Characterization of diesel-degrading, hydrolytic enzymes-producing Streptomyces spp. isolated from fuel-oil polluted soils

Abstract Thirty-four different soil Streptomyces isolates previously recovered from soils of different gas stations that were historically polluted with fuel oil for long time were analyzed for their ability to produce five different hydrolytic enzymes, as well as for their potential to use diesel fuel as a sole carbon source for growth, in addition to the PCR amplification of the alkane hydroxylase gene (alkB). Results showed that 97.68% were producing at least one of the five hydrolytic enzymes, pectinase, carboxymethyl cellulase (CMCase), lipase, chitinase or amylase. Three isolates (1a, 5e, and 12a) were only producing all the tested enzymes and were able to use diesel fuel as a sole carbon source for growth as assessed by biochemical enzymatic assay and dry weight measurements. The capability of these isolates to use diesel as a sole carbon source for growth was obvious by changing the colourcolour of the reaction mixture within 6 hours, and the increase in dry weight after incubation for 28 days. Although the three isolates showed diesel degrading capability, they tested negative for harbouring the alkB gene by the PCR assay. alkB gene might not be the only gene responsible for alkanes degradation, and the isolates that grew on diesel and showed no PCR product might devoid the alkB gene.


Introduction
Excessive use of petroleum hydrocarbons (PHs) may lead to their seepage and leakage to adjacent areas leading to soil and water pollution (Song, Wu, Song, Shi, & Zhang, 2021).Several physical and chemical technologies (Khalifa, Khan, & Akhtar, 2023;Xue, Chen, Xiang, Zhou, & Wang, 2023) and biological based methods (Ambaye et al., 2022;Khalifa et al., 2023;Lv et al., 2023) had been investigated to treat petroleum contaminated soils.The bioremediation process was reported to be an effective method (Kalia et al., 2022) less expensive (Rakesh, Saini, Gupta, Neelam, & Rahi, 2023), easy to handle (Jabbar, Alardhi, Mohammed, Salih, & Albayati, 2022), safe and reliable technology (Rakesh et al., 2023) and environmentally friendly (Cabral et al., 2022) compared to chemical, thermal, and conventional methods in the degradation of PHs and soil remediation process.In bioremediation processes, microorganisms such as bacteria, fungi, and algae, are usually used to remove PHs from petroleumcontaminated soils (Kalia et al., 2022).Diesel-degrading bacteria and fungi species were reported to play an important role in effective bioremediation of polluted soil and aquatic sites (Ambaye et al., 2022;Mohapatra, Dhamale, Saha, & Phale, 2022).Through the biodegradation process microorganisms completely biodegrade and mineralize the PHs to simple inorganic compounds such as CO 2 and H 2 O (Jabbar et al., 2022;Kalia et al., 2022).
Streptomyces is a large genus of actinobacteria in the order of Actinomycetales.Members of this genus are Gram-positive and aerobic with diverse metabolic and nutritional characteristics.They are well known for antibiotics productivity and their ability to decompose organic matter (Shepherdson, Baglio, & Elliot, 2023).Several reports demonstrated the capability of strains Streptomyces to biodegrade diesel and PHs (Chaudhary, Sharma, Singh, & Nain, 2011;Lv et al., 2023;Peng et al., 2013;Sivakumar, Sundaram, Krishnasamy, & Rao, 2019).However, only a few of these studies were based on strains isolated from PHs-contaminated sites (Soumeya, Allaoueddine, & Hocine, 2022).The degradation of diesel in pure cultures or in soils were reported to vary significantly according to the Streptomyces strains.For example, Soumeya et al. (2022) were able to isolate Streptomyces ginkgonis KM-1-2 from a waste oil storage site, which is capable of degrading 76.4% of used motor oil and emulsification index of 82.6%.
Streptomyces species can produce hydrolytic enzymes (Soumeya et al., 2022) as well as biosurfactants (Ferradji et al., 2014).These two key properties enhance the process of diesel and other petroleum hydrocarbons biodegradation.Streptomyces species vary in their degradation of PHs using different extracellular enzymes that mediate and contribute to interconnected metabolic pathways (Rakesh et al., 2023).Interestingly, most of these strains produce different enzymes and each enzyme has a specific hydrocarbon substrate, which allows for degradation of all types of PHs.For instance, Soumeya et al. (2022) observed that Streptomyces ginkgonis degraded a wide range of aromatic compounds, which points to its ability to produce a wide variety of enzymes.In our laboratories, several Streptomyces were isolated from polluted soils, for their potential to produce different enzymes such as xylanases, cellulases, pectinases and chitinases (Saadoun, Rawashdeh, Dayeh, Ababneh, & Mahasneh, 2007, Saadoun, Al-Omari, Jaradat, & Ababneh, 2009, Saadoun, Dawagreh, Jaradat, & Ababneh, 2013, Saadoun, Ananbeh, Ababneh, & Jaradat, 2017).Moreover, Elnahas, Hou, Wall, and Majumder (2021) found that Streptomyces sp.MOE6 strain can produce extracellular polysaccharide during the biodegradation of petroleum hydrocarbon.They suggested that extracellular polysaccharide could be used in bioremediation and cleaning-up contaminated sites.
Generally, only few studies were reported the role of indigenous Streptomyces species in degrading PHs in polluted environments.For instance, different strains of bacteria capable of degrading short chain alkanes and diesel were isolated from oil-polluted soil (Saadoun, 2002(Saadoun, , 2004)).Moreover, Saadoun and Alawawdeh (2019) recovered different Streptomyces species from soils chronically exposed to fuel petroleum oil contamination.However, these studies did not investigate the ability of these organisms to produce hydrolytic enzymes.Thus, this work aimed to investigate the capability of Streptomyces species from soils historically exposed to fuel petroleum oil contamination to produce hydrolytic enzymes, as well as their potential to use diesel fuel as a sole carbon source for growth.

