Pengoptimuman Penjerapan Acinetobacter baumannii UKMP-12T pada Aerogel Selulosa dan Aplikasi dalam Degradasi Hidrokarbon

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1 Undergrad. Res. J. Biomol. Sci. Biotech., 207 Undergraduate Research Journal for Biomolecular Sciences and Biotechnology ISSN Pengoptimuman Penjerapan Acinetobacter baumannii UKMP-2T pada Aerogel Selulosa dan Aplikasi dalam Degradasi Hidrokarbon Nur Aida Syazwana Che Zakaria, Siti Khatijah Shafie & Ainon Hamzah* Program Mikrobiologi, Pusat Pengajian Biosains dan Bioteknologi, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia * mel-e pengarang rujukan: antara@ukm.edu.my Abstrak: Penggunaan aerogel selulosa dipilih sebagai satu kaedah penyimpanan bakteria untuk bioremediasi. Objektif kajian ini adalah untuk pengoptimuman penjerapan Acinetobacter baumannii UKMP-2T pada aerogel selulosa menggunakan kaedah permukaan gerak balas (RSM) dan aplikasi aerogel terjerap bakteria dalam degradasi hidrokarbon secara kultur goncangan dan lapangan. Selulosa diekstrak menggunakan 4% NaOH dan.7% NaClO 2. Tiga parameter yang dioptimumkan untuk penjerapan adalah suhu, ph dan masa penjerapan. Bacaan ketumpatan optik (OD) diambil bagi melihat penjerapan bakteria. Penentuan degradasi hidrokarbon dilakukan secara kultur goncangan dan lapangan menggunakan biojisim berbeza aerogel selulosa yang telah terjerap bakteria. Hasil kajian RSM menunjukkan kombinasi suhu 25 o C, ph 7 dan masa penjerapan 5 jam memberikan hasil penjerapan bakteria paling optimum. Bilangan koloni bakteria yang terjerap pada aerogel basah adalah CFU/mL/g. Degradasi hidrokarbon secara kultur goncangan dalam medium garam mineral (MSM)+ % (i/b) minyak mentah Tapis adalah paling tinggi apabila 0.6 g aerogel selulosa digunakan iaitu 88.63% pada hari ke 7 eraman. Manakala bagi kaedah lapangan, penggunaan 2.25 g aerogel selulosa terjerapbakteria untuk 500 g tanah + 0% (i/b) minyak mentah Tapis menghasilkan degradasi TPH sebanyak 89.2%. pada hari ke-28. Kata kunci: Acinetobacter baumannii; aerogel selulosa; penjerapan; bioremediasi Abstract: Cellulose aerogel was chosen as a biocarrier for bacterial storage and bioremediation. The objectives of this study are to optimize the adsorption of Acinetobacter baumannii UKMP-2T onto cellulose aerogel using response surface methodology (RSM) and the application of aerogel adsorbed bacteria for hydrocarbon degradation using shake flask method and field study. Cellulose extraction was carried out using 4% NaOH and.7% NaClO 2. Three optimization parameters used were temperature, ph and absorption time. Optical density (OD) was taken to observe the bacterial adsorption. Hydrocarbon degradation was performed by shake flask method and field study using different weight of cellulose aerogel that had been adsorbed by bacteria. The RSM results showed the combination of a temperature at 25 o C, ph 7 and 5 hours of adsorption time produced the optimal bacterial adsorption. The number of bacterial colonies adsorbed to the wet aerogel was CFU/mL/g. Hydrocarbon degradation by shake flask method in salt mineral medium (MSM) + % of Tapis crude oil was the highest when 0.6 g of aerogel adsorbed-bacteria was used with 88.63% of TPH degraded on the 7 days of incubation. Meanwhile, for field study, usage of 2.25 g of cellulose aerogel adsorbed-bacteria for 500 g soil+0% of Tapis crude oil produced 89.2% TPH degradation on the 28 day incubation. Keywords: Acinetobacter baumannii; cellulose aerogel; adsorption; bioremediation 83

