GENOTYPIC AND PHENOTYPIC CHARACTERIZATION OF Alcaligenes javaensis JG3 POTENTIAL AS AN EFFECTIVE BIODEGRADER
Downloads
Utilization of glycerol by lipase producing bacteria offers great benefits for fat and oil waste degradation and waterwaste treatment. Nevertheless, there have been lack of reports about the availability of non-pathogenic, lipase producing bacteria, which could naturally degrade glycerol produced from the lipolysis process by lipase. This study reported a newly identified species of rhizobacteria, Alcaligenes javaensis JG3, which is not only able to produce high level of lipase, but also able to degrade glycerol molecules. Identification of strain JG3 was carried out using SEM (Scanning Electron Microscope), BD Phoenix 100 Automated Microbiology System and 16S rRNA gene analysis to determine its taxonomy status. The ability of the strain to metabolize glycerol was investigated both genotypically and phenotypically using degenerate PCR and a glycerol minimal medium. Identification test results showed that strain JG3 belongs to genus Alcaligenes, with the closest relationship with A. faecalis and A. aquatilis (96% nucleotide similarity maximum). Degenerate PCR resulted in a 248-bp sequence showing 93% similarity with glpK of Candidatus Sodalis pierantonius SOPE, a key gene involved in glycerol metabolism. In vitro glycerol utilization test result showed that Alcaligenes sp. JG3 was able to grow on glycerol aerobically, but not anaerobically. It is concluded that Alcaligenes sp. JG3 possesses genes coding for glycerol metabolism and this trait is phenotypically expressed, thus making the strain potential to be used as an effective fat and oil biodegrader.
Allen ME. 2005. MacConkey agar plates protocol. Washington (US): American Society for Microbiology. p. 1-4.
Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res 25:3389–402.
Chaerun SK, Hasni S, Sanwani E, Moeis MR. 2012. Mercury (Hg)-resistant bacteria in Hg-polluted gold mine sites of Bandung West Java Province. Indonesia Microbiol 6:57-68.
Charulatha R, Prabhakar S, Sivamani P, Kandeepan C, Tiagarajan V. 2012. Characterization of clinical application of extracellular lipase by microorganisms from soil. Int J Curr Sci 3:61-6.
Drancourt M, Bollet C, Carlioz A, Martelin R, Gayral JP, Raoult D. 2000. 16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J Clin Microbiol 38:3623-30.
Ethica SN, Hammi MK, Lestari P, Semiarti E, Widada J, Raharjo TJ. 2013a. Amplification of Azospirillum sp. JG3 gene fragment using degenerate primers glpD generated by web-based tools. J Microbiol Biotech Food Sci 3:231-4.
Ethica SN, Nataningtyas DR, Lestari P, Istini I, Semiarti E, Widada J, Raharjo TJ. 2013b. Comparative evaluation of conventional versus rapid methods for amplifiable genomic DNA isolation of cultured Azospirillum sp. JG3. Indo J Chem 13:248-53.
Ethica SN, Semiarti E, Widada J, Oedjijono, Raharjo TJ. 2017. Characterization of moaC and a non-target gene fragments of food-borne pathogen Alcaligenes sp. JG3 using degenerate colony and arbitrary PCRs. J Food Saf 37:e12345. Available from: https://doi.org/10.1111/jfs.12345
.
Felsenstein J. 1985. Phylogenies and the comparative method. Am Nat 125(1):1-15.
Gadberry MD, Malcomber ST, Doust AN, Kellogg EA. 2005. Primaclade - a flexible tool to find conserved PCR primers across multiple species. Bioinformatics 21:1263-4.
Garrity GM, Bell JA, Lilburn T. 2005. Class I. Alphaproteobacteria class. nov. In: Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s Manual of Systematic Bacteriology. Boston (US): Springer. 574 p.
Holmberg C, Beijer L, Rutberg B, Rutberg L. 1990. Glycerol catabolism in Bacillus subtilis: nucleotide sequence of the genes encoding glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (glpD). J General Microbiol 136:2367-75.
Hucker GJ. 1921. A new modification and application of the Gram stain. J Bacteriol 6:395-7.