Growth conditions and adaptation of Streptomyces on diesel
Streptomyces isolates that were isolated from the contaminated soils and were able to produce all tested enzymes were adapted on diesel and grown in conditions as described previously by Saadoun and Alawawdeh (2019).Jacobs, Prior, and Dekock (1983) method was followed for the biodegradation of diesel and as previously reported by Saadoun and Alawawdeh (2019).The potential of Streptomyces isolates to enzymatically degrade diesel was tested by heating the cells at 90 C for 10 min or the removal of NAD þ followed by the inability of degradation by the isolate demonstrated by keeping the original colour of the reaction mixture.The colour change was compared to four controls: (i) Control 1 did not contain the substrate (diesel), (ii) control 2 did not contain NAD þ , (iii) control 3 had no cells, and (iv) control 4 was based on heating the cells at 90 C for 10 minutes.

PCR assay
2.4.1.Growth conditions, extraction, and quantitation of genomic DNA from pure Streptomyces isolates, estimation of DNA purity, primers design for detection of alkane hydroxlyase gene (alkB), PCR amplification, and electrophoresis The Streptomyces isolates that produced all tested enzymes and degraded diesel were cultured to extract, quantitate, and estimate the purity of their genomic DNA.Partial PCR amplification of alkane hydroxlyase gene (alkB) was done as described previously by Saadoun and Alawawdeh (2019) and Saadoun, Alawawdeh, Jaradat, and Ababneh (2020).

Characterization of Streptomyces spp
Morphological and physiological characterization of the recovered Streptomyces isolates that produced all tested enzymes and were able to degrade diesel were performed as reported by Saadoun and Alawawdeh (2019) and according to the International Streptomyces project (ISP) (Shirling & Gottlieb, 1966).

