ISOLATION AND SCREENING OF PRODUCING …

1 , (1)2013:44-50 ISSN2229 644144 ISOLATION AND SCREENING OF -AMYLASE AND GLUCOAMYLASEPRODUCING FUNGI AND THEIR APPLICATION IN BIOETHANOLPRODUCTION1 Ominyi, ,2 Ogbonna, ,1 Nwoba, ,1 Nwagu, ,3 Ukachi, of Biotechnology, Ebonyi State University, 053 Abakaliki of Microbiology, University of Nigeria, Pathology Department, Federal Teaching Hospital Abakaliki,Ebonyi State of -amylase and glucoamylase PRODUCING fungal strains was investigated. Samples (120 in number) werecollected aseptically from rice mill industrial areas, cassava processing grounds, potato farms, corn processingindustriesand refuse dumping sites within Abakaliki metropolis in Ebonyi State of Nigeria. The samples were first grown on solidagar (PDA) and sub-cultured to get their pure cultures. The pure cultures were then grown for 3 days on PDA and screenedforstarch hydrolysis using Iodine-potassium iodide method. Out of the 120 cultures, 26 showed some degrees of starchhydrolysis.

2 The 26 positive fungal isolates were further screened for enzymatic activities which were measuredquantitatively with spectrophotometric methods. However, only 3 isolates (2a, 3 and 6b) were finally selected based ontheir high -amylase and glucoamylase production by co-cultivation of the selected isolatesshowed higher activities than single cultures. For instance, the co-culture of isolate 2a and 3 was higher ( + ) than individual activities of isolate 2a ( + U/ml) or isolate 3 ( + U/ml). bioethanol productionwas achieved by Simultaneous Saccharification and Fermentation (SSF) process using the selected fungal isolates andSaccharomyces bioethanol concentrations were measured quantitatively with boiling/iodometric 3 with the yeast gave the highest concentration of the ethanol ( + g/L) after 96hours followed by isolate2a and yeast ( + g/L) and then isolate 6b and yeast ( + g/L).

3 Finally, the selected fungal isolates wereidentified asAspergillus species(isolate 3),while isolates 2a and 6b wereMucorandRhizopus :Alpha-amylase, Glucoamylase, bioethanol , Fungi, ISOLATION , are of great importance in fermentation andfood industries for hydrolysis of starch and other relatedoligosaccharides (Akpanet al, 1999; Pederson andNielson, 2000). They are enzymes produced by variety oforganisms, ranging from bacteria to plants and and fungi secrete amylases to the outside of theircells to carry out extracellular digestion. Amylasesdegrade starch and other related polymers to yieldproducts characteristic of individual amylolytic is a glucose polymer linked together by -1,4 and -1,6 glycosidic bonds. Amylases act by hydrolyzing theglycosidic bonds between adjacent glucose units of thepolymer (Bernfeld, 1951).Based on the points of attack on the glucose polymerchain, alpha-amylases can be classified into twocategories, liquefying and saccharifying (Nigam andSingh, 1995).

4 The fungal -amylase belongs to thesaccharifyingcategory and attacks the second linkage ofthe straight chain of starch molecule from the non-reducing terminal ( end), resulting in the splitting offof two glucose units at a time (Fadel, 2000). The resultingproduct is a disaccharide called maltose. The starch chainsare thus broken down into smaller units. Glucoamylase(amyloglucosidase) consequently hydrolyses -1, 4-glycosidic bonds from non-reducing ends of starchmolecules, resulting in the production of glucose. To alesser extent, glucoamylase also has the ability tohydrolyze -1, 6 linkages, also resulting in glucose as theend product (Mertens and Skory, 2006).Fungal -amylase and glucoamylase may be used togetherto convert starch to simple sugars, which in turn serve as afeedstock for production of bioethanol or in the productionof high fructose syrups (Sevieketal, 2006). Whilefructose syrups are important sweetener in food processingindustries like beverages, bakeries and confectionaries, bioethanol has great potential asa renewable, non-toxicand clean alternative fuel that reduces dependency onfossil energy.

5 Fermentation of sugars derived from energycrops and grains is an economical and efficient method forbioethanol production. The use of these sugars forproducingbioethanol leads to opportunities for farmers byincreasing demand for their products, resulting in a boostin rural economies (Olfertet al, 2007). bioethanol as analternative source of energy has received special attentionover the world due to depletion of fossil fuels. Accordingto United States Department of Energy, for every unit ofenergy put towards ethanol production, units arereturned (Hilletal, 2006).Ethanol has the advantages ofbeing renewable, cleaner burning and produces nogreenhouse gases (Alt ntaset al, 2002).Yeast cells (Saccharomyces cerevisiae) are facultativeanaerobes and under anaerobic conditions can fermentglucose to cerevisiaeis ideal forethanol production due to several properties including fastIsolation and SCREENING of -amylase and glucoamylase PRODUCING fungi45growth rates, efficient glucose repression, effectiveethanol production and tolerance for environmentalstresses, such as high ethanol concentration and lowoxygen levels.

