Numerical Taxonomy for Detecting the Azotobacterial Diversity

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1 Numerical Taxonomy for Detecting the Azotobacterial Diversity Enny Zulaika 1 *, Maya Shovitri 2 and N.D. Kuswytasari 3 1,2,3. Department of Biology-Faculty of Mathematics and Natural Sciences, ITS-Surabaya *Corresponding author s. Tel: , enny@bio.its.ac.id Abstract Azotobacter was a non-symbiont of bacteria that were abundant in the land and it able to bind free nitrogen. The bacterial diversity required a holistic integration with a variety of methods, both at the level of the community, or functional structure of the bacteria. Each member of the bacterial population had varying responses to environmental factors and phenotypic variation in accordance with it genotype, so that required a grouping or classification to facilitate the study of bacterial diversity. Through a numerical taxonomy approach will hopefully reveal the diversity of Azotobacter and it similarity relations thus it enable bundling its potential in usage as bioremoval, or as eco-friendly biofertilizer for critical land. Azotobacter was isolated from soil of Institut Teknologi Sepuluh Nopember (ITS) Eco Urban Farming, using selective media for Azotobacter. The Azotobacterial isolates were characterized by utilizing a numerical taxonomy method based on phenotypic characters, including morphological, biochemical and physiological characters. Based on the numerical taxonomy analysis the isolates separate into two different phenotypic groups at similarity level of 79-85%. Species members in one cluster included A5, A6, A7 that had a colony of white-beige and the other cluster with its members consist of A1a, A3 and A9 with their colonies of yellow-brown. Keyword: Azotobacter, diversity, numerical taxonomy, similarity Introduction Azotobacter was a non-symbiont genus of bacteria that were abundant in the land and it able to bind free nitrogen, some of its species were siderophore bacteria, phytohormones producer (Kizilkaya, 2009), and thus it potentially used as biofertilizer. Diversity of Azotobacter in it habitat affected by the physical and chemical properties of soil as well as interactions among microorganisms in the soil (Lenart, 2012). Some members of the Azotobacter was high salinity tolerant (Akhter et al., 2012) and resistant to mercury (Aleem et al., 2003), so it also potentially used as a mercury bioremediation on high salinity area. Mercury was one of heavy metals that were highly toxic and dangerous even in very low concentrations, may accumulate in the cells or tissues of organisms food chain (Oehmen et al., 2009). A minimum threshold of mercury in Indonesia based on the Government regulation of the Republic of Indonesia No. 82 in 2001 was mg/l. Bacteria were microorganisms that live in various habitats, either in normal or extreme environments. Inorganic mercury 5 µg/l shall be inhibited the growth of microorganisms, but naturally there were bacteria that able to live in these conditions, it was called Mercury Resistant Bacteria or MRB. MRB members were enzymatic mechanism that may turn toxic anorganic mercury (Hg 2+ ) into volatile mercury (Hg 0 ) so that mercury did not poison the cells (Brown et al., 2002). Characterization and identification of the bacteria was not as easy to identify specimens of plants or animals, therefore bacterial cell structure were microscopic, simple and difficult to distinguish just based on the visualization only. Study of bacterial diversity August 2014, Korea University, Seoul, Korea Page 1

2 required a holistic integration with a variety of methods, both at the level of the community, or functional structure of the bacteria itself (Schleifer, 2009). Each member of the bacterial population had varying responses to environmental factors and phenotypic variation in accordance with it genotype, so that required a grouping or classification to facilitate the study of bacterial diversity (Selvakumaran et al., 2008). Given the importance role of bacteria in nature for biogeochmical cycles within the ecosystem, it was necessary to understand the diversity of bacterial communities deeply and its role in habitats. Through a numerical taxonomy approach will hopefully reveal the diversity of Azotobacter and it similarity relations, thus it enable bundling its potential in usage as mercury bioremoval, bioremediation or as eco-friendly biofertilizer for critical land. Material and Methods Isolation of bacteria Using composite method, Azotobacter was isolated from soil of the Institut Teknologi Sepuluh Nopember (ITS) Eco Urban Farming. Selective media (Himedia )- HgCl 2 0,1 mg/l used to grow its inoculum and incubated at room temperature for hours. The grown colony was a mercury resistant, isolate Azotobacter, a colony which had different colors and shapes then reisolated and replanted again. Identification, characterization, and generic assignment All the selected isolates characterized based on morphological, physiological and biochemistry reaction, then followed by generic assignment used profile matching to determine the key characters of the genus Azotobacter. Verification refers to the Bergey s Manual of Determinative Bacteriology (Holt et al., 1994). All methods of morphology, physiological characterization and biochemistry reaction above resulted from modification of several manuals laboratory (Harley & Prescott, 2002). Collection of data Data were obtained from morphological characterization of phenotypes, physiology and biochemistry reaction. Those data were include how colonies morphology on flat agar media (diameter, color, surface, shape, transparency, elevation, and the edges of the colony), colonies morphology on upright and oblique agar media (growth and a colony form), the cell morphology (shape, Gram staining characteristics, and the presence of cysta). It resisted to metals (Hg, Cd, Pb), antibiotics and NaCl osmotic pressure. Physiological reactions and biochemistry included oxygen demand (aerobic and anaerobic), carbohydrate metabolism (glucose, sucrose, maltose, galactose, fructose, manitol and sorbitol). The characters of the morphology, physiology and biochemistry reaction obtained from the characterization, then it converted with a positive value (+) or negative (-). Data tabulated into a matrix n x t, with n as the number of isolate Azotobacter and t as number of characters, then it inserted into Programmer's File Editor (PFE) August 2014, Korea University, Seoul, Korea Page 2

