SCREENING OF FIBROLYTIC ENZYME PRODUCING BACTERIA TO IMPROVE THE NUTRITIVE VALUE OF PALM KERNEL CAKE. Remi, E. and Wong, C. M. V. L.

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1 SCREENING OF FIBROLYTIC ENZYME PRODUCING BACTERIA TO IMPROVE THE NUTRITIVE VALUE OF PALM KERNEL CAKE Remi, E. and Wong, C. M. V. L. Biotechnology Research Institute Universiti Malaysia Sabah Jalan UMS, 884 Kota Kinabalu ABSTRACT A total of one thousand one hundred and forty six numbers of isolates were isolated from various EFB compost samples, effective microbe solution and raw EFB. Out of that number, a total of six hundred and twenty seven bacteria, two hundred and nineteen actinomycetes, one hundred and one yeast and one hundred and ninety nine fungi had been isolated and screened for galactomannanase, xylanase, cellulase and lipase activity. Fifteen isolates with good screening profile were assayed using quantitative assay for measurements of mannanase, cellulase and xylanase activity. The assay indicated that three isolates of the Bacillus sp. exhibited significantly higher activity of mannanase, cellulase and xylanase compared to the other twelve bacteria isolates. The isolates were also identified through 6S rdna sequencing. Ten different Bacillus sp., Micromonospora sp., Streptomyces sp. and Thermoactinomyces sp. were identified by comparing the partial 6S rdna sequences using the Basic Alignment Search Tool (BLAST) of PKC-based feed. These microbes could be useful in treating PKC and increase the feed nutritive value in the future after more detailed research have been conducted. INTRODUCTION Malaysia is the world s largest producer and exporter of palm oil. In the year 2, Malaysia produced.84 million tones of palm oil which accounted for 49.9% of the world palm oil production (Hishamuddin, 2). Therefore an abundant amount of by-product is produced. Palm kernel cake (PKC), empty fruit bunches (EFB), fibre, shell and potato ash are among the major by-products generated in the palm oil extraction process. Palm kernel cake is an important palm oil by-product. More than 6% of PKC is composed of cell wall components. The cell wall consists of 58% mannan, 2% cellulose and 4% xylan (Daud and Jarvis, 992). PKC is widely used as animal feed especially ruminants and poultry. However, due to the high fibrous components, PKC is more suitable for ruminants than nonruminants. Non-ruminants lack the ability to digest the fibrous components. One way to overcome this problem is to remove those fibrous anti-nutritive agents in PKC. Thus this study was carried out to isolate and screen for potential enzymes producing bacteria. The microbes were isolated from empty fruit bunch (EFB) compost and were screened for galactomannanase, xylanase, cellulase and lipase activites. EFB compost, an oil palm by-product was selected as the source of microbes because the diversity of microbes in the composting stages and the high incubation temperature of compost. These microbes with the ability to produce fibrolytic enzymes could be used to treat PKC.

