Collection of samples

From various places around the Al-Dora oil plant, Baghdad, Iraq. 18 soil samples were taken. The soil samples were kept cold by being placed in sterile polythene bags. In order to conduct additional studies, these samples were taken to the lab and kept there at 4°C.

Primary screening of xylanase production

A soil sample of 1 g was combined with 99 ml of distilled water, shaken for 30 min, and then allowed to settle for 30 min. From dilutions of 10^-2 to 10^-6, we made successively. To generate xylanolytic bacterial colonies, each sample was inoculated onto nutritional agar medium containing 0.5% of corn cob xylan and incubated at 30°C for 48 h. For the purpose of determining the location of the xylan hydrolyzing zone surrounding the bacterial growth on xylan agar plates, plates were stained with 0.1% Congo red solution and left for 1 hour. The plates were then washed with 1M NaCl for 15 minutes for zoon analysis.

Identification of xylanase producers

Morphological and biochemical studies were run on the colonies that had already been created as explained by [7]. To confirm these isolates, the Vitek 2 technology is also used.

Secondary screening for xylanase production

The chosen isolates were cultured at 30°C for 24 hours after being infected with nutritional broth that had been supplemented with 5% corn cob xylan. After 30 minutes of centrifugation at 8000 rpm, xylanase activity was assessed.

Xylanase activity and protein contents

The substrate solution was created by mixing 0.3M of phosphate buffer with 1% corncob xylan at pH 7. To create a 1mL reaction mixture, 0.5mL of the substrate solution and 0.5 ml of each bacterial supernatant were added to an assay tube. The reaction mixture was then incubated in assay tubes for 15 minutes in a water bath at 55°C. Following the incubation, 1.5mL of 3, 5-dinitrosalicyclic acid solution (DNS) was added to each test tube, and the reaction was stopped by boiling the tubes at 100 °C for 10 minutes. Each bacterial supernatant's release of reducing sugar during hydrolysis was quantified using an absorbance value of 540nm. The amount of xylose released per milliliter of enzyme per minute [8] was used to calculate one unit (IU) of xylanase activity. Using a bovine serum albumin standard and the Bradford method with the absorbency measured at 595nm, protein content was determined [9].

Purification of xylanase

By making a few adjustments to the procedure described in [10], the xylanase was purified. The clear supernatant from centrifugation was then provided to DEAE-cellulose chromatography after the chosen isolate had been cultured on the optimal production medium and fractionated using ammonium sulfate at 20–70% saturation. The dialysis-concentrated active pooled sample was put onto sephadex G-150, which equilibrated before being eluted with 0.3M phosphate buffer at pH 7. L-xylanase activity and protein concentration for the obtained portions were noted.

Xylanase's removal of hydrocarbons from polluted soil

20 gm of hydrocarbon-contaminated soil was mixed with 40 millilitres of pure xylanase. As a control, distilled water was used in place of the xylanase, which was incubated at 30°C for 24 hours. After being centrifuged for 10 minutes at 6000rpm, the soil's created liquid solution was given back. Every three days, the same amounts of soil and toluene were combined to determine the amount of hydrocarbons in the soil after interaction with pure xylanase vs the control. After centrifugation, the toluene with hydrocarbon dissolved in it was measured at 410um absorbance [10].

Results

Figures & Tables

Discussion

The results in figure 1 showed that 7 isolates of Pseudomonas were found out of the 18 collected samples after cultivating hydrocarbon-contaminated soils on nutrient broth supplemented with 5% corn cob xylan and identifying the growing colonies. The diameter of clear zones on agar plates varied between 13 and 24mm according to the qualitative screening for xylanase production, with Pseudomonas putida isolates showing the best productivity, as indicated in table (1). In order to select Pseudomonas putida isolates as the greatest xylanase producers. Pseudomonas, Acinetobacter, and Mycobacterium are just a few of the bacterial strains that have been found to be capable of breaking down petroleum hydrocarbons [7]. This ability is attributed to the presence of genes and enzymes that use chemical complexes found in petroleum as important energy sources. Because of the greater molasses levels, Pseudomonas stutzeri, an enzyme that breaks down xylan to get nutrients, may not have been created by the bacteria. that breaks down xylan to obtain nutrients. Microbes typically prefer to use a simpler molecule when there are two or more sources of carbon in the medium [11]. Pseudomonas stutzeri demonstrated strong xylanolytic activity in a medium containing 2% (w/v) birchwood xylan and a temperature of 37°C; this resulted from the release of the xylanase enzyme, which breaks down xylan in the liquid media [12]. The medium including xylan, glucose, and meat extract, which was further adjusted by adjusting the component concentrations, promotes the growth of Pseudomonas sp. XPB-6 [13].Through the use of ammonium sulfate fractionation, DEAE-Sepharose CL-6B, Toyopearl HW-50S, and Butyl-Toyopearl 650M column chromatography, an endo-1,3-beta-D-xylanase was isolated from the culture fluid of Pseudomonas sp. PT-5 [14]. The xylanase has showed homogeneity in the gel electrophoresis by following a methodical procedure for the isolation and purification of xylanase from Pseudomonas flu using the techniques (NH)2SO4 fractionation and Sephede x G100 gel column [15].

Numerous microbial cellulolytic enzymes are necessary for the complicated process of cellulosic material degradation [16]. Cellulose and hemicellulose can be broken down either chemically (with acids or alkalis) or enzymatically. In the industry, lignocellulose degradation has been accomplished via a number of acid hydrolysis processes using sulfuric or hydrochloric acid at various concentrations, temperatures, and pressures [17]. The depolymerization of the polysaccharide components into sugar monomers, which occurs during the breakdown of lignocellulosic materials, is a difficult process that calls for a variety of enzymes [10]. Cellulose is broken down into glucose monomers by enzymes referred to as cellulases [16]. The type and number of microorganisms present in contaminated environments directly affect the efficacy of the applied restoration approach. The availability of the free contaminant and its ability to permeate the organism’s membrane affect how quickly the pollutant can be absorbed by the microorganism once PHC removal has started [18].

Pseudomonas putida could be the best producer foe xylanase than other species. Purified xylanase led to removal of hydrocarbons from hydrocarbon-contaminated soil. These findings can encourage using of xylanase as an alternative bioremediator for cleaning up of oil-contaminated areas.

No conflict of interest

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