2 Undergrad. Res. J. Biomol. Sci. Biotech., 207. Pengenalan Pencemaran tanah oleh hidrokarbon sering berlaku disebabkan oleh tumpahan minyak, kebocoran tangki di loji-loji pemprosesan minyak serta pembuangan minyak yang tidak terkawal. Selain itu, pembuangan minyak daripada bengkel serta pusat servis kereta juga merupakan penyebab kepada berlakunya pencemaran tanah dan air sungai. Walaupun terdapat banyak kaedah yang boleh merawat hidrokarbon, tetapi kaedah bioremediasi sering digunakan kerana kecekapannya serta kosnya yang rendah (Liu et al. 20). Bioremediasi merupakan satu kaedah rawatan bahan tercemar dengan menggunakan mikroorganisma. Kaedah ini dapat mengurangkan berlakunya risiko pencemaran. Bagi merawat pencemaran ini, jumlah mikroorganisma yang besar diperlukan. Penyediaan kultur bakteria penting bagi merawat kawasan pencemaran. Tetapi, penyediaan kultur bakteria segar mengambil masa yang lama, menggunakan kos yang tinggi dan tidak efektif kerana kultur segar tidak boleh disimpan dalam jangka masa yang panjang. Oleh itu, satu kaedah baru dengan menggunakan aerogel selulosa dipilih sebagai satu kaedah penyimpanan bakteria. Selulosa diekstrak daripada pelepah kelapa sawit kerana kandungannya yang tinggi dengan selulosa, mudah didapati serta murah kerana ianya merupakan bahan buangan yang tidak digunakan. Aerogel selulosa dapat menyimpan bakteria dalam jumlah yang besar, mudah untuk diaplikasikan pada kawasan pencemaran dan menggunakan kos yang rendah. Objektif bagi kajian ini adalah untuk mengoptimumkan penjerapan Acinetobacter baumannii pada aerogel selulosa menggunakan kaedah permukaan gerak balas (RSM) dan menguji keberkesanan degradasi hidrokarbon secara kaedah kultur goncangan dan lapangan. 2. Bahan dan Kaedah 2. Pengekstrakan selulosa daripada pelepah kelapa sawit dan sintesis butiran aerogel Pelepah kelapa sawit diambil daripada Hutan Pendidikan Alam, Universiti Kebangsaan Malaysia, Bangi Selangor. Pengekstrakan pelepah kelapa sawit dilakukan berasaskan kaedah Chan et al. (203) dengan pengubahsuaian. Pelepah dipotong dan dikisar menggunakan pengisar kepada saiz cm. Sampel dijalankan rawatan alkali dengan 4% sodium hidroksida (NaOH) dan direndam pada suhu 80 C di dalam pengukus air selama 2 jam, dibilas dengan air dan diulang sebanyak 3 kali. Rawatan pelunturan dilakukan dengan menggunakan.7% sodium klorit (NaClO 2) pada ph 4.5 dan dimasukkan ke dalam pengukus air yang bersuhu 80 C selama 4 jam, dibilas dengan air dan diulang sebanyak empat kali. Seterusnya selulosa yang diekstrak dikeringkan pada suhu 80 C dan ditimbang. Pelarutan selulosa dilakukan berdasarkan kaedah daripada Qin et al. (203) dengan pengubahsuaian. Selulosa pelepah kelapa sawit dimasukkan ke dalam larutan tersebut pada kepekatan 