2 METHODOLOGY Sampling and isolation of microorganisms Samples were obtained from Mahbina Sdn. Bhd., Shah Alam. Three different samples were received; 3 batches of raw empty fruit bunch (EFB), 3 batches of effective microbe (EM) solution, 4 days turnover EFB compost, 7 days turnover EFB compost and samples taken during composting. Isolations were done using dilution plate method (Cappuccino & Sherman, 983) on Nutrient agar (NA), yeast extract peptone glucose agar (YEPD), actinomycete isolation agar (AIA) and potato dextrose agar (PDA) to isolate bacteria, yeast, actinomycete and fungi respectively. In addition, starch casein nitrate agar (SCN) and Czapek-dox yeast extract casamino acid agar (CYC) were used to maintain mesophilic and thermophilic actinomycetes respectively. The plates were incubated overnight for bacteria, 3-7 days for actinomycetes and fungi and 2-5 days for yeast isolates at 3 C for mesophilic microorganisms and 55 C for thermophilic microorganisms. Screening for enzyme activities Isolates were screened for their galactomannanase, xylanase, cellulase and lipase activity. Substrate agar (. g/l peptone,. g/l yeast extract,.5 g/l KH 2 PO 4,.5 g/l MgSO 4.7H 2 O, 5. g/l agar and. g/l substrate ) was used for screening of different enzymes. Commercially available substrates; Azo-carob-galactomannan, Azo-xylan (oat) and Azo-CMcellulose were used to screen for galactomannanase, xylanase and cellulase respectively. Clearing zone around the colony was observed as having the ability to degrade the respective substrates. Sobitan monolaurate (Tween 2) was used as the lipid substrate (Hankin & Anagnostakis, 975). Lipase activity was observed by a visible precipitate due to the formation of crystals of the calcium salt of the lauric acid liberated the enzyme, or as a clearing of a such precipitate around a colony due to complete degradation of the salt of the fatty acid. Enzyme production by fifteen bacterial isolates Fifteen potential fibrolytic enzymes (galactomannanase, xylanase and cellulase) producing bacterial isolates were furthered screened by growing them in liquid medium and assaying enzyme activity from the cell-free culture supernatant liquid. The growth medium used contained.5% substrate,.% yeast extract,.% polypepton,.% NH 4 NO 3,.4% KH 2 PO 4,.2% MgCl 2 and % tap water (Abe et. al, 994). Locust bean gum (LBG), oat-spelt xylan and carboxymethyl cellulose (CMC) were used as substrates for mannanase, xylanase and cellulase activity respectively. The medium was sterilized by autoclaving at 2 C (5 psi) for 5 minutes. This medium was inoculated with % of an overnight culture as the starter culture and incubated at 3 C for mesophilic bacteria and 55 C for thermophilic bacteria in a water bath at 2 rpm for 24 hours. Sampling was done every two hours for 24 hours. Before assay, the cells were separated by centrifugation at, rpm. The clear supernatant was used as crude enzyme preparation. Enzyme assay Mannanase, cellulase and xylanase activity was assayed by measuring the release of reducing sugar from locust bean gum (LBG), oat-spelt xylan and carboxymethyl cellulose (CMC) respectively following the dinitrosalicylic acid (DNS) method (Miller, 959). Six hundred µl of substrate suspension which consist mixture of % LBG and buffer, ph 7. was aliquoted into.5ml eppendoff tubes. The substrate was prewarmed at 3 C for 5 minutes before

3 adding 6µl of the culture supernatant. The mixture of substrate suspension and culture supernatant was mixed and incubated at 3 C. The reaction was stopped after 3 minutes by adding 3µl dinitrosalicylic acid (DNS) reagent. The mixture was mixed and then boiled for 5 minutes. Absorbance was measured spectrophotometrically at 54 nm against a reagent blank where 6µl phosphate buffer was used instead of the culture supernatant. One unit of enzyme activity was defined as amount of enzyme that released µmol reducing sugar equivalent to mannose in minute. Xylanase and cellulase activity were determined by the same procedure as the mannanase, but using oat-spelt xylan and CMC as substrates respectively. Protein concentration was determined using the BioRad Protein Assay Kit which is based on the Bradford method. Identification of potential enzymes producing microbes Fifteen fibrolytic enzymes producing bacteria were subjected to DNA extraction. DNA extraction was done using the phenol-chloroform-isoamyl alcohol (PCI) method. Primers used for amplification of DNA were Com (5 -CAGCAGCCGCGGTAATAC-3 ) and Com 2 (5 - CCGTCAATTCCTTTGAGTTT-3 ). The PCR mixture consisted of mm dntps, 25mM MgCl 2, x PCR Buffer,.5mM of each primer and 2U/ l Taq DNA polymerase. The PCR conditions were; 94 C for 3 min, 94 C for min, 6 C for 3 sec, 72 C for 45 sec and 72 C for min. The PCR product was analyzed on agarose gel and was purified using HiYield Gel/ PCR DNA fragments extraction kit. Purified PCR product was sequenced, and compared to DNA sequences in the GeneBank ( RESULT AND DISCUSSION A total of one thousand one hundred and forty six numbers of isolates were isolated from EFB complete compost, effective microbe (EM) solution, raw EFB and samples taken during the composting of EFB. Out of that number, a total of six hundred and twenty seven bacteria, two hundred and nineteen actinomycetes, one hundred and one yeast and one hundred and ninety nine fungi had been isolated as presented in the pie chart in Figure. Microorganisms isolated from different samples Fungi, 99 Yeast, Bacteria, 627 Actinomycetes, 29 Figure : An overview of isolated microorganisms from different samples