Kim YJ. 1991. Modeling and application of aqueous 2-phase system in an a-b-e fermentation process. Master Thesis. Maryland (US): University of Maryland. Advisor Weigand WA.
Lestari P, Handayani SN, Oedjijono. 2009. Biochemical properties of crude extracellular lipase from Azospirillum sp. JG3. Molekul 4:73-82.
Lestari P, Raharjo TJ, Matsjeh S, Haryadi W. 2016. Partial purification and biochemical characterization of extracellular lipase from Azospirillum sp. JG3 bacteria. AIP Conference Proceedings 1755(1):080003.
Lesuisse E, Schanck K, Colson C. 1993. Purification and preliminary characterization of the extracellular lipase of Bacillus subtilis 168, an extremely basic pH-tolerant enzyme. Eur J Biochem 216:155-60.
Litsanov B, Brocker M, Bott M. 2012. Glycerol as a substrate for aerobic succinate production in minimal medium with Corynebacterium glutamicum. Microb Biotechnol 6:189-95.
Matsuoka H, Miura A, Hori K. 2009. Symbiotic effects of a lipase-secreting bacterium, Burkholderia arboris SL1B1, and a glycerol-assimilating yeast, Candida cylindracea SL1B2, on triacylglycerol degradation. J Biosci Bioeng 107:401–8.
Murarka A, Dharmadi Y, Yazdani SS, Gonzalez R. 2008. Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals. Appl Environ Microbiol 74:1124-35.
Oedjijono, Ryandini D, Permiarti. 2003. Formulasi biofertilizer dari kultur campuran bakteri pemfiksasi nitrogen dan pelarut fosfat pada medium onggok dan dedak [Research report, unpublished]. Retrieved from Fakultas Biologi Universitas Jenderal Soedirman.
Pettigrew DW, Ma DP, Conrad CA, Johnson JR. 1988. Escherichia coli glycerol kinase: cloning and sequencing of the glpK gene and the primary structure of the enzyme. J Biol Chem 263:135-9.
Prasad MP, Manjunath K. 2012. Effect of media and process parameters in the enhancement of extracellular lipase production by bacterial isolates from industrial effluents. Int J Microbiol Res 4:308-11.
Promega. 2010. Wizard® Genomic DNA purification kit technical manual (TM050), Section 3.G. Isolating genomic DNA from gram-positive and gram-negative bacteria. Madison (US): Promega Corporation. Revised December.
Saitou N, Nei M. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406-25.
Schauder R, Schink B. 1989. Anaerovibrio glycerini sp. nov., an anaerobic bacterium fermenting glycerol to propionate, cell matter and hydrogen. Arch Microbiol 152:473-8.
Sya'di YK, Wahyuni ET, Rahayu ES, Cahyanto MN. 2017. Cellulose produced by Gluconacetobacter xylinus BTCC B796. IJST 5(6):98-103.
Tamura K, Nei M, Kumar S. 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. PNAS 101:11030-5.
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA 6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725-9.
Thompson JD, Gibson T, Higgins DG. 2002. Multiple sequence alignment using ClustalW and ClustalX. Curr Protocols Bioinformatics:2–3.
Turner S, Pryer KM, Miao VP, Palmer JD. 1999. Investigating deep phylogenetic relationships among cyanobacteria and plastids by small subunit rRNA sequence analysis. J Euk Microbiol 46:327-38.
William SG, Greenwood JA, Jones CW. 1994. The effect of nutrient limitation on glycerol uptake and metabolism in continuous cultures of Pseudomonas aeruginosa. Microbiology 140(11):2961-9.
Yazdani SS, Gonzalez R. 2007. Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry. Curr Opin Biotechnol 18:213-9.
Copyright (c) 2018 BIOTROPIA - The Southeast Asian Journal of Tropical Biology

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Authors who publish with this journal agree with the following terms:
- Authors retain copyright and grant the journal right of first publication, with the work 1 year after publication simultaneously licensed under a Creative Commons attribution-noncommerical-noderivates 4.0 International License that allows others to share, copy and redistribute the work in any medium or format, but only where the use is for non-commercial purposes and an acknowledgement of the work's authorship and initial publication in this journal is mentioned.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).




