Isolation of potential Zn solubilizing bacteria from corn rhizosphere

Deficiency of micronutrients is a limiting factor in crop productivity. In terms of micronutrient deficiencies, zinc (Zn) deficiency is considered the most common. Obstacles to the availability of Zn in plants are not caused by the low total concentration of Zn, but by the low solubility of Zn in the soil. Therefore, a feasible approach is to exploit the capacity of soil microorganisms, especially bacteria, to convert the insoluble form of Zn to the soluble form of Zn, thereby increasing its availability and ability of plants to absorb it. This study aims to isolate various bacteria with the potential to dissolve Zn. Bacterial isolates obtained from the rhizosphere of corn plants were tested on insoluble zinc compounds, such as ZnCO3, ZnO, and Zn3(PO4)2 separately. Dissolving potential was assessed qualitatively by observing the formation of transparent zones. Of the 56 types of bacteria tested, only 6 isolates formed transparent zones. The results showed variations in the efficiency of dissolving insoluble Zn compounds between bacteria and the Zn sources used. Isolate Btg.2.3 showed the widest transparent zone in Zn media (ZnO, ZnCO3, and Zn3(PO4)2) with sizes of 20.0 mm, 24.0 mm and 15.7 mm, while isolate Bn.1.7 showed the smallest transparent zone with sizes 14.0 mm, 14.3 mm and 8.7 mm. The area of the transparent zone indicates the level of solubility and can be related to the dissolution of soluble Zn compounds produced by bacterial isolates. Of the Zn sources used, ZnCO3 showed the highest formation of transparent zones, followed by ZnO and Zn3(PO4)2.


Introduction
There are essential nutrients needed by plants in relatively small amounts which are referred to as micronutrients.Micronutrients have a specific function in plant growth and development, and cannot be completely replaced by other nutrients.However, if these micronutrients are excessive, they can be toxic to plants.One of the micronutrients that is considered important is zinc (Zn) [1].Zn in plants has a key role as a structural constituent or regulatory cofactor for various types of enzymes in many important biochemical pathways [2].As a short-term strategy to increase Zn availability, Zn fertilization in plants is important.This fertilization helps build Zn reserves needed for uptake and translocation in plants.
However, often the application of chemical fertilizers has low efficiency, where plants can only absorb a small amount of Zn dissolved in the soil.As a result, the decrease in the efficiency of using Zn chemical fertilizers is still a problem, especially in the long term.Zn deficiency not only hinders crop productivity, but also significantly reduces Zn content in crops such as seeds or cobs, negatively impacting their nutritional quality.Although chemical fertilizers can easily and quickly correct Zn deficiencies, their use is unsustainable and increases production costs [3].In addition, to overcome Zn deficiency in the long term, the use of chemical fertilizers alone is not enough, because most (96-99%) of the Zn used will turn into unavailable forms such as carbonate, oxide, or sulfate, which can eventually damage the plant.Environment [4].Therefore, an alternative that can be considered is to utilize rhizosphere bacteria which have the potential to dissolve the fixed form of Zn found in the soil into a form that is available biologically, so that it can meet the Zn needs of plants [5].
Microorganisms in the natural environment constitute a mixed population of various species.They can be found in soil, water, air, food, and in the bodies of animals and plants.To study these types of microorganisms, including bacteria, it is necessary to separate them for the purpose of studying their culture, morphology, physiology and characteristics.This separation process is called isolation, which involves purification.Bacterial isolation refers to the process of taking bacteria from their medium or 1255 (2023) 012017 IOP Publishing doi:10.1088/1755-1315/1255/1/012017 2 environment, then growing them in an artificial medium to obtain pure cultures.This allows further research on the isolated bacteria.In this context, further research on the potential of rhizosphere bacteria in dissolving Zn (zinc) is necessary based on the information previously mentioned [6].Based on the background above, further research is needed regarding the potential of rhizosphere bacteria on Zn dissolution.

Research method
The research was carried out at the Chemistry and Soil Fertility Laboratory, Department of Soil Science, Faculty of Agriculture, Hasanuddin University and the Molecular Biology Laboratory of the Cereal Crops Research Institute (Balitsereal) in September -December 2022.Bacterial isolates obtained from isolation of the rhizosphere of corn plants were tested on various non-soluble zinc compounds separated from three sources namely ZnO, ZnCO3 and Zn3(PO4)2.Dissolving potential was assessed qualitatively.The ability to dissolve Zn from the bacterial isolates obtained was evaluated using mineral salt media (glucose: 10 gr, (NH4)2SO4: 1 gr, KCl: 0,2 gr, K2HPO4: 0,1 gr, MgSO4: 0,2; gr, 1000 mL double distilled water and buffered to pH 7) added with 2.0 gr of insoluble Zn, separated from three sources namely, ZnO, ZnCO3 and Zn3(PO4)2.After being autoclaved at 121°C for 20 minutes, it was transferred to a sterilized petri dish.One full loop (10 µL) of bacterial isolates cultured for 24 hours were inoculated into petri dishes and incubated at 28°C for 48 hours and repeated for 3 times.The characteristic observed was the presence of a clear zone (halo zone) which was determined after 24 hours to 7 days [7].

Results
In this study, 56 types of bacteria were isolated from the rhizosphere of corn plants.The ability of bacterial isolates to dissolve Zn qualitatively was indicated by the presence of a clear zone that formed around the bacterial colonies growing on the selective medium.Only 6 out of 56 bacterial isolates consistently formed clear zones on insoluble Zn mineral sources.The results showed variations in the formation of the clear zone by each isolate isolated from the selective medium, as shown in table 1.