4 Table : Screening profiles of the fifteen bacterial isolates (Halo zone size; + less than mm, ++ between mm to 2mm, +++ more than 2mm) Isolate Group Enzyme activity Xylanase Cellulase Mannanase Lipase 7DB Bacteria DU3 Bacteria DB3 Bacteria DB6 Bacteria DB8 Bacteria DB3 Bacteria DU3 Bacteria EFB(B)B6 Bacteria EFB(B)TB3 Thermophilic Bacteria D(B)TB8 Thermophilic bacteria D(C )TB4 Thermophilic bacteria D(C )TB6 Thermophilic bacteria NNB4 Actinomcyete D(A)A Actinomycete D(A)TA3 Thermophilic actinomycete All of the isolated microorganisms were screened for galactomannanase, xylanase, cellulase and lipase activity where fifteen potential fibrolytic enzymes producing bacteria were choosen based on their ability to degrade more than one substrate. Also, all fifteen bacterial isolates were able to degrade galactomannan. Due to the objectives of this study, further studies were done on their ability to produce mannanase, xylanase and cellulase. Table shows the screening profiles of the fifteen bacterial isolates. Enzyme production on locust bean gum During the mannanase production studies, the growth pattern of the fifteen isolates were observed every two hours for twenty four hours in the liquid medium containing.5% locust bean gum as a carbon source in 25ml conical flask. A quick glance at the data collected reveals that the fifteen bacterial isolates were able to grow in liquid media containing LBG as a carbon source. The isolates could be grouped into three different groups based on their growth; fast, moderate and slow bacterial growth, when cultured in liquid medium containing LBG as shown in Figure 2.

5 Fast growing bacterial isolates when cultured in medium containing LBG O.D. (6nm) DU3 4DB3 4DB8 4DB3 EFB(B)B6 EFB(B)TB Figure 2(a): Fast growing bacterial isolates when cultured in liquid medium containing LBG Bacterial isolates with moderate bacterial growth rates when cultured in medium containing LBG O.D. (6nm).6.4 2(B)TB8 28D TB4 7DB 4DU Figure 2(b): Moderately growing bacterial isolates when cultured in liquid medium containing LBG

6 Slow growing bacterial isolates when cultured in medium containing LBG O.D. (6nm).8.6 NNB4 24D(A)A 35D(A)TA3 4DB6 24D(C)TB Figure 2(c): Slow growing bacterial isolates when cultured in liquid medium containing LBG Time course of mannanase production of the fifteen isolates were shown in Figure 3(a), (b) and (c); grouped according to their growth rate. The graphs showed that all of the fifteen bacterial isolates excreted a certain amount of mannanase when cultured in liquid medium using LBG as sole carbon source. Eight isolates; 4DB3, 7DU3, EFB(B)B6, 4DB3, 4DU3, NNB4, 24D(A)A and 4DB6, exhibit significantly higher mannanase activity compared to the other seven isolates. Maximum mannanase activity of isolates 4DB3, 7DU3 and EFB(B)B6 were achieved at 8 hours of incubation in liquid medium containing LBG with specific activities.2446,.22 and.88 μmol/min/μg respectively. Isolate 4DB3 reached its maximum enzyme activity (.4 μmol/min/μg) at 6 hours of incubation. Interestingly, all four isolates exhibit an enzyme production curve similar to the bacterial growth curve (Figure 3(a)). Also, mannanase was produced during the log phase stage for all the four isolates where cells display the fastest rate of growth with cell division. Seven isolates, 4DB8, EFB(B)TB3, 7DB, 2D(B)TB8, 28D(C)TB4, 24D(C)TB6 and 35D(A)TA3, showed very low mannanase production throughout the incubation period. All of the isolates produced less than.5 μmol/min/μg of mannanase up till 24 hours of incubation in the liquid medium containing LBG making the isolates less significant compared to the other eight isolates.

7 .35 Mannanase activity produced by fast growing bacterial isolate during cultivation in LBG liquid medium.3 Mannanase activity (micromol/min/microgram) DU3 4DB3 EFB(B)B6 4DB3 4DB8 EFB(B)TB3 Figure 3(a): Mannanase production of fast growing bacterial isolates during cultivation in liquid medium containing LBG as sole carbon source.45 Mannanase production by bacterial isolates with moderate growth rates during cultivation in LBG liquid medium.4 Mannanase activity (micromol/min/microgram) D(B)TB8 28D(C )TB4 7DB 4DU3 Figure 3(b): Mannanase production of moderately growing bacterial isolates during cultivation in liquid medium containing LBG as sole carbon source