Characterization of Streptomyces isolates
Thirty-four different soil Streptomyces-isolates were previously recovered from chronically fuel-oil-polluted soil samples on SCNA medium.Testing the recovered Streptomyces isolates for hydrolytic enzyme production (CMCase, pectinase, chitinase, lipase and amylase) showed that most of them (97.68%) were producing at least one of the five hydrolytic enzymes.In addition, 2.94% of the 34 isolates were producing one enzyme, 8.80% produced two enzymes, 41.20% produced three enzymes, 35.30% produced four enzymes, and 8.82% of these isolates were producing five enzymes.However, one isolate (2.94%) did not produce any of these enzymes (Figure 1).
Results indicated that 81.4% of the isolates were producing CMCase, whereas 74.40% produced lipase, 67.45% produced amylase, and 51.1% produced pectinase and chitinase (Figure 2).Activities of the recovered isolates to produce the enzymes relatively ranged from weak, moderate to strong as indicated by the width of the clear zone formed around the colonies in the screening process (Figure 2).
Data indicated that only three namely 1a, 5e, and 12a produced all tested enzymes.They were phenotypically characterized into three-colour series (Table 1) and were with either spiral, biverticillus, or rectus sporophore and isolate 5e was the only one able to produce soluble and melanin pigments.The three isolates exhibited distinctive reverse side colour.Moreover, sugar utilization test revealed that all isolates were unable to utilize rhaffinose and sucrose, but they were able to utilize glucose, fructose, xylose, arabinose, and maltose.Morphological characteristics of the Streptomyces isolates are detailed in Table 1.

Growth of Streptomyces on diesel
Growth of Streptomyces on diesel was assessed by measuring the dry weight of the isolates after the specified incubation period.The growth response of 1a, 5e, and 12a Streptomyces isolates on diesel fuel is demonstrated with a percentage increase of 24.60%, 27.21%, and 22.30% in the dry weight (Figure 3) after 28 days of incubation as compared to zero time, respectively.The three isolates showed similarity in growth on diesel as compared to each other.

Enzymatic assay
Results in Table 2 show that three hydrolyticenzymes producing Streptomyces isolates were capable of degrading diesel demonstrated by changing the colour of 2, 6-dichlorophenolindophnol (DCPIP) from blue to yellow after incubation for 6 h.

PCR amplification and detection of alkB gene sequence in Streptomyces isolates
Extraction of genomic DNA was performed using a commercial DNA isolation kit.The bands of the extracted DNA were of good quality when tested by agarose gel electrophoresis (data not shown).Results of the alkB gene PCR analysis for three isolates (1a, 5e, and 12a) that are producing the five enzymes and able to grow on diesel are shown in Figure 4. None of these three isolates harboured the alkB gene PCR as judged by the absence of PCR product following agarose gel electrophoresis analysis.

Discussion
This study is focussed primarily on examining the hydrocarbon-polluted soil streptomycetes and their ability to produce hydrolytic enzymes due to the absence of any information about those organisms in hydrocarbon-polluted soils as apparent from the literature.

Screening of Streptomyces isolates for amylase, lipase, chitinase, CMCase, and pectinase activities
In this study, all the 34 Streptomyces isolates were assessed for amylase, lipase, chitinase, CMCase, and pectinase activities (Figure 1).CMCase was the most prevalent enzyme among the tested isolates (81.4%), followed by lipase with 74.4% (Figure 2).Few studies have been conducted on cellulytic active Streptomyces isolated from Jordanian soil (Jaradat et al., 2008;Saadoun et al., 2007); however, studies on lipolytic active Streptomyces species are lacking.CMCases hydrolyze cellulose into sugars (Shatta, El-Hamahmy, Ahmed, Ibrahim, & Arafa, 1999), while lipases hydrolyze lipids into acylglycerol (Arpigny & Jaeger, 1999;Sharma, Chisti, & Banerjee, 2001) which, are also hydrolyzed by lipases (Jaeger & Eggert, 2002).Lipases are thus considered as one of   the most important groups of biocatalysts for industrial applications, such as manufacturing of detergents, production of food ingredients, treatment of wastewater, paper processing, production of pharmaceuticals, fine chemical synthesis and manufacturing of pesticides, cosmetics as well as single cell proteins (Arpigny & Jaeger, 1999;Sharma et al., 2001).In addition, lipases are important as catalysts for synthesis of esters and for trans-esterification of the oil to produce biodiesel (Gulati et al., 2005;Gunstone, 1999;Poonam et al., 2005).Similar screening for lipase production by bacterial isolates obtained from oil-contaminated habitats revealed the isolation of 20 bacterial isolates with the identification of the isolate LP10 as Streptomyces exfoliates to be the best lipase producer (Aly, Tork, Al-Garni, & Nawar, 2012).
The recovered Streptomyces isolates were producing five different hydrolytic enzymes with the majority to be CMCase followed by lipase.Optimizing the conditions for maximum enzyme production of CMCase and lipase by these Streptomyces isolates and their characterization deserve studying in future.

