Highlights in BioScience ISSN:2682-4043 DOI:10.36462/H.BioSci.202404 Research Article Open Access 1 Department of Biotechnology Research, Micro- biology Laboratory, Kyaukse, Myanmar. 2 Department of Zoology, Mandalay University, Mandalay, Myanmar. * To whom correspondence should be addressed: khinthaemarrmm@gmail.com Editor: Alsamman M. Alsamman, International Center for Agricultural Research in the Dry Areas (ICARDA), Cairo, Egypt. Reviewer(s): AbdulAziz Ascandari, Chemical and Biochemical Sciences-Green Process Engineering, University Mohammed VI Polytechnic, BenGuerir, Morocco. Jean Legeay, Université de Lorraine, INRAE, UMR IAM - Interactions Arbres-Microorganismes, France. Received: June 4, 2024 Accepted: October 11, 2024 Published: December 25, 2024 Citation: Marr KT, Wilbur ND, Yu SS, Ei SL. Evaluation on multi-trait activities of Azotobacter spp. from various water samples in Mandalay environments. 2024 Dec. 25;7:bs202404 Copyright: © 2024 Marr et al.. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and supplementary materials. Funding: The authors have no support or funding to report. Competing interests: The authors declare that they have no competing interests. Evaluation on multi-trait activities of Azotobacter spp. from various water samples in Mandalay environments Khin Thae Marr1 >< �, San San Yu1 >< �, Naw Dolly Wilbur2 >< �, Shun Lai Ei1 >< � Abstract Fourteen water samples were collected from different locations of Mandalay and Kyaukse Township. Among them, eight bacterial isolates named A1 to A8 were nominated as Azotobacter according to their colonial morphology, microscopic morphology, and pigment production. According to the biochemical characteristics and sequence analysis of isolated bacteria, they were Azotobacter chroococcum, Azotobacter vinelandii, and Azotobacter beijerinckii. The phosphate solubilizing of these isolates was observed from 2.3 to 2.6 SI, and A4 had the highest solubility index. The potassium decomposing of these isolates was observed 2.5 to 3.8 SI, and A6 and A8 had the highest K-decomposing activity. The zinc solubilizing of these isolates was observed at 2.3 to 3.5 SI, and A3 had the highest zinc solubilizing index. Screening methods showed that all eight strains have nitrogen fixing activity and indole acetic acid (IAA) producing activity. The antagonistic activity of all isolated strains was also found against Pythium sp. and Fusarium oxysporum. The isolated Azotobacter sp. can be used as biofertilizer in the agriculture sector, which can increase crop yields and enhance soil fertility according to their plant growth promoting activities. Keywords: Isolation, characterization, 16S rRNA gene, plant growth, Azotobacter, biofertilizer. Introduction Azotobacteraceae are heterotrophic gram-negative bacteria and a group of aerobic. The main characteristic of Azotobacteraceae is the ability to fix nitrogen in the natural atmosphere, which is vital in the nitrogen cycle. Azotobacter species are generally spherical on LB medium or oval on glucose nitrogen free medium and motile (G-NFMM) [1]. Azotobacter can live as cysts and produce enormous amounts of capsular slime. They can grow on any medium that has a suitable pH and an organic carbon source [2]. Azotobacter produces blue white fluorescent, green, and yellow green pigments [3]. The optimum temperature range of Azotobacter is between 20 and 30 oC, and the optimum pH of Azotobacter is 6.5 in neutral to alkaline soil [4]. They can be found in the soil of a number of crop plants and vegetative plants [5]. Azotobacter species is the preferred organism because they are non-pathogenic and can stay as cysts in soil for many years [6]. Beijerinck studied Azotobacter chroococcum isolated from the rhizosphere in Holland in 1901, and it was the first species. Azotobacter agilis was also studied by Beijerinck [7]. Lipman found Azotobacter vinelandii in 1903 and Azotobacter beijerinckii in 1904 from the rhizosphere [8]. Azotobacter vinelandii has the ability to produce a yellow-green pigment that gives fluorescence under ultraviolet light. In alkaline soils, marine sediments, and marsh water, Azotobacter can be generally found [9]. Biofertilizers, also known as organic preparations or bioinoculants, contain microorganisms that promote nitrogen fixation, phosphate solubilizing, and potassium decomposing for plant growth [10]. Azotobacter, Azospirillum, and Rhizobium are