4% (b/i). Sebatian disejukkan pada -20 C selama 0 minit dan dikisar selama 6 minit dengan menggunakan pengaduk Wisemix HG-5A. Seterusnya, sebatian tersebut dibekukan pada - 20 C selama 24 jam bagi proses pembentukan gel. Penghasilan butiran selulosa dilakukan berdasarkan kaedah daripada Krishnan et al. (200) dengan pengubahsuaian. Selepas dibekukan selama 24 jam, sebatian dikeluarkan dan dicairkan pada suhu bilik. Seterusnya, agen pembentuk liang, kalsium karbonat (CaCO 3) ditambah dengan nisbah : (g) ke dalam larutan tersebut dan dikacau rata. Dengan menggunakan picagari, sebatian dititiskan ke dalam asid hidroklorik pada kepekatan 0% (i/i) pada jarak -2 cm daripada permukaan pelarut. Butiran aerogel yang terhasil dikeluarkan dan direndam di dalam air ternyahion selama 48 jam sebelum dikeringkan bersama butiran gel silika pada suhu 60 C selama 24 jam. 2.2 Penjerapan bakteria pada aerogel selulosa Penyediaan inokulum piawai bakteria dilakukan mengikut kaedah Ainon et al. (200). Rekabentuk percubaan menggunakan kaedah permukaan gerak balas (RSM) digunakan untuk mengoptimumkan penjerapan bakteria pada selulosa. Tiga parameter digunakan iaitu masa penjerapan (4 jam hingga 84

3 Undergrad. Res. J. Biomol. Sci. Biotech., jam), ph (5.5 hingga 7) dan suhu (20 o C hingga 25 o C). Kelalang Erlenmeyer 50 ml ditambah 20 ml inokulum piawai dan butiran selulosa sebanyak 6% (.2 g) dan dieram di dalam penggoncang orbital mengikut rekabentuk RSM. Bacaan ketumpatan optik (OD) diambil mengikut masa yang yang ditetapkan menggunakan spektrofotometer dan bilangan koloni bakteria (CFU/mL) dikira dengan kaedah piring sebaran. 2.3 Degradasi Hidrokarbon Secara Kultur Goncangan Jenis minyak mentah yang digunakan adalah minyak mentah Tapis yang dibekalkan oleh Petronas Research Sdn. Bhd. Sebanyak % (i/i) minyak mentah Tapis ditambah ke dalam setiap kelalang yang mengandungi 20 ml medium garam mineral (MSM) pada ph 7.5 dan ditambah dengan jumlah aerogel selulosa yang berbeza (Jadual ). Semua kelalang dieram di dalam penggoncang orbital pada suhu 30 C, pada 00 rpm selama 7 hari. Pada hari ke 7 bilangan bakteria ditentukan dengan kaedah piring sebaran dan ekstraksi minyak dilakukan mengikut kaedah Ainon et al. (200). Ujikaji dilakukan dalam 3 replikat. Jadual. Reka bentuk kajian menggunakan kaedah kultur goncangan Rawatan Kandungan A MSM + % (i/i) minyak mentah (kawalan) B MSM + % (i/i) minyak mentah + % (b/i) aerogel selulosa C MSM + % (i/i) minyak mentah + 2% (b/i) aerogel selulosa D MSM + % (i/i) minyak mentah + 3% (b/i) aerogel selulosa 2.4 Degradasi Hidrokarbon Secara Kaedah Lapangan Tanah yang digunakan adalah tanah merah dan pasir dalam nisbah 2:. Tanah ditapis dengan menggunakan penapis yang bersaiz liang 2 mm. ph tanah diselaras sehingga ph dengan menggunakan larutan kalsium karbonat (5% b/i). Kelembapan tanah diukur dengan alat pengukur kelembapan dan suhu. Kelembapan tanah dikekalkan kepada 20% kandungan isipadu air (VMC). Setiap plot mengandungi 500 g tanah, ditambah dengan 0% (i/b) minyak mentah dan digaul rata. Seterusnya, tanah tersebut dibiarkan selama 24 jam supaya minyak meresap sepenuhnya ke dalam tanah. Tanah tersebut ditambah dengan aerogel selulosa yang telah dijerap dengan A. baumannii. Kajian ini menggunakan 00 dan 50 butiran aerogel selulosa iaitu 20% dan 30% daripada berat tanah yang bersamaan dengan.5 g dan 2.25 g aerogel selulosa (Jadual 2). Setiap rawatan dijalankan secara triplikat. Jadual 2. Reka bentuk kajian lapangan Rawatan Kandungan A B C 500 g Tanah + 0% (i/b) minyak mentah (kawalan) 500 g Tanah + 0% (i/b) minyak mentah +.50 g aerogel selulosa 500 g Tanah + 0% (i/b) minyak mentah g aerogel selulosa 85

4 Undergrad. Res. J. Biomol. Sci. Biotech., 207 Persampelan dilakukan setiap tujuh hari bermula daripada hari 0 untuk menentukan bilangan koloni bakteria (CFU/mL) dengan kaedah piring sebaran dan degradasi minyak mentah ditentukan dengan kromatografi gas (GC-FID) dan diukur dalam petroleum hidrokarbon jumlah (TPH). 2.5 Analisis statistik Data yang diperolehi daripada kajian ini dianalisis secara statistik dengan menggunakan perisian Design-Expert Hasil dan Perbincangan 3. Pengekstrakan selulosa dan sintesis butiran aerogel Sampel selulosa yang dirawat bertukar menjadi warna putih kerana hemiselulosa dan lignin telah dibuang. Berat purata selulosa yang dapat diekstrak daripada 0 g pelepah kelapa sawit adalah sebanyak g bersamaan 0.4 g selulosa/g pelepah (Rajah a). Purata berat bagi butiran aerogel selulosa yang disintesis adalah 0.0 g bagi aerogel yang kering manakala 0.02 g bagi aerogel yang basah (Rajah b). Tasasao (205) telah mengekstrak selulosa daripada tandan kosong kelapa sawit dan menghasilkan 0.3 g selulosa /g tandan kosong kelapa sawit, iaitu 0. g kurang daripada yang diekstrak dalam kajian ini. (a) (b) Rajah. Hasil (a) selulosa selepas rawatan alkali dan pelunturan (b) butiran aerogel. 3.2 Pengoptimuman penjerapan bakteria pada aerogel selulosa dengan menggunakan kaedah permukaan gerak balas (RSM) Dalam kajian ini, terdapat tiga faktor yang tidak bersandar telah diuji iaitu A = suhu ( o C), B = ph dan masa (jam) manakala gerak balas bagi kajian ini ialah bacaan OD. Larutan 0.5 inokulum piawai Acinetobacter baumannii telah memberikan bilangan koloni bakteria sebanyak CFU/mL. 86

5 Undergrad. Res. J. Biomol. Sci. Biotech., 207 Jadual 3. Kesan suhu, ph dan masa terhadap penjerapan A. baumannii pada aerogel selulosa Rawatan Suhu ( o C) ph Masa (jam) OD Berdasarkan hasil yang diperolehi, kombinasi suhu 25 o C, ph 7 dan masa selama 5 jam menghasilkan penjerapan tertinggi bakteria pada aerogel selulosa dengan bacaan OD sebanyak (Jadual 3). Bilangan optimum bakteria yang terjerap pada aerogel selulosa basah adalah sebanyak CFU/mL/g. Dalam kajian Rochex et al. (2004), penjerapan optimum bagi Pseudomonas putida pada gentian selulosa adalah pada ph berasid iaitu ph 5 dan pada masa dan suhu yang sama (5 jam, 25 o C). Bilangan bakteria yang terjerap pada gentian selulosa adalah sebanyak CFU/mL/g. Hal ini adalah disebabkan oleh penggunaan bakteria yang berbeza genus dan spesis. Selain itu, kajian Ortiz et al. (200) dalam pelekatan bakteria P. putida pada gentian agav menunjukkan pelekatan optimum adalah masa 5 jam, ph 7 dan suhu 40 o C. Bilangan bakteria yang terjerap pada gentian agav adalah sebanyak CFU/mL/g. Melalui kajian ini menunjukkan bahawa ph dan masa pelekatan optimum yang digunakan adalah sama, manakala suhu optimum yang digunakan adalah berbeza. Ini adalah mungkin disebabkan oleh penggunaan gentian yang berbeza di mana gentian ini lebih sesuai dan tahan pada suhu yang tinggi, manakala selulosa tidak tahan pada suhu yang tinggi. 3.3 Analisis data pengoptimuman penjerapan bakteria pada aerogel selulosa Bagi model pengoptimuman profil penjerapan A. baumannii pada aerogel selulosa, didapati bahawa nilai P bagi model ini adalah signifikan dengan nilai kerana tidak melebihi 0.05, manakala nilai Lack of fit dilihat tidak signifikan iaitu melebihi nilai Bagi nilai R 2 pula menunjukkan nilai melebihi Nilai ini menunjukkan model yang baik (fit). 87

6 Undergrad. Res. J. Biomol. Sci. Biotech., 207 Jadual 4. Analisis ANOVA bagi pengoptimuman profil penjerapan bakteria A. baumannii pada aerogel selulosa Sumber Darjah kebebasan Jumlah kuasa dua Min kuasa dua Nilai F Nilai P Kuadratik E E A 5.506E E B 2.944E E C.054E E A E E B E E C E E AB 3.25E E AC.52E E BC 3.62E E Residual E E-005 Lack of fit E E Ralat tulen E E-005 Jumlah E-003 Analisis ANOVA berkenaan faktor tunggal iaitu suhu (A) menunjukkan nilai 0.000, ph (B) menunjukkan nilai dan masa (C) menunjukkan nilai (Jadual 4). Nilai yang ditunjukkan ketiga-tiga faktor tersebut adalah signifikan dan tidak melebihi Ini menunjukkan bahawa ketiga-tiga faktor ini memberikan kesan yang signifikan terhadap penjerapan A. baumannii pada aerogel selulosa. Manakala, analisis bagi perkaitan antara faktor-faktor tersebut juga menunjukkan kesan yang signifikan. Hal ini ditunjukkan oleh perkaitan faktor suhu dan ph (AB) mempunyai nilai , suhu dan masa (AC) mempunyai nilai dan ph dan masa (BC) mempunyai nilai Nilai yang ditunjukkan oleh perkaitan antara faktor-faktor ini adalah signifikan iaitu tidak melebihi Analisis ini menunjukkan bahawa terdapat perkaitan yang ditunjukkan oleh kombinasi ketiga-tiga faktor tersebut. Interaksi yang ditunjukkan pada plot 3-Dimensi adalah interaksi antara suhu dan ph, suhu dan masa dan ph dan masa. Dalam analisis ini menunjukkan penjerapan pada suhu 25 o C, ph 7 dan masa selama 5 jam adalah penjerapan pada tahap tertinggi yang memberikan bacaan OD paling rendah iaitu berdasarkan plot 3-Dimensi ini (Rajah 2). 88

7 Undergrad. Res. J. Biomol. Sci. Biotech., 207 (a) (b) (c) Rajah 2. Interaksi antara (a) suhu dan ph, (b) suhu dan masa dan (c) ph dan masa terhadap penjerapan A. baumanni pada aerogel selulosa. 3.4 Penentuan nilai optimum bagi setiap faktor Hasil analisis ANOVA memberikan cadangan penyelesaian pengoptimuman bernombor untuk penjerapan A. baumannii pada aerogel selulosa iaitu suhu 25 o C, ph 7 dan masa selama 5 jam bagi penjerapan yang paling tinggi dengan bacaan OD adalah Hasil uji kaji cadangan ini telah memberikan bacaan OD adalah Di dalam kajian ini menunjukkan penjerapan A. baumannii pada aerogel selulosa adalah lebih tinggi daripada yang dicadangkan. 