6 Glucose is broken down to form pyruvatein most organisms via the glycolytic pathway and thispyruvate can result in the production of ethanol underanaerobic conditions. The energy for growth of cellsduring ethanol production is provided by the glycolyticand fermentation pathways (Piskuret al., 2006).The efficiency of bioethanolproduction largely dependson the availability of suitable substrate, yeast strain andmethod employed. Starchy substrates are promising due totheir economic viability, availability and renewability(Coughlan, 1985).Starch, a macromolecular polymer ofglucose units, is a significant component of domestic andcommercial waste and a useful resource that can beconverted into ethanol. Yeast is unable to consume rawstarch and hence, the starch must first be broken down intosimple sugars (Birolet al., 1998). This task is achieved bytwo enzymes: -amylase, which hydrolyzes -1, 6linkages and glucoamylase, which hydrolyze -1,4linkages in starch molecules.

7 These enzymes areexpensive and contribute significantly to bioethanolproduction enzymes are not yet producedin Nigeria, making them too expensive for our so, in Ebonyi State, Abakaliki precisely, muchresearch work are yet to be done in the area of amylaseproduction. Meanwhile, fungal organisms are known fortheir ability to produce extracellular enzymes, -amylaseand glucoamylase capable of hydrolyzing starch substrates(Syu and Chen, 1997). These challenges were actually thedriving forces that necessitated this research, to isolate andscreen for effective amylase PRODUCING fungal strains fromour environment. And so doing, we could thereforeexplore the possibility to ameliorate the cost of amylaseenzymes for our industries and thus, develop a cheaperprocess for conversion of starch materials to AND METHODSM aterials used include: Agar media {Potato DextroseAgar (PDA)}, distilled water, petridishes, autoclave,thermometer, centrifuge, refrigerator, water bath,incubator, pH-meter, weighing balance, Bunsen burner,wire loops, test tubes andracks, bijou bottles, universalcontainers, Erlenmeyer flask, spectrophotometer and otherlaboratory collectionSamples were aseptically collected from cassavaprocessing sites, rice milling industries, corn processingareas, potato farms and refuse dumping grounds withinAbakaliki metropolis in Ebonyi State, South-East ofNigeria.

8 The upper layers of the sites werescraped offwith a sterilized spatula and the samples beneath werecollected using aseptic bags. These were immediatelytransported to the laboratory and stored at of Culture MediaPotato Dextrose Agar (PDA) was prepared according tothe manufacturer s specification. That is, 39g of agarmedia powder was dissolved in Erlenmeyer flaskcontaining 1L (1000ml) distilled water. The flask wascovered with cotton-plug and foil and sterilized byautoclaving at 121oC for 15 minutes. After coolingtoabout 45oC, about 12 ml of the media was poured intodifferent sterile petridishes and then left undisturbed untilthe agar solidified. The plates were maintained at Fungal OrganismsThe samples from different sources were suspended in 10ml of sterilized distilled water, followed by serial dilutionof each sample into four different flasks. A loopful ofeach sample from the diluents was streaked on thesolidified PDA media plates and incubated at 37oC for 72hours for the fungal growth.

9 The pure cultures wereidentified by their morphology and colony characteristicsand sub-cultured. The organisms were maintained on PDAslants and stored at of Isolates for Starch HydrolysisThe isolates were screened forstarch hydrolyzing fungal isolates were inoculated on 1 % starch PDAplate. After 3-4 days of fungal growth, the plates wereflooded with iodine solution. Starch reacted with iodine toform a dark blue starch-iodine complex that covered theentire agar. When starch was broken down into sugars,there were clear zones surrounding streaked lines whichindicate starch hydrolysis (Alfred, 2007). SCREENING of Isolates for Amylase Enzymes ProductionThe isolates were screened quantitatively for theproduction of -amylase and glucoamylase in a liquidculture medium constituted of (in gram per litre) solublestarch, 20; KH2PO4, 14; NH4NO3, 10; KCl, ; , ; , This was adjustedto About 100ml of the medium was distributed toeach 250ml Erlenmeyer flasks and sterilized byautoclaving at 121oC for 15 minutes.

10 They were allowedto cool down to room temperature (25oC). The isolateswere inoculated into the prepared liquid medium andincubated with shaking at 37oC for 3-5 days. The entirecontent of the culture were centrifuged and filteredaseptically to get the crude enzymes as culture were used to hydrolyze fresh starch solution andassayed for -amylase and glucoamylase of Alpha-amylase activityThe -amylase activities of the isolates were assayed usingthe Caraway-Somogyi, Iodine-Potassium Iodide (IKI)method according to Cheesbrough (2005).Measurement of Glucoamylase activityThe glucoamylase activity of each of the isolates wasdetermined by the method ofTrinder,(1959) knownas theglucose oxidase/peroxidase (GOD/POD) of Mixed-Culture of the Fungal Isolates onAlpha-amylase and Glucoamylase ProductionThe three selected fungal isolates were co-cultivated (twoisolates in one culture) in a liquid medium to compare theefficacies of mixed and single cultures of the isolates onthe production of -amylase and of Selected FungalIsolatesThe selected fungal isolates were identified bymorphological characteristics according to the taxonomickey of Alexopouloset al.