beneficial microbes for crop production to use as biofertilizers [11; 12]. Nowadays, many countries use biofertilizers that give advantages to economic activity. The chemical fertilizers threaten environmental pollution and human health. The research was designed to isolate Azotobacter sp. from various water samples, to characterize biochemical tests, identify the isolates by 16S rRNA sequencing analysis, and study the multi-trait activities of isolated bacteria. Highlights in BioScience Page 1 of 8 December 2024|Volume 7 https://doi.org/10.36462/H.BioSci.202404 https://creativecommons.org/licenses/by/4.0/ mailto:khinthaemarrmm@gmail.com https://orcid.org/0009-0005-9920-9385 mailto:sakurasan2007@gmail.com https://orcid.org/0000-0003-2465-2866 mailto:2929852582@qq.com https://orcid.org/0009-0004-4759-6638 mailto:shunlai.ei@gmail.com https://orcid.org/0000-0003-2444-2439 http://bioscience.highlightsin.org/ Marr et al., 2024 Evaluation on multi-trait activities of Azotobacter spp. Materials and Methods Study Area and Sample Collection The bottles of water samples were collected from different places in Mandalay Environs and Kyaukse Township, Myanmar (Figure 1). The water samples were taken from 5 to 12 cm of water depth. The plastic bottle (1 liter) was used to collect water samples. Then, the water bottles were carried to the department of biotechnology research to be studied. The samples were named S1 to S14 (Table 1). The strains isolated from these samples were named A1 to A8. Their sampling sites and different locations of bacterial isolates were shown in Table 2. Table 1. Water samples collected from different locations in Mandalay and Kyaukse Township. No. Samples Collection site Township 1 S1* Ayeyarwady river jetty3 Mandalay 2 S2 Kandawgyi lake site1 Mandalay 3 S3* Kandawgyi lake site2 Mandalay 4 S4* Kandawgyi lake site3 Mandalay 5 S5* Fish pond in Mahar Muni Image Mandalay 6 S6* Taw-dwin Lake Kyaukse 7 S7* Tone-long (southern fish lake) Kyaukse 8 S8* Tone-lone (northern fish-lake) Kyaukse 9 S9 SunYe Loch site1 Kyaukse 10 S10 SunYe Loch site 2 Kyaukse 11 S11 SunYe Loch site3 Kyaukse 12 S12* SunYe Loch site 4 Kyaukse 13 S13 Thamote Canal Kyaukse 14 S14 Zawgyi River Kyaukse Isolation of Bacteria To isolate bacteria, 5 ml of water was added to the sterile test tube, and 5 ml of 0.9% NaCl solution was mixed with it. The tubes with the water sample were kept standing for about 30 minutes, and 100 µl of water was spread on G-NFMM [13]. After inverting the GNFMM plates, they were incubated at 35 oC for 48 hours. The bacterial colonies that appeared on G-NFMM were purified by subculturing and incubated at 35 oC for 2 days. The shapes and sizes of isolated bacteria were characterized by colonial and microscopic morphology. Microscopic morphology was checked by using gram staining method [14]. The single isolated colony was stained and examined with a drop of oil immersion under a microscope at 100 magnifications to check purity. Biochemical Characteristics of Isolated Bacteria The isolated bacteria were characterized by using some bio- chemical tests. Biochemical characteristics of selected bacteria were studied according to Bergey's manual of determinative bac- teriology [15]. These tests include the motility test, the catalase test, the starch hydrolysis test, the citrate utilization test, the methyl red test, the Voges-Proskauer test, the indole test, and the triple sugar iron agar test. Figure 1. Location map of study area, Mandalay environments, and Kyaukse Township. (Source: Myanmar information management unit and google earth pro) Molecular Identification of Bacterial Isolates The purified bacterial isolates were sent to a DNA sequenc- ing service, Eurofins company in Germany, and the sequence data of individual isolates were queried using BLAST in the na- tional center for biotechnology information (NCBI) and GenBank databases. Sequence alignment results showed the maximum identities of each isolate and their relevant accession number. Muscle was used to align sequences that were identified, and a phylogenetic tree was constructed based on the neighbor join- ing (NJ) and the number of differences method algorithms using the MEGA 11 software. To acquire accession numbers, the nu- cleotide sequences of isolated Azotobacter species were then sent to GenBank. For further application, all isolates were maintained at the microbiology laboratory at the department of biotechnology research. Screening on Nitrogen Fixing Activity by Indophenol Method The bacterial strains were injected in the bottles that contain G-NFMM broth and incubated for one week at 35oC. Then 1 ml of sample was centrifuged at 10000 rpm for 20 minutes. Am- monium concentration was measured by indophenol method by taking the supernatant. Firstly, 0.5 ml of EDTA solution was added to supernatant. Then 0.5 ml of p-buffer was added. After adding 2.5 ml of nitroprusside reagent, 2.5 ml of hypochlorite solution was added to the supernatant in a small bottle. The reaction mixture was placed at room temperature for 3 hours and ammonium was estimated by the color of the sample. Highlights in BioScience Page 2 of 8 December 2024|Volume 7 http://bioscience.highlightsin.org/ Marr et al., 2024 Evaluation on multi-trait activities of Azotobacter spp. Table 2. Bacteria isolates from different locations in Mandalay environments and Kyaukse Township. No. Samples Strain Collection Site pH Temperature 1 S1 A1 96°03 21.745" E and 21°58 13.576" N 7.8 25°C 2 S3 A2 96°03 21.709"E and 21°57 07.664" N 7.3 25°C 3 S4 A3 96°03 21.470" E and 21°56 49.277"N 7.4 25°C 4 S5 A4 96°04 48.758" E and 21°57 09.017"N 7.6 25°C 5 S6 A5 96°07 44.652" E and 21°32 32.639" N 8.2 25°C 6 S7 A6 96°07 34.727" E and 21°33 40.007"N 8.4 25°C 7 S8 A7 96°07 34.588" E and 21°33" 40.333" N 8.3 25°C 8 S12 A8 96°13 28.758" E and 21°40 36.516" N 8.2 25°C In this method, 0 mg/ml of ammonium concentration will show yellow color, and 3 mg/ml ammonium concentration will show green color. Screening on Phosphate Solubilizing Activity To determine the phosphate solubilizing activity of isolated bacteria, national botanical research institute phosphate (NBRIP) media was used. The single colony of bacteria was placed on NBRIP media. After five days of incubation at 35oC, the colony showed a zone of clearance around it. The halo-zone diameter was recorded by the phosphate solubilizing index [16]. The solubilizing index (SI) can be determined using the following formula: S I = colony diameter + halozone colony diameter Screening on Potassium Decomposing Activity To determine the decomposing of potassium, the eight bac- terial strains were tested on K decomposing medium by plating method. Pure colonies were spotted on K medium and incubated at 35oC. After five days, the clear zone around the colony was observed, and the diameters of the clear zone were recorded [17]. The solubilizing index (SI) was calculated using the same formula as the phosphate solubilizing activity. Screening on Zinc Solubilizing Activity To determine the solubilizing of zinc, zinc solubilizing medium was used to test the eight bacterial strains. The single colony of bacteria was dotted on medium at 35oC. After incubating them for five days, clear zones around the colonies were recorded [18]. The SI was measured and calculated using the above formula. Screening on IAA Producing Activity The production of IAA by the isolated strains was determined calorimetrically using Salkowski's reagent mixture [35% per- chloric acid (50 ml); 0.5 M FeCl3 (1 ml)]. To test IAA quality, isolated strains were aseptically cultured in G-NFMM plates containing 0.5 mg L-tryptophan per ml, and then incubated at 35oC for 3 days. After incubating the plates, 2 ml of Salkowski's reagent and one drop of orthophosphoric acid were added into the plate medium at room temperature. The color turned pink, which indicated IAA production [19; 20]. Table 3. Biochemical characterization of isolates Biochemical Test A1 A2 A3 A4 A5 A6 A7 A8 C Gram Staining - - - - - - - - - Catalase + + + + + + + + - Starch Hydrolysis - - - - - - - - - Citrate Utilization + + + + + + + + - Motility + + + + + + + + - Methyl Red + + + - - + - - - Voges-Proskauer + + + + + + + + - Indole - + - - - - - - - TSI agar glucose + + + + + + + + - lactose + + + + + + + + - sucrose + + + + + + + + - gas - - - - - - - - - H2S - + - + + - + - - Solubility Index Phosphate 2.5 2.5 2.3 2.6 2.3 2.3 2.3 2.3 0 Potassium 2.5 3 3 3 3.5 3.8 3.5 3.8 0 Zinc 2.8 2.3 3.5 2.3 2.3 2.3 2.3 3 0 Screening on Antagonistic Activity To determine the antagonistic activity, a total of eight bacte- rial isolates were tested with Pythium sp. and Fusarium oxyspo- rum on potato dextrose agar (PDA) medium by plating method. Dual cultures (bacteria and fungus) were spotted separately on opposite sides on potato dextrose agar medium