3 Settings and data processing Data in table n x t copied and inserted in the PFE as a program editor. Files edited in accordance the procedure of PFE, then it stored in a format.mvs that can be opened using MVSP (Multi Variate Statistical Package). Matrix n x t file that has been edited with PFE then processed with MVSP. So that it can be entered MVSP isolates coded. For Taxonomical Operational Unit (OTU), each isolate was codified with A, B, C and so on, as for the character code-named AA, AB, AC, and so on according to the number of characters used. Simple Matching Coeficient (S SM ) used to specify the similarity between isolates Azotobacter. Grouping was done using Unweighted algorithm Paired Group Method with Averages (UPGMA) arithmatic in form of a dendorgram (Sneath & Sokal, 1973). Result and Discussion Phenotypic characterization The results obtained by 79 isolates and only 6 isolates were resisted to HgCl 2 10 mg/l, which were A1a, A3, A5, A6, A7, A9, and those isolate then were characterized. All isolates had small colonies, only isolates A7 was punctiform and A1a isolates that had a large colony. Colonies were normally grouped into 4 colors, white or cream (A5, A6 and A7), brown (A1a), yellowish white (A9) and yellow (A3). They had circular form for colonies, except A9 and A1a with irregular shape (Figure 1.) A1a A3 A5 A6 A7 A9 Figure 1. The colony of Azotobacterial isolates The elevation varies between colonies drive from flat, convex into a pulvinate. They had colony margin such as entire, undulate, and erose. They also had all smooth colony texture, bright and opaque appearance (most were an opaque). Its growth of oblique and upright agar media were filiform and echinulate August 2014, Korea University, Seoul, Korea Page 3

4 According to Aquilanti et al. (2004), Azotobacter chroococcum colonies appear after 24 hours of incubation with the color white and wet colonies turned into dark brown after 3-5 days, Azotobacter paspali after 48 hours of incubation, colonies were yellow in center, Azotobacter agilis was yellowish white. The Azotobacter can be cysta forming that to protect the cell from a state of extreme environments, Azotobacter was an obligate anaerobic bacteria although it could grow under low O 2 conditions (Jímenez et al., 2011). All isolates were able to perform the metabolism of glucose, fructose, sucrose, maltose, galactose, manitol and sorbitol (Table 1.). The genus Azotobacter were able to ferment carbohydrates, types of carbohydrate which fermented depending on the species (Boone et al., 2005), Tabel 1. The Azotobacterial metobolism of carbohydrat Carbohydrate Isolates of Azotobacter A1a A3 A5 A6 A7 A9 Glucose Fructose Sucrose Maltose Galactose Manitol Sorbitol (+) positive test; (-) negative test All Azotobacter isolates resisted to HgCl 2, CdCl 2 dan PbCl 2 10 mg/l, moreover isolate A5, A6, and A7 resisted to HgCl 2 up to 20 mg/l. All isolates were also resistant to the antibiotic ampisilin, tetracycline, and chloramphenicol 30 µg either to NaCl salinity 5%. The mechanism of bacterial resistance to metal or heavy metals varies depending on the genera/species/strains of bacteria, the metal concentration and duration of contacts took place. The majority of bacterial resistant to heavy metals through active efflux transport mechanism or system involving the role of genes in the operon. Mercury involved mer-operon, on cadmium-involved cad-operon and at cuprum (copper) involves a cop-operon (Brown et al., 2002). The mechanisms of bacterial resistance toward heavy metal can be through bioadsorption by Exo Polysacharide (EPS) that located in the outer layer of the cell wall, had a covalent bond and formed a polysaccharide slime that easy off. Bioadsorption process with heavy metals by the EPS was a non-metabolic mechanism because of the ties between the organic acid residue ligand which has negatively charged with functional groups that has the positively charged metal (Aquilanti et al., 2004). All isolates of Azotobacter from soil in Eco Urban Farming had shown resistant to ampsilin, chloramphenicol and 30 µg tetracycline. All isolate Azotobacter were also tolerant to salinity (NaCl) 5%. On the research of Zulaika et al. (2012), Azotobacter S8 of the Kalimas Surabaya resistant to 10µg ampisilin, and 30µg chloramphenicol, tetracycline. And also tolerant of 10% NaCl August 2014, Korea University, Seoul, Korea Page 4

5 Numerical taxonomy The species concept used in this study was through the taxospesies approach. Based on numerical taxonomy analysis produced a dendogram that describes the similarity between Azotobacter isolates, dendogram clumped into two clusters were cluster A and B (Figure 2.). Cluster A Cluster B Figure 2. Dendogram which showed similarity phenotypic between isolates Azotobacter. The scale indicates the level of similaritas (S SM ). Numerical taxonomy used as a system of classification with a politetic phenentic character. Classification by numerical taxonomy of politetic phenentic was widely used and successfully explained isolates representing the diversity of natural habitats included soil, water and could decipher large and heterogeneous taxa (Priest & Austin, 1996). Based dendorgram on Figure 2., Azotobacter isolate form two clusters that included A cluster of isolates; A5, A6, A7 and cluster B isolates that encompassing A1a, A3, A9. Isolates members of A cluster and the cluster B had similarity > 70%, it was assumed that member within of A cluster of isolates be the same species, as well as the members of the cluster B (Table 2) Table 2. Index of Similarity Isolates Isolates A1a A3 A5 A6 A7 A9 A1a 1,000 A3 0,758 1,000 A5 0,613 0,694 1,000 A6 0,597 0,613 0,855 1,000 A7 0,677 0,661 0,839 0,887 1,000 A9 0,839 0,823 0,710 0,597 0,645 1,000 In accordance with the concept of taxospecies, which belongs to the strains of a species must have a similarity > 70% (Goodfellow & O Donnel, 1993). On cluster A had a similarity > 85% and cluster B > 79%. Members in A cluster had similarities in color August 2014, Korea University, Seoul, Korea Page 5

6 white-beige colony and that was capable to use sorbitol and galactose as carbon source and resistant to HgCl 2 for up to 20 mg/l. Members of cluster B had similarity in yellowbrown colour colonies and resistant to HgCl 2 only up to 10 mg/l. Between cluster A and B had similarity only 65%, according to the concept of taxsospecies, isolates in cluster A differed to isolates in cluster B Conclusion The genus of Azotobacter from ITS Eco Urban Farming were consist of isolates A1a, A3, A5, A6, A7 and A9 that resisted to HgCl 2 10 mg/l. Based on concept of taxospecies, the isolates had derived into two species clusters. Species members in A cluster included A5, A6, A7 that had a colony of white-beige and cluster B with its members consist of A1a, A3 and A9 with their colonies of yellow-brown. Acknowledgement We would like to thank to ITS for the financial support, with Contract Number: /IT2.7/PN.01.00/2014 We specially thanked to our students Kusnul Khotimah, Anjar Lulu Sakinah and Afina Dhuhaini who helped us conducting the experiments, they worked with their excellent scientific enthusiasm performing the laboratory works during the hard days. References Akhter, M.S., S.J. Hossain, S.A. Hossain, and R.K. Datta Isolation and characterization of salinity tolerant Azotobacter sp. Greener Journal of Biological Sciences. 2: Aleem, A., J. Isar, and A. Malik Impact of long-term application of industrial wastewater on the emergence of resistance traits in Azotobacter chrococcum isolated from rhizosperic soil. Bioresource Technology, 86: Aquilanti, L., F. Favili, and F. Clementi Comparison of different strategies for isolation and preliminary identification of Azotobacter from soil samples. Soil Biology & Biochemistry, 36: Brown, N., Y. Shih, C. Leang, K. Glendinning, K., Hobman, J. and Wilson, J. (2002) Mercury Transport and Resistance.International Biometals Symposium, Biometals, Boone, R.D., R.W. Castenholz, D.J. Breener, N.R. Krieg, and J.T. Staley Bergey s Manual Systematic and Bacteriology 2 nd edition. USA: Springer. Goodfellow, M. and A. G. O Donnell, Handbook of New Bacterial Systematics. Academic Press, Inc. Harley, J.P. & Prescott, L.M Laboratory Exercises in Microbiology. 4 th Edition. The McGraw-Hill Companies: New York Holt, J.G., Krieg, N.R., Sneath, H.A., Staley, J.T. and Wiliam, S.T Bergey s Manual of Determinative Bacteriology 9 th Ed, William & Wilkins, Baltimor. Jiménez D.J., J.S. Montaña, and M.M. Martínez Characterization of Free Nitrogen Fixing Bacteria of the Genus Azotobacter in Organic Vegetable-grown Colombian Soils. Brazilian Journal of Microbiology, 42: August 2014, Korea University, Seoul, Korea Page 6

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