microbial community analysis of leachate

characterize the bacterial community

1. MICROBIAL COMMUNITY ANALYSIS OF LEACHATE FROM JERAM AND JABOR LANDFILL FOR POTENTIAL BIOREMEDIATION Supervisor : Assoc. Prof. Dr. Noor Faizul Hadry Nordin Co-supervisor : Prof. Dr. Hamzah Mohd Salleh Assoc. Prof. Dr. Husna Ahmad Tajuddin Student : Siti Marhamah Drahaman Institution : INHART, International Islamic University Malaysia

2. INTRODUCTION

3. Illustration for microbial community analysis Beneficial for microorganism that has specific growth requirements ( Ogram et al., 2007) and easy to manipulate based on specific required condition ( Süß et al., 2004). C ultivation has an important impact on soil degradation, biological activities and soil microbial community (Ghimire et al. 2014). One of the effective and low-cost method for the characterization of bacterial populations, taxonomical analysis and species identification (Cardenas & Tiedje 2008).

4. OBJECTIVE

5. METHODOLOGY Sample collection & processing Using sampling device Leachate samples stored at 4 ℃ Cultivation Leachate was centrifuged at at 12,000 rpm for 5 min The pellet was resuspended in 1 mL of sterile dH 2 O 100 µL of the solution was spread on M9 media supplemented with 10 ppm of PCB, incubated at 37 ℃ for 48 hours. Extraction Using Nucleospin Soil kit ( Macherey -Nagel) as per kit instruction in the manual. Amplification Polymerase chain reaction (PCR) amplification using 16S rRNA universal primers set Restriction Fragment Length Polymorphism (RFLP) Two different restriction enzymes: Fermentas FastDigest ® Hinf1 for Jeram leachate Fermentas FastDigest ® EcoR1 for Jabor leachate The mixture was incubated at 37 ◦ C for 16 hour and the digested PCR products were separated using 1% agarose gel electrophoresis. The results were analyzed using NTsys software before sent for sequencing by the sequencing service provider Phylogenetic analysis Basic Local Alignment Search Tool (BLAST) was performed for similarity searches. Phylogenetic tree was constructed using software MEGA version 11.0 based on Neighbour -joining method.

6. RESULT 1: PCR amplification Figure 1 M; Smobio 1 Kb DNA ladder, 1-40; Jeram PCR product Figure 2 M; Quick-Load 1KB extend DNA ladder, 1-47; Jabor PCR product 1.2 Jabor PCR product 1.1 Jeram PCR product

7. PCR amplification using universal primer of 27F and 1492R was conducted and purified before sent for sequencing The annealing temperature for the amplification was determined as 56℃ in order to get an intense band For Jeram and Jabor leachate, the purified PCR product with size of ~1500 bp were 29 and 40 bands respectively.

8. RESULT 2: RFLP RFLP phenogram for purified PCR product of Jeram leachate (A) and Jabor leachate (B) 2.0 RFLP phenogram B A

9. (RFLP) is a molecular biology technique for profiling of microbial communities based on the position of a restriction site . The PCR product was digested with Hinf1 for Jeram leachate and EcoR1 for Jabor leachate. There were 20 and 4 groups of isolated with the same restriction profile for Jeram and Jabor leachate respectively. For Jeram , J1, J8, J18, J20, J9, J13, J14, J17, J21, J23, J34, J2, J4 and J6 were selected for further sequencing, while JB2, JB37, JB10, JB6 and JB15 for Jabor sample was selected.

10. RESULT 3: Phylogenetic analysis The highest sequence similarity based on BLAST Sequence Alignment for Jeram leachate (A) and Jabor leachate (B) 3.1 BLAST Similarity search Isolates Organism Sequence length (bp) Max Identity (%) Accession number J1 Alcaligenes faecalis strain SCNAH10 16S ribosomal RNA gene, partial sequence 1163 74 KT327202.1 J2 Alcaligenes faecalis strain RY1 16S ribosomal RNA gene, partial sequence 1528 77 MT950274.1 J4 Bacillus sp . H59 16S ribosomal RNA gene, partial sequence 540 78 AY074897.1 J6 Bacillus subtilis strain CUMB TP-08 16S ribosomal RNA gene, partial sequence 1257 71 OK605298.1 J8 Alcaligenes faecalis subsp. phenolicus strain WZS013 16S ribosomal RNA gene, partial sequence 1437 78 MH497605.1 J9 Bacterium MPBA40 16S ribosomal RNA gene. Partial sequence 1136 84 EU476009.1 J13 Burkholderia metallica strain HA38 16S ribosomal RNA gene, partial sequence 1461 97 MT565308.1 J14 Alcaligenes sp . P21 16S ribosomal RNA gene, partial sequence 1110 94 KT004378.1 J17 Alcaligenes faecalis subsp. phenolicus strain DHN33 16S ribosomal RNA gene, partial sequence 1431 74 MN83358.1 J18 Alcaligenes sp. strain L3102 16S ribosomal RNA gene, partial sequence 1143 92 OM920553.1 J20 Alcaligenes faecalis subsp. phenolicus strain DHN33 16S ribosomal RNA gene, partial sequence 1431 75 MN833581.1 J21 Alcaligenes faecalis strain IVN61 16S ribosomal RNA gene, partial sequence 1152 97 KT254063.1 J23 Alcaligenes faecalis strain AN-13 16S ribosomal RNA gene, partial sequence 1506 92 ON328334.1 J34 Burkholderia sp. strain DIN3 16S ribosomal RNA gene, partial sequence 1388 97 MH756747.1 Isolates Organism Sequence length (bp) Max Identity (%) Accession number JB2 Burkholderia ambifaria strain CK2_2_36 16S ribosomal RNA gene, partial sequence 1165 97 MN691467.1 JB37 Priestia flexa strain FS-3 16S ribosomal RNA gene, partial sequence 1422 83 OL440766.1 JB10 Burkholderia metallica strain HA38 16S ribosomal RNA gene, partial sequence 1461 99 MT565308.1 JB6 Burkholderia cenocepacia strain AWP-17 16S ribosomal RNA gene, partial sequence 1395 97 OM943810.1 JB15 Propionibacterium freudenreichii strain CB129slpB chromosome, complete genome   2684198 78 CP030279.1 A B

11. 3.2 Phylogenetic tree Jeram & Jabor 16S rRNA sequences of selected isolates were aligned and compared in the National Centre for Biotechnology Information (NCBI) website. For Jeram leachate, bacterial community was dominated by Alcaligene sp . (J1, J2, J8, J14, J17, J18, J20, J21, J23) , followed by Burkholderia sp. (J13, J34) and Bacillus sp . (J4, J8). F or Jabor leachate, the bacterial community was dominated by Burkholderia sp. (JB2, JB10, JB6).

12. DISCUSSION

13. CONCLUSION

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