and incubated at 25oC. After three weeks of incubation, an antagonistic effect was observed. The zone diameter of inhibition on fungal growth was determined and recorded. Results Study Area and Sample Collection Collection sites and township of S1 to S14 samples are shown in Table 1, and the results of strains A1 to A8 from 14 samples Highlights in BioScience Page 3 of 8 December 2024|Volume 7 http://bioscience.highlightsin.org/ Marr et al., 2024 Evaluation on multi-trait activities of Azotobacter spp. Figure 2. Colonial and Microscopic Morphology of Isolated Strains. are shown in Table 2. The samples showed a pH range of 7-9 and a temperature of 25°C. Isolation and Biochemical Characterization of Bacteria The purified bacteria were observed for their colonial mor- phology and microscopic morphology such as color, shape and size (Figure 2). The isolated colonies were checked by gram staining method under microscope that revealed and character- ized as Azotobacter species. They were pale-yellow and mucoid according to their morphology and colony sizes were 2 to 4 mm. According to gram straining, they were gram negative, cocci and their sizes were 2 to 4 µm. The isolated bacteria, A1 to A8 were identified by biochemical characterization tests shown in Table 3. Identification of Azotobacter spp. by 16S rRNA gene sequencing Detection of 16S rRNA genes of eight isolated bacteria by gel electrophoresis was shown in Figure S1. The isolated strains be- longed to the genus Azotobacter according to molecular analysis. The strain A1 showed 100% identity with Azotobacter chroococ- cum in NCBI BLAST search while the strain A2 showed 99.77% identity with the Azotobacter chroococcum, respectively. The strain A5 revealed 100% similarity with Azotobacter vinelandii strain IAM 15004 (NR 041039.1) (Figure 3). The 16S rRNA gene sequences of A6 showed 99.52% similarity with Azotobac- ter vinelandii, A7 showed 98.87% with Azotobacter beijerinckii, Figure 3. Phylogentic tree of 16S rRNA gene sequences of the bacterial eight isolated strain from water samples. A8 showed 99.58% with Azotobacter chroococcum and A3 ex- hibited 99.50% with Azotobacter chroococcum, respectively. The accession numbers were MT658735 for A1, MT658739 for A3, MT658736 for A5, MT658737 for A6 and MT658738 for A7, re- spectively, after submitting to the GenBank. The closely related sequences were aligned the number of differences method by using MEGA 11 and the neighbor-joining tree was created [21]. The bootstrap (1000 replication) was used for a statistical support for the nodes in phylogenetic tree constructed with the 16S rRNA similarities (%). Figure 3 showed that the phylogenetic tree analysis involved 26 nucleotide sequences was united through Muscle using MEGA 11 software. Bootstrap values below 50% are not indicated, fewer than 5% alignment gaps. A total of 757 positions in the final data were involved. Screening on Nitrogen Fixing Activity by Indophenol method When the activity of isolated strains that fix nitrogen was screened by culturing in the G-NFMM broth, all isolates changed the color (Figure 4). The potential of nitrogen fixation was indicated by their color changing of all isolates. Screening on Phosphate Solubilizing Activity A1, A2, A4 gave higher halo zone diameter than A5 in NBRIP media. Strain A3, A5, A6, A7, and A8 showed the same clear zone in phosphate solubilizing activity test (Figure 5). Screening on Potassium Decomposing Activity In K-decomposing test, strain A6, A8 exhibited 3.8 mm clear zone and the strains A5, A7 showed the same 3.5 mm clear zone. Highlights in BioScience Page 4 of 8 December 2024|Volume 7 http://bioscience.highlightsin.org/ Marr et al., 2024 Evaluation on multi-trait activities of Azotobacter spp. Figure 4. Screening of Azotobacter species nitrogen fixing activity. Figure 5. Screening on phosphate solubilizing activity of Azotobacter. Strains A2, A3, A4 showed 3 mm clear zone strain A1 showed 2.5 mm clear zone (Figure 6). Screening on Zinc Solubilizing Activity All strains showed clear zone around their colonies on zinc agar plates after 5 days of incubation at temperature 35°C. Among eight strains, A3 showed the maximum clear zone, and followed by A8 and A1. Strains A2, A4, A5, A6, and A7 showed the same clear zone (Figure 7). Screening on IAA Producing Activity IAA producing activities of all isolated strains were screened by using GNFMM containing 0.5 