3.5 Degradasi Hidrokarbon Secara Kultur Goncangan Pertumbuhan bakteria di dalam MSM yang ditambah % minyak Tapis menunjukkan peningkatan bilangan bakteria selepas tujuh hari eraman (Jadual 5). Rawatan yang mengandungi 0.6 gm aerogel menunjukkan jumlah bakteria yang paling tinggi iaitu.0 0 cfu/ml dengan penurunan hidrokarbon petroleum jumlah (TPH) sebanyak 88.63% dan degradasi pristana sebanyak 8.29%. Perbandingan dengan kajian Raja Farzarul (202) yang menggunakan kultur segar sebanyak 0% (i/i) inokulum piawai A. baumannii di dalam MSM yang ditambah % (i/i) minyak mentah Tapis juga secara kultur goncangan selama 7 hari menunjukkan hasil degradasi TPH yang lebih rendah iaitu sebanyak 76.24% dengan bilangan bakteria cfu/ml. Hal ini disebabkan oleh penggunaan aerogel selulosa terjerap-bakteria mungkin membantu pelepasan bakteria ke dalam medium secara perlahan, oleh itu bakteria dapat mandiri dalam tempoh masa yang lebih lama sekaligus dapat menghasilkan enzim yang berterusan yang membantu degradasi hidrokarbon dengan lebih tinggi. Lin et al. (203) menggunakan Acinetobacter sp. HC8-3S yang dipegunkan pada serat kapas untuk mendegradasi 0.5 g minyak mentah di dalam 50 ml larutan nutrien tambahan steril. Hasil yang didapati selepas 5 hari menunjukkan degradasi hidrokarbon tepu yang lebih efisien iaitu 89

8 Undergrad. Res. J. Biomol. Sci. Biotech., 207 sebanyak 30% lebih tinggi daripada sel bakteria yang terampai. Menurutnya, serat kapas membantu degradasi hidrokarbon dengan menyerap minyak di dalam larutan dan memberikannya kepada bakteria yang terjerap pada serat kapas tersebut untuk didegradasi. Dalam kajian ini, aerogel selulosa dapat menyerap minyak yang berada di dalam medium MSM dan menyalurkannya secara terus kepada bakteria yang terjerap sebagai sumber karbon. Jadual 5. Kesan peratusan aerogel yang berbeza terhadap degradasi hidrokarbon selepas tujuh hari eraman di dalam medium MSM + % minyak mentah Tapis Peratus aerogel (%) Purata bilangan koloni bakteria (CFU/mL) Degradasi TPH (%) Degradasi Pristana (%) X X X Degradasi Hidrokarbon Secara Kaedah Lapangan Dalam kaedah lapangan, tanah yang ditambah dengan 2.25 g aerogel selulosa terjerap-bakteria menunjukkan peningkatan bakteria yang paling tinggi sehingga hari ke-28 iaitu cfu/g berbanding dengan hari permulaan sebanyak cfu/g (Rajah 3a). Bakteria indigenus pada hari ke 28 adalah cfu/g. Penurunan TPH yang paling tinggi adalah 89.2% pada hari ke-28 dengan penggunaan 2.25 g aerogel selulosa, iaitu bersamaan dengan 7.3% lebih daripada degradasi TPH bagi rawatan kawalan yang hanya mempunyai mikrob indigenus sahaja (Rajah 3b). Degradasi pristana paling tinggi adalah bagi penggunaan 2.25 g aerogel selulosa iaitu 95% (Jadual 6). Liang et al. (2009) yang menggunakan karbon aktifan sebagai bahan pembawa bagi bakteria pencurai hidrokarbon menunjukkan penggunaan bahan pembawa membantu dalam meningkatkan degradasi minyak mentah di dalam tanah dengan degradasi TPH sebanyak 48% dan peningkatan jumlah bakteria daripada 0 3 cfu/g sehingga 0 8 cfu/g pada hari yang ke-33. Menurutnya, penggunaan bakteria yang dipegun pada bahan pembawa dapat memberikan perlindungan dan nutrien kepada bakteria tersebut. Bahan pembawa juga dapat meningkatkan interaksi antara substrat dengan sel yang telah dipegunkan yang menyebabkan peningkatan degradasi (Wilson & Bradley 996). (a) (b) Rajah 3. Kesan berat aerogel yang berbeza selama 28 hari di dalam tanah yang mengandungi 0% (i/b) minyak mentah Tapis (a) bilangan koloni bakteria dan (b) degradasi TPH (%). 90

