MICROBIAL COMMUNITY ANALYSIS IN ANAEROBIC PALM OIL MILL EFFLUENT (POME) WASTEWATER BY DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE)

Abstract

Microorganisms play a key role in wastewater bio-treatment processes and understanding the microbial community structure is of great importance to improve treatment performance. Denaturing gradient gel electrophoresis (DGGE) was used to monitor succession of the microbial community and thus predominant bands were sequenced to reveal the microbial community composition inside palm oil mill effluent (POME) wastewater.DNA bands from DGGE gels were excised with a sterile blade and placed in 1.5 ml eppendorf tube containing 50 μl deionized water (ddH2O). Tubes were incubated overnight at 4C to elute the DNA. Eluted DNA was purified using QIAquick gel extraction kits (QIAGEN, Inc., Valencia, CA) and was frozen and thawed three times.Microbial DNA successfully excised and purified from DGGE was amplified using polymerase chain reaction (PCR).Five micro liters of the supernatant were used as a template to re-amplify the DNA using 16s rDNA primers,341f (with no GC-clamp) (5'- cct-acg-gga-ggc-agc-ag-3') and reverse(r) primers 907r (5'-ccc-cgt-caa-ttc-att-tga-gtt-t-3'). Amplification was repeated referring to the steps in ‘PCR amplification of 16s rDNA’. PCR products from agarose gels were cut and purified using QIAquick Gel Extraction Kit (QIAGEN, Inc., Valencia, CA), which were similar to the purification steps after recovery of DNA from DGGE, and sequenced in both directions with the same primers (with no GC-clamp) as used in PCR. Moreover, start-up is an important step in establishing proper community structure in all kinds of biological treatment processes. In anaerobic POME wastewater, 6 sequences of Firmicutes, 5 sequence of Proteobacterium and 2 sequences of Bacteroidetes were found through denaturing gradient gel electrophoresis (DGGE) results. Sequence closely related to Rummeliibacillus suwonensis strain G20 was detected grows at band BE10, BE11, BE12, BE15, BE16, BE17 and BE18. Meanwhile BE25 and BE26 were found at ban d after treatment process done.Rummeliibacillus suwonensis is an aerobic, Gram-positive, rod shaped, round-spore-forming bacteria which were isolated from aerobic condition.