mg/ml L-tryptophan and incu- bated at 35oC for 3 days. After incubating the plates, Salkowski's reagent was added into the culture plates. IAA producing activity was indicated by the development of pink color (Figure 8). Screening on Antagonistic Activity The antifungal activity of isolated strains A1 to A8 showed good antifungal activity against Pythrium sp. and did not show Figure 6. Screening on zinc solubilizing activity of Azotobacter. Figure 7. Screening on potassium decomposing activity of Azotobacter. distinct activity against Fusarium sp. after 3 weeks incubation (Figure S2). Discussion Azotobacter can be found in the rhizosphere of vegetables and plantation crops [22]. All plants need nitrogen, phosphate, potassium and zinc for its growth. However, plants can absorb ammonia converted from nitrogen. Azotobacter is proficient to convert nitrogen into ammonia, making it readily available for plant absorption [23]. Azotobacter is beneficial nitrogen fixer. Azotobacter bio-fertilizer benefits in agriculture to integrate ni- trogen management. Rhizobium, Azotobacter, and Azospirillum enrich nitrogen nutrition in N-deficient soils [24]. After the death of the Azotobacter cell, cell proteins are mineralized in soil and contribute to the nitrogen accessibility of crop plants [25]. Gib- berellic acid and IAA produced by Azotobacter encourage plant growth development. Antimicrobial compounds produced by some species of Azotobacter are able to inhibit plant pathogens and prevent economic losses [26]. In this research, eight iso- lated Azotobacter strains were obtained from 14 water samples. The bacteria were characterized by 16S rRNA gene sequencing method to know exactly about the genus of Azotobacter sp. They were A. chroococcum, A. beijerinckii,and A. vinelandii. Eight strains were cultured in G-NFM medium containing BTB as in- dicator and showed changing of the color from green to blue that was an indication of nitrogen fixing activity. The isolated strains A1 to A8 showed nitrogen-fixing activity on the nitrogen- free medium. All eight strains showed phosphate solubilization activity on NBRIP medium. The widest clear zone diameter was found in A. chroococcum of all isolated strains in phosphate solubilization. To convert insoluble phosphorus to an accessible form, orthophosphate, the capacity of some microorganisms is an important trait in a PGPR for increasing crop yields. Azotobacter can also convert insoluble phosphate to orthophosphate that is soluble for plants to absorb [27]. Insoluble inorganic phosphate can be absorbed by the roots of plants with the help of bacteria that can solubilize phosphate [28]. Eight isolates showed the clear zone around the colonies in potassium decomposition. A. vinelandii A6 and A. chroococcum A8 showed the highest potassium solubilizing activity among eight strains. The potas- sium is available in four forms i.e., K solution, K as mineral, K as transferable metal and K as non-exchangeable metal. Min- eral K held tightly on the surface of clay. Non-exchangeable Highlights in BioScience Page 5 of 8 December 2024|Volume 7 http://bioscience.highlightsin.org/ Marr et al., 2024 Evaluation on multi-trait activities of Azotobacter spp. Figure 8. Indole acetic acid (IAA) producing activity of Azotobacter sp. K is a primary factor in determining soil and tightly bound in the crystal lattice. Exchangeable K is loosely bound water in the soil. Soluble K could be measured in a water extract of soil [29; 30]. Zinc solubilizing bacteria showing clear halo zone around their colonies were found in all eight strains. Azotobacter chroococcum A3 showed the widest clear zone among the eight strains. A very slight portion of total zinc is present as soluble form in soil solution. The majority of zinc in soil is found in insoluble compounds and minerals [31]. One of the important hormones which promote plant growth is IAA. A positive cor- relation was detected between tryptophan concentrations and IAA production. All strains produced IAA in screening of IAA production, using tryptophan as carrier. Fusarium oxysporum and Pythium fungi were found to cause severe diseases in vari- ous crops [32]. Pythium fungi are water molds, and their spores attack living plants, causing issues such as seedling decay, root and crown rot, and leaf blight [33; 34]. Fusarium oxysporum is a fungal pathogen that infect banana, tomato, many other vegeta- bles and