9 Undergrad. Res. J. Biomol. Sci. Biotech., 207 Jadual 6. Kesan berat aerogel yang berbeza terhadap degradasi hidrokarbon selepas 28 hari eraman di dalam tanah + 0% (i/b) minyak mentah Tapis Aerogel selulosa (g) Degradasi Pristana (%) 0.0 (kawalan) Kesimpulan Bakteria yang terjerap pada aerogel selulosa mampu bermandiri dalam masa yang lebih lama dan mendegradasi hidrokarbon lebih baik. Aerogel selulosa dapat melindungi bakteria yang terjerap daripada kekeringan tanah. 5. Penghargaan Kami ingin mengucapkan ribuan terima kasih kepada UKM kerana memberi geran penyelidikan DLP untuk projek ini. Rujukan Ainon Hamzah, Amir Rabu, Raja Farzarul Hanim Raja Azmy & Noor Ainni Yussoff Isolation and characterization of bacteria degrading Sumandak and South Angsi Oils. Sains Malaysiana 39: Chan, H.C., Chia, C.H., Sarani, Z., Ishak, A. & Dufresne, A Production and characterisation of cellulose and nano-crystalline cellulose from kenaf core wood. BioResources 8(): Krishnan, V., Sasikumarb, Dass, F.P. & Vijayaraghavanb, R Effect of pore forming agents on the physical characteristics and release kinetics of levofloxacin hemihydrate from floating alginate drug delivery system - an in vitro study. Trends in Biomaterials & Artificial Organs 24(3): Liang, Y., Zhang, X., Dai, D. & Li, G Porous biocarrier-enhanced biodegradation of crude oil contaminated soil. International Biodeterioration & Biodegradation 63: Lin, M., Liu, Y., Chen, W., Wang, H. & Hu, X Use of bacteria-immobilized cotton fibers to adsorb and degrade crude oil. International Biodeterioration & Biodegradation 88: 8-2. Liu, P.W.G., Chang, T.C., Whang, L.M., Kao, C.H., Pan, P.T., & Cheng, S.S. 20. Bioremediation of petroleum hydrocarbon contaminated soil: Effects of strategies and microbial community shift. International Biodeterioration and Biodegradation. 65: Ortiz, J.R.R., Arreola, D.E.R., Gomez, C., Reynoso, O.G., & Nunez, R.G Bacterial immobilization by adhesion onto agave-fiber/polymer foamed composites. Bioresource Technology. 0: Qin, X., Lu, A. & Zhang, L Gelation behaviour of cellulose on NaOH/urea aqueous system via crosslinking. Cellulose 20: Raja Farzarul Hanim Raja Azmy Degradasi minyak mentah Tapis oleh bakteria pencilan tempatan. Tesis S. Sn, Universiti Kebangsaan Malaysia. Rochex A., Lecouturier D., Pezron I., Lebeault J.M Adhesion of a Pseudomonas putida strain isolated from a paper machine to cellulose fibres. Applied Microbiology and Biotechnology. 65: Tasaso, P Optimization of reaction conditions for synthesis of carboxymethyl cellulose from oil palm fronds. International Journal of Chemical Engineering and Applications. 6 (2): Wilson N.G. & Bradley, G Enhanced degradation of petroleum (sloven diesel) in an aqueous system by immobilized Pseudomonas fluorescens. Journal of Applied Microbiology 80:

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