8 Mannanase production of slow growing bacterial isolates during cultivation in LBG liquid medium.3 Mannanase activity (micromol/min/microgram) NNB4 24D(A)A 35D(A)TA3 4DB6 24D(C )TB6 Figure 3(c): Mannanase production of fast growing bacterial isolates during cultivation in liquid medium containing LBG as sole carbon source Enzyme production on oat-spelt xylan During the xylanase production study, two growth patterns were observed when the fifteen isolates were cultured in liquid medium containing oat-spelt xylan as the sole carbon source. Thus the isolates were grouped into groups depending on their growth rates; fast growing and slow growing bacteria. Isolates that were able to grow directly or after two hours of incubation and were actively growing (log phase) between 2- hours of incubation were grouped into the fast growing bacteria. Isolates that are in their log phase for a longer period of time were grouped into the slow growing bacteria. Growth rates of the fifteen bacterial isolates when cultivated in liquid medium containing oat-spelt xylan are shown in Figure 4. Fast growing bacterial isolates when cultured in oat-spelt xylan medium.8.6 O.D. (6nm) DB3 4DU3 EFB(B)B6 7DU3 2D(B)TB8 EFB(B)TB3 Figure 4(a): Fast growing bacterial isolates when cultured in oat-spelt xylan liquid medium

9 Slow growing bacterial isolates when cultured in oat-spelt xylan.2.8 O.D. (6nm) DB6 24D(C )TB6 NNB4 24D(A)A 35D(A)TA3 4DB3 7DB 4DB8 28D(C)TB4 Figure 4(b): Slow growing bacterial isolates when cultured in oat-spelt xylan liquid medium Time course of xylanase production of the fifteen isolates were shown in Figure 5(a), (b) and (c); grouped according to their growth. The graphs showed that all fifteen isolates were able to produce extracellular xylanase when cultured in liquid medium containing oat-spelt xylan as sole carbon source. However each isolate exhibit different pattern of xylanase production ability. Isolates 7DU3, EFB(B)TB3, 4DB3, 4DU3, 4DB8,4DB3, 4DB6, 7DB, 24D(C)TB6, 35D(A)TA3 and 24D(A)A were to able produce significantly higher amount of extracellular xylanase among the fifteen isolates. Isolates EFB(B)B6, 2D(B)TB8, 28D(C)TB4 and NNB4 showed very low xylanase production throughout the incubation period. All of the isolates produced less than.3 μmol/min/μg of xylanase up till 24 hours of incubation in the liquid medium containing oat-spelt xylan making the isolates less significant compared to the other eight isolates..8 Xylanase production of fast growing bacterial isolates during cultivation in oat-spelt xylan liquid medium.6 Xylanase activity (micromol/min/microgram) DB3 4DU3 EFB(B)B6 7DU3 2D(B)TB8 EFB(B)TB3 Figure 5(a): Xylanase production of fast growing bacteria when cultured in liquid medium containing oat-spelt xylan

10 .2 Xylanase production of slow growing bacterial isolates during cultivation in oat-spelt xylan liquid medium Xylanase activity (micromol/min/microgram) DB6 24D(C )TB6 NNB4 24D(A)A 35D(A)TA3 4DB3 7DB 4DB8 28D(C )TB4 Figure 5(b): Xylanase production of slow growing bacteria when cultured in liquid medium containing oat-spelt xylan Enzyme production on CMC During the cellulase production study, it was observed that the growth of the fifteen bacterial isolates could be grouped into three distinct groups; fast, moderate and slow growing during cultivation in liquid medium containing CMC as sole carbon source. Isolates 7DU3, 4DB3, 4DU3 and 28D(C)TB4 were grouped into the fast growing bacterial isolates as their growth increased steadily between 2-8 hours of incubation time before entering death phase when cultured in liquid medium containing CMC. Bacterial isolates with moderate growth rates were isolates experiencing log phase between 2-4 hours of incubation in CMC liquid medium. This include isolates 7DB, 4DB3, 4DB8, EFB(B)B6, EFB(B)TB3 and 2D(B)TB8. It was also observed that the isolates reached their maximal growth between -4 hours of incubation but different isolates have different optical density when measured at absorbance 6nm. Slow growing bacterial isolates when grown in liquid medium containing CMC were isolates 4DB6, 24D(C)TB6, NNB4, 24D(A)A and 35D(A)TA3. Growth rates of the fifteen bacterial isolates when cultivated in liquid medium containing oat-spelt xylan are shown in Figure 6.

