4, 5, 6, 7, and 8. For cellulase intervention temperature consequence was tested by utilizing the temperature is 25 oC, 30oC, 35oC, 40oC, 45oC and 50oC. Treatment of the consequence of revolutions per minute is 100, 125.150, 175, and 200.

3.9 Preparation for Production Etanol Using Saccharomyces cerevisiae

Oil thenar empty fruit Bunches are made into fibres by utilizing a patrol car machine to destruct so that a small-sized fibres and fibres are added to a concentration of 2 % sulphuric acid with a ratio of 1: 10 for 1 gm fibre and 10 milliliter of sulphuric acid. fibres and sulphuric acid put into 250 milliliters erlenmeyer sized and inserted into the sterilizer with temperature 121oC for 75 proceedingss. after completion, the blend of fibres and sulphuric acid is filtered utilizing Whatman filter paper No.1. liquid separated from solids. solids or residue that is taken to be a substrate for subsequent proving but before solids have to neutralisation by utilizing NaOH until obtained pH 6 – 7.

For enzymatic saccharification OPEFB from the acerb hydrolysis procedure was soaked a 100 milliliter of ethanoate buffer solution ( pH 4.8 ) and assorted with cellulase ( 70 FPU mL-1 from micro-organism at temperature of 48oC and rate of agitation 150 revolutions per minute for 48 hr. The OPEFB hydrolysate obtained was used for agitation.

3.9.1 Inoculums Preparation

Saccharomyces cerevisiae was ab initio grow on yeast-peptone-glucose ( YPG ) and incubated at a temperature of 35oC and a rate of agitation 150 revolutions per minute for 18 to 24 hr. In this survey, YPG medium consisted of ( gL-1 ) ; 10 g barm infusion, 20 g peptone and 20 g glucose. After incubation period, the cells were so harvested by centrifugation at 3000 revolutions per minute at 4oC for 15 minute. The pellet was so rinsed twice with sterilised saline solution before being re-suspended in sterilised saline solution to give an Optical Density ( OD ) of 1.0 at 600nm. The standardised S. cerevisiae was used for subsequent survey.

3.9.2 Ethanol Production

Agitation of Residues of acerb hydrolysis pretreatment procedure was carried out by utilizing S. cerevisiae. A sum of 150 milliliter of residues from enzymatic saccharification was prepared in a 250 milliliter conelike flask. A sum of 10 % ( v/v ) of standardised 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 revolutions per minute. OPEFB hydrolysate was harvested every 12 to 24 hr interval. The harvested samples were filtered utilizing a 0.45 µm membrane filter and so the filtered samples were put in 2.5 milliliter phials prior to being analyzed ( Mohd. Kassim et al. , 2011 ) .

Oil thenar empty fruit clump ( OPEFB )

Input signal on Cruise Machine

OPEFB fibre

Mix OPEFB with H2SO4 ( 1g OPEFB: 10ml H2SO4 )

Add H2SO4, 2 %

Input signal on Autoclave, 121oC for 75 proceedingss

Filtrated by Whatman paper No.1

Liquor Hydrolyzates

( Xylose, glucose )

Solid Hydrolyzates

( residues )

Figure 9. Flow Chart Preparation Acid Hydrolysis Residue from OPEFB

Samples

Plantation Palm Oil Field

Factory of Palm Oil

Incubation on OPEFB substrate + Mineral Medium, 30oC for 7 yearss

Incubation on CMC substrate + mineral medium,

30oC for 10 yearss

Incubation on Acid Hydrolysis Residues substrate + Mineral Medium, 30oC for 7 yearss

Microorganisms

Single settlement of isolates micro-organisms

Put on CMC Media,

Checking clear zone by Congo ruddy

The best isolates of micro-organisms

Preparation growing micro-organism curve

Preparation Glucose criterion curve

Activity enzym assay

The Best Condition trial:

pH, temp, clip, revolutions per minute

Production Ethanol utilizing S. cerevisiae

Figure 10. Flow Chart of Hydrolytic Microorganisms Associated with Palm Oil Industry

Incubation on CMC substrate + mineral medium,

30oC for 7 yearss

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