Growth on diesel
By measuring the dry weight of the tested Streptomyces isolates after the incubation period, we were able to identify the isolate with the highest potential to grow on diesel.It is of importance to report that diesel degradation is not linear during all the time intervals.Moreover, the biochemical technique used here in this study enabled us to show the ability of the examined isolates to degrade diesel as demonstrated by changing the colour of the reaction mixture from blue to yellow.
Results revealed that all tested isolates were able to change the colour of 2, 6-dichlorophenolindophnol (DCPIP) from blue colour to yellow after 6 h of incubation.The ability of Streptomyces to degrade diesel enzymatically was tested by either the removal of NAD þ from the reaction mixture or heating the cells for 10 min at 90 C with the subsequent inability of the isolate to perform degradation as indicated by the no change in the final colour of the reaction mixture.

PCR amplification and detection of alkB gene sequence in Streptomyces isolates
Alkane hydroxylase gene (alkB) is an important gene reported to be in Streptomyces spp.and associated  with degradation of hydrocarbons (Saadoun et al., 2008).Although the three Streptomyces isolates (1a, 5e, and 12a) produced all five enzymes and were able to grow on diesel, none of them showed any PCR product.It is well known that diesel is a complex hydrocarbon that contains many hydrocarbon components other than alkanes, thus, harbouring of these three Streptomyces isolates to alkB gene does not imply that they are able to degrade diesel.This has been explained before by Saadoun and Alawawdeh (2019) that the alkB gene is not the only gene that is responsible for the degradation of alkanes.In addition, the isolates that grew on diesel as a sole carbon source and showed no PCR product might devoid the alkB gene.

Conclusion
This study highlights the existence of streptomycetes in soils historically exposed to fuel-oil pollution and the potential of Streptomyces flora prevailing in such hydrocarbon-polluted environment to produce five different fibre hydrolytic enzymes with the majority to be CMCase followed by lipase.The alkB gene in Streptomyces isolates is not the only gene that is responsible for the degradation of alkanes and presence of this gene in Streptomyces isolates does not imply that they are able to degrade diesel.This study highlights the existence of streptomycetes in soils historically exposed to fuel-oil pollution and the potential of Streptomyces flora prevailing in such hydrocarbon-polluted environment to produce five different fibre hydrolytic enzymes with the majority to be CMCase followed by lipase.Additionally, these bacteria can degrade diesel.It is important to note while the alkB gene is one of the genes responsible for the degradation of alkanes, it is not the only gene involved in this process.The presence of this gene in Streptomyces isolates does not necessarily imply their ability to degrade diesel.

Figure 2 .
Figure 2. Percentage distribution of hydrolytic enzymes production by Streptomyces isolates.Amylase production on SCNA plate, chitinase production on chitin agar plate, CMCase production on carboxy methyl cellulose agar plate, lipase production on egg-yolk agar plate, and pectinase production on pectin agar plate.

Figure 3 .
Figure 3. Growth response of different Streptomyces isolates on diesel fuel as indicated by the increase in dry weight (mg/ml) and O.D at (540 nm) reading.A; 1a, B; 5e, C; 12a.

Table 1 .
Morphological characteristics of the Streptomyces isolates.

Table 2 .
Action of different Streptomyces isolates on diesel fuel indicated by colour change a .
a Color change from dark blue to other colours at different time intervals by each Streptomyces isolate: Y ¼ yellow; