Sains Malaysiana 45(5)(2016): 811–815

Rapid Screening Method for Isolation of Glycerol-consuming Bacteria for Ethanol Production

(Kaedah Saringan Pesat untuk Pengasingan Bakteria Penggunaan-Gliserol untuk

Penghasilan Etanol)

SHERIL NORLIANA SUHAIMI¹, NUR AMELIA AZREEN ADNAN¹, PHANG LAI YEE¹*, SURAINI ABD-AZIZ¹ & TOSHINARI MAEDA²

¹Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia, 43400 Serdang, Selangor Darul Ehsan, Malaysia

²Department of Biological Functions and Engineering, Graduate School of Life Sciences

and Engineering, Kyushu Institute of Technology, 2-4 Hibikino, Wakamatsu-ku, Kitakyushu-shi, Fukuoka 808-0196, Japan

Received: 25 September 2014/Accepted: 20 November 2015

ABSTRACT

Large numbers of glycerol-consuming bacteria are present in nature; hence bioconversion of glycerol into biofuel which is bioethanol is one of the interests. The effective screening procedure is needed to screen and isolate broad ranges of bacteria from environment. The screening method was modified based on enzymatic oxidation of ethanol, which is correlated to reduction of 2,6-dichlorophenol-indophenol dye that resulted in the formation of yellow zone. Approximately 300 colonies were able to grow on minimal media using glycerol as sole carbon. Only about 70 isolates showed positive result when using the modified ethanol production assay after pre-screening stage. The formation of decolourized zone was apparent using modified assay containing 5 mL/L of 0.05M 2,6-dichlorophenol-indophenol, 10 mL of reaction mixture and 500 μl/L of enzyme, respectively. The ethanol production capability of the isolates was further proven by anaerobic fermentation as a quantitative method. This modified method is applicable in screening for ethanol producer from glycerol as carbon source allows rapid and more bacteria can be screened.

Keywords: 2,6-dichlorophenol-indophenol; ethanol production; glycerol-utilization; screening and isolation

ABSTRACT

Bio-penukaran gliserol kepada bioetanol menjadi satu kepentingan kerana sejumlah besar bakteria di dalam alam sekitar didapati berupaya untuk menggunakan gliserol. Oleh yang demikian, kaedah penyaringan yang berkesan diperlukan untuk menyaring dan memencilkan bakteria dalam jumlah yang banyak dari alam sekitar. Kaedah penyaringan telah diubah suai berdasarkan pengoksidaan enzim etanol yang berkait rapat dengan pemudaran pewarna 2,6-diklorofenol-indofenol dan seterusnya menghasilkan zon kuning. Kira-kira 300 koloni bakteria didapati dapat hidup apabila media minimum digunakan dengan gliserol sebagai sumber karbon utama. Hanya 70 daripada koloni tersebut menunjukkan keputusan positif daripada kaedah analisis etanol yang diubah suai pada peringkat pra-saringan. Pembentukan zon warna yang pudar dapat dilihat dengan jelas apabila menggunakan komposisi yang telah diubah suai iaitu 5 mL/L 0.05M 2,6-diklorofenol-indofenol, 10 mL campuran tindak balas dan 500 μl/L enzim. Keupayaan bakteria pencilan tempatan tersebut dibuktikan dengan fermentasi anerobik sebagai kaedah kuantitatif. Kaedah yang diubah suai ini boleh diguna pakai untuk menyaring lebih banyak bakteria yang berpotensi sebagai pengeluar etanol daripada gliserol sebagai sumber karbon dengan lebih cepat.

Kata kunci: Menyaring dan memencilkan; penggunaan gliserol; penghasilan etanol; 2,6 diklorofenol-indofenol

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*Corresponding author; email: phanglaiyee@upm.edu.my