Discussion
From the results obtained, it shows that each bacterial isolate has a different ability to form clear zones around colonies inoculated on selective media and the formation of clear zones formed after 24 to 72 hours of incubation.The formation of a clear zone around the colony indicates the ability to dissolve Zn and if no clear zone is formed then the bacteria do not have the ability to dissolve Zn.Zn-solubilizing bacterial isolates have the ability to enhance plant growth and yield due to the diverse direct and indirect mechanisms involved to influence growth.Direct mechanisms involve nutrient or mineral solubilization, stimulation of biological nitrogen fixation, siderophore production, ACC deaminase activity, and phytohormone production, while indirect mechanisms involve antibiotic production, exopolysaccharides and fungal activity [7].Zn-dissolving bacterial strains solubilize unavailable forms of Zn by producing chelating ligands, secreting organic acids, vitamins, and phytohormones and through oxide-reductive and proton-extrusion systems [8].Organic acid production by bacterial strains is the main mechanism used for Zn solubilization.
The Zn bacterial clear zone refers to the area around the disc or discs containing zinc (Zn) compounds used in antibiotic sensitivity tests.When a disk containing Zn compounds is placed on an agar medium that has been planted with bacteria, the Zn compounds can diffuse from the disc into the surrounding environment.If the bacteria growing around the disk are not sensitive to Zn compounds, they will continue to grow normally and form colonies throughout the agar medium, including around the disk.The area around the disc that has no bacterial growth is called the clear zone.The clear zone indicates that the Zn compound has an antibacterial effect on the bacteria around it.The larger the clear zone formed, the more sensitive the bacteria are to Zn compounds.These results are used to evaluate the antibiotic activity and sensitivity of bacteria to these compounds.However, it should be noted that the clear zone of Zn bacteria only shows sensitivity to the tested Zn compound and cannot be used directly to identify a particular type or species of bacteria.For this purpose, additional methods are needed such as morphological-based bacterial identification tests, Gram staining, or biochemical tests.
Bacteria can dissolve micronutrients such as zinc (Zn) through a process called solubilization.Some bacteria have the natural ability to secrete organic compounds or organic acids into their environment.These organic compounds are able to dissolve zinc bound in soil or organic matter so that it becomes a form that can be taken by plants.Several mechanisms of dissolving zinc by bacteria are Production of organic acids: Some bacteria have the ability to produce organic acids such as citric acid, oxalic acid, or aminopolycarboxylic acids.These acids are capable of dissolving zinc bound in soil or organic matter, forming zinc-organic complexes that are easily taken up by plants.Excretion of siderophore group compounds: Bacteria can also secrete siderophore group compounds, which play a role in binding iron but are also able to bind zinc.Siderophores help dissolve zinc in the soil and make it available to microorganisms and plants [9].Production of phytase enzymes: Some bacteria can secrete phytase enzymes, which are capable of hydrolyzing complex phosphate organic compounds in soil.This process can increase the availability of zinc in a form that can be taken up by plants.It is important to note that the ability of bacteria to solubilize zinc can vary depending on the type and species of bacteria, as well as environmental conditions.Several bacteria that are generally known to have the ability to solubilize zinc include Pseudomonas, Bacillus, and Rhizobium.However, for agricultural applications, it is important to carry out further research and evaluation when using bacteria as a micronutrient addition in soil [9].
The formation of a clear zone by Zn-solvent bacteria can refer to the ability of certain types of bacteria to dissolve or reduce the concentration of zinc (Zn) ions in their surrounding environment.This process is known as bioremediation, in which microorganisms such as bacteria are used to remove harmful contaminants from soil or water.Some Zn solubilizing bacteria that have this ability include several species from the genera Acidithiobacillus, Thiobacillus, and Pseudomonas.These bacteria have special metabolic properties that allow them to oxidize sulfur or iron compounds in certain biochemical processes.During this process, they may also secrete organic acids or other metabolic compounds that can dissolve or bind zinc ions [10].When Zn solubilizing bacteria are placed in a medium containing a zinc source, they can produce organic acids such as citric acid, oxalic acid, or amino acids which can dissolve zinc ions in aqueous complex forms.At the same time, these bacteria can also produce enzymes or other compounds that help in the dissolving process.The result of this process is the formation of a clear zone or area around the bacterial colony where the concentration of zinc ions decreases.This can be observed in the form of clear or transparent areas around bacterial colonies on agar media or in field bioremediation systems.It is important to note that the formation of clear zones by Zn solubilizing bacteria is not a common phenomenon in nature.Several types of bacteria have this ability, but not all Zn solubilizing bacteria can form clear zones.In addition, bioremediation processes using Zn solubilizing bacteria are still an active area of research, and their use in practical applications is still under development.

Conclusion
The results showed variations in the efficiency of dissolving insoluble Zn compounds between bacteria and the Zn sources used.Isolate Btg.2.3 showed the widest transparent zone in Zn media (ZnO, ZnCO3, and Zn3(PO4)2) with sizes of 20.0 mm, 24.0 mm and 15.7 mm, while isolate Bn.1.7 showed the smallest transparent zone with sizes 14.0 mm, 14.3 mm and 8.7 mm.The area of the transparent zone indicates the level of solubility and can be related to the dissolution of soluble Zn compounds produced by bacterial isolates.Of the Zn sources used, ZnCO3 showed the highest formation of transparent zones, followed by ZnO and Zn3(PO4)2.

Table 1 .
Clear zone area of bacterial isolates on selective mediaThe formation of the clear zone by the bacterial isolates on ZnO selective medium ranged from 14.0 to 20.0 mm, with the bacterial isolate Btg.2.3 showing the largest clear zone and bacterial isolate Bn.1.7 has the smallest clear zone.On ZnCO3 selective medium, the formation of clear zones ranged from 14.3 to 24.0 mm, with isolate Btg.2.3 having the largest clear zone and isolate Bn.1.7 having the smallest.Meanwhile, on Zn3(PO4)2 selective medium, bacterial isolates were able to form clear zones between 8.70 and 15.7 mm, with bacterial isolates Btg.2.3 having the largest clear zones and isolate Bn.1.7 having the smallest clear zones.