ornamentals [35; 36]. In the present study, all strains noticeably showed fungal growth inhibition against the tested pathogen (Pythium). The antifungal activity of A. chroococcum A1, A3 and A4, A. vinelandii A5 and A6, A. beijerinckii A7 were occurred against Pythium sp. A. chroococcum A2 and A8 were fair antifungal activity against Pythium sp. after 3 weeks. A. chroococcum A4 and A8, the other strains were observed as moderate antifungal activity. As for antifungal activity against Fusarium oxysporum, all isolated strains exhibited minimal antag- onistic activity, as indicated by the small zone areas. According to the data, eight isolated strains showed good antifungal activity against the Pythrium sp. although they have no good activity against Fusarium sp. Azotobacter can be used as a bio-fertilizer without any significant limitations or challenges, except that it should not be used in conjunction with chemical pesticides and herbicides. The optimum conditions such as pH and temperature of isolated bacteria should be explored for their optimum growth and plant growth promoting activities. As for future research, isolated Azotobacter strains would be used in combination with other bio-fertilizers and would be used as antifungal and bio-fertilizers in agricultural fields. Bio- fertilizers in Myanmar using Azotobacter have already been used in agriculture [37]. Azotobacter can fix nearly 20 kg N/ha per year to use as a substitute for fertilizer in crop production [38; 39]. Therefore, it can reduce the cost for mineral fertilizers. It has economic impact and safety in farmers. Conclusion Eight bacterial isolates were obtained and identified as Azo- tobacter vinelandii, Azotobacter beijerinckii and Azotobacter chroococcum. Their existence of spherical form is about 2 µm and that of oval form is about 3-4 µm. Their shapes were ranging from cocci which occurred single, in pairs or irregular clusters and some were arranged in chains of variable lengths. They were motile, gram negative and cysts formed. All eight strains showed nitrogen fixing, phosphate solubilizing, potassium decomposing, zinc solubilizing and IAA producing activities. They also have antifungal activity against Pythium and Fusarium oxysporum fungi. Therefore, Azotobacter strains from this study could be used to develop bio-fertilizer which can enhance soil fertility due to their plant growth promoting activities. Acknowledgement We are greatly indebted to Dr. Thant Zin, Professor, Head of the Department of Zoology, University of Mandalay, Ministry of Education for allowing us to conduct the research. Our special thanks to Dr. Aye Aye Khaing, Director General, Department of Biotechnology Research, Ministry of Science and Technology for allowing us to conduct the study in the laboratory. Thanks to Dr. Tin Mar Lynn and all the researchers of Microbiology Laboratory, Department of Biotechnology Research, Kyaukse for their kind help and sharing their experiences. Supplementary Figure S1: Detection of 16S rRNA gene from isolated bacte- ria by gel electrophoresis. Figure S2: Screening on antagonistic activity of Azotobacter against A. Pythium sp. and B. Fusarium sp. Reference 1. Jnawali A, Ojha R, Marahatta S. Role of Azotobacter in soil fertility and sustainability–a review. Advances in Plants and Agricultural Research. 2015;2(6):1-5. 2. Ramadhan ZK, Issa FA. 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Highlights in BioScience Page 8 of 8 December 2024|Volume 7 http://bioscience.highlightsin.org/ Abstract Introduction Materials and Methods Study Area and Sample Collection Isolation of Bacteria Biochemical Characteristics of Isolated Bacteria Molecular Identification of Bacterial Isolates Screening on Nitrogen Fixing Activity by Indophenol Method Screening on Phosphate Solubilizing Activity Screening on Potassium Decomposing Activity Screening on Zinc Solubilizing Activity Screening on IAA Producing Activity Screening on Antagonistic Activity Results Study Area and Sample Collection Isolation and Biochemical Characterization of Bacteria Identification of Azotobacter spp. by 16S rRNA gene sequencing Screening on Nitrogen Fixing Activity by Indophenol method Screening on Phosphate Solubilizing Activity Screening on Potassium Decomposing Activity Screening on Zinc Solubilizing Activity Screening on IAA Producing Activity Screening on Antagonistic Activity Discussion Conclusion Supplementary