11 Fast growing bacterial isolates during cultivation in CMC containing media.2.8 O.D. (6nm) DU3 4DB3 28D(C )TB4 4DU3 Figure 6(a): Fast growing bacterial isolates during cultivation in liquid medium containing CMC as sole carbon source.2 Bacterial isolates with moderate growth rates during cultivation in liquid medium containing CMC.8 O.D. (6nm) DB 4DB8 4DB3 EFB(B)TB3 2D(B)TB8 EFB(B)B6 Figure 6(b): Moderately growing bacterial isolates during cultivation in liquid medium containing CMC as sole carbon source

12 Slow growing bacterial isolates during cultivation in liquid medium containing CMC O.D. (6nm) DB6 24D(C )TB6 NNB4 24D(A)A 35D(A)TA3 Figure 6(c): Slow growing bacterial isolates during cultivation in liquid medium containing CMC as sole carbon source Time course of CMC-ase production of the fifteen isolates were shown in Figure 7(a), (b) and (c); grouped according to their growth rate. The graphs showed that all fifteen isolates were able to produce CMC-ase when cultured in liquid medium containing CMC as sole carbon source. Isolates 7DU3, 4DB3, 4DB6 and 4DB3, 35D(A)TA3, EFB((B)TB3 and EFB(B)B6 were able to produce CMC-ase significantly higher among the fifteen bacterial isolates. Although all of the fifteen bacterial isolates were able to grow in liquid medium containing CMC, but the rate of their enzyme production were rather low compared to mannanase and xylanase activity. With the exception of isolate 7DU3, all isolates produced CMC-ase activity between.-.5 μmol/min/μg. Isolate 7DU3 reached its maximum CMC-ase activity (.734 μmol/min/μg) at 24h. Low detection of CMC-ase activity throughout the incubation period could indicate that longer incubation period is required for the monitoring of the bacterial isolates CMC-ase activities.

13 .2 Cellulase production of fast growing bacterial isolates during cultivation in liquid medium containing CMC.8 Cellulase activity (micromol/min/microgram) DU3 4DB3 28D(C )TB4 4DU3 Figure 7(a): Cellulase production of fast growing bacterial isolates during cultivation in liquid medium containing CMC as sole carbon source.6 Cellulase production of bacterial isolates with moderate growth rates during cultivation in CMC liquid medium.5 Cellulase activity (micromol/min/microgram) DB 4DB8 4DB3 EFB(B)TB3 2D(B)TB8 EFB(B)B6 Figure 7(b): Cellulase production of moderately growing bacterial isolates during cultivation in liquid medium containing CMC as sole carbon source

14 .6 Cellulase production of slow growing bacterial isolates during cultivation in liquid medium containing CMC.5 Cellulase activity (micromol/min/microgram) DB6 24D(C )TB6 NNB4 24D(A)A 35D(A)TA3 Figure 7(c): Cellulase production of slow growing bacterial isolates during cultivation in liquid medium containing CMC as sole carbon source Identification of the fifteen bacterial isolates Ten different Bacillus sp., two Bacterium sp., Micromonospora sp., Streptomyces sp. and Thermoactinomyces sp. were identified by comparing the partial 6S rdna sequences using the Basic Alignment Search Tool (BLAST) of PKC-based feed. A phylogenetic dendrogram based on the 6S rdna sequence data was constructed using MEGA version 4 (Tamura et al., 24) indicating different strains of Bacillus species as shown in Figure 8. 7DB 4DB3 4DB3 7DU3 4DB8 4DU3 EFB(B)B6 4DB6 2D(B)TB8 24D(C)TB6 28D(C)TB4 EFB(B)TB3 35D(A)TA3 NNB4 24D(A)A Figure 8: Phylogenetic tree of fifteen bacterial isolates constructed using the Neighbourjoining method.

15 CONCLUSION Three isolates of the Bacillus sp. (Isolates 7DU3, 4DB3 and EFB(B)B6) exhibited significantly higher activity of mannanase compared to the other twelve bacteria isolates during cultivation in liquid medium containing LBG. Isolates 7DU3, 4DB8 and EFB(B)TB3 exhibited significantly higher xylanase activity compared to the other twelve bacterial isolates during cultivation in liquid medium containing oat-spelt xylan. Isolates 7DU3, 4DB3 and 4DB3 exhibited significantly higher cellulase activity compared to the other twelve bacterial isolates during cultivation in liquid medium containing CMC. These microbes with the ability to produce one or more fibrolytic enzymes could be used to treat PKC to increase the feed nutritive value. REFERENCE Cappuccino,J. G. & Sherman, N Microbiology: A Laboratory Manual. Addision- Wesley,Reading, Massachusetts, pp Daud, M.J. and Jarvis, M.C Mannans of oil palm kernels. Phytochemistry 3(2), Hankin, H. & Anagnostakis, S. L. (975). The use of solid Media for detection of enzyme production by fungi. Mycologia. 67, Hishamuddin Mohd Aspar. 2. Malaysian palm kernel cake as animal feeding. Journal of Palm Oil Development 34, 4-6. Tamura K, Dudley J, Nei M & Kumar S (27) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.. Molecular Biology and Evolution 24: (Publication PDF at