Isolation and identification of biosurfactant-producing bacteria from oil contaminated soil

Introduction Zinc is one of the imperative micronutrients required relatively in small concentrations in tissues for healthy growth and reproduction of plants. Zinc deficiency in plants leads to reduced membrane integrity and synthesis of carbohydrates, auxins, nucleotides, cytochromes, and chlorophyll and develops susceptibility to heat stress. The solubility of Zn is highly dependent upon soil pH and moisture and hence arid and semiarid areas are often zinc-deficient. The use of microorganisms with the aim of improving nutrients availability for plants is an important practice and necessary for agriculture. Zinc-solubilizing microorganisms can solubilize zinc from inorganic and organic pools of total soil zinc and can be utilized to increase zinc availability to plants. Therefore, the present study was carried out to isolate and characterize native zinc-solubilizing bacteria from Zea mays rhizosphere and evaluate their zinc-solubilizing potential and the effect of zinc solubilizing isolate on Zea mays growth.
Materials and Methods In vitro zinc solubilization assay of isolates was done using 0.1% zinc from zinc oxide in both plate and broth assays. Actively growing cultures of each isolates were spot-inoculated (7 µL) onto the agar and plates were incubated at 28°C for 120 h. The clearing zone around colony was recorded. Quantitative study of zinc solubilization was studied in 150 mL conical flasks containing 50 mL of liquid mineral salt medium. The broth was inoculated with 500 µL of overnight grown bacterial inoculum and incubated for 120 h at 160 rpm in an incubator shaker at 28°C. After incubation, the culture broth was centrifuged and the concentration of Zn in the supernatant was estimated in atomic absorption spectrophotometer. Among these isolates, 18 isolates with a solubility index of 1 and higher were selected based on morphological, biochemical and physiological characteristics for further studies. An isolate with more ability to dissolve zinc, phosphorus, potassium and auxin production was selected to investigate the effect of isolate on Zea mays growth. Maize seeds of cultivable variety were surface sterilized with 10% sodium hypochlorite for 10 min and washed several times with sterile distilled water. Seeds were treated with inoculum containing 108 cfu·ml−1 of isolate. A factorial experiment in a completely randomized design with five replications was conducted. The treatments included two levels of bacteria B1 (control), B2 (stenotrophomonas maltophilia) and zinc sulfate fertilizer at three levels of Zn0 (control), Zn20 (20 kg/ha) and Zn40 (40 kg/ha). After 20 days of sowing, plants were removed from the tubes carefully and biometric parameters like, Chlorophyll index, root length, shoot length and dry mass of plants were recorded as the indicative of plant growth.
Results and Discussion A total of 50 bacterial isolates were isolated from corn rhizosphere. Of all, sixteen isolates showed solubilization halo on plate agar medium. Among the cultures, Z1, Z3 and Z16 showed the highest solubilisation zone in ZnO amended medium with maximum solubility index (1.3). Quantitative assay for zinc solubilisation revealed that Z13 was able to dissolve 44.8 ppm from ZnO in liquid medium while solubility index of this isolate was lower than above the mentioned isolates (1). Of all, isolate Z13 with the highest zinc solubilisation by broth assay was characterized and identified as Stenotrophomonas species based on Gram-negative reaction, endospore-forming cells, and other biochemical and physiological properties. This isolate was able to produce auxin and dissolve insoluble phosphorus and potassium from the source tricalcium phosphate and vermiculte, respectively. One of these strains (Z13), Stenotrophomonas, was used as inoculum in corn culture.
Seed bacterization of maize with zinc solubilising Stenotrophomonas enhanced the plant growth significantly after 15 days. Results indicated a significant interaction effect of bacterium and fertilizer on shoot dry weight and chlorophyll content (P<0.01). The maximum spad index and wet weight of aerial part was obtained in the presence of bacterium and without using of zinc sulfate. The main effect of bacterium on wet and dry weight of root and wet weight of aerial part, root length and shoot height was significant (P<0.01).The highest chlorophyll index and aerial part dry weight were related to bacterial treatments, which showed an increase of 34.7% and 87%, respectively, compared to control (without fertilizer and bacteria).
Conclusion PGPR is known as a group of useful rhizospheric bacteria that increase plant growth. Today, the increasing use of PGPRs in agriculture is an alternative to chemical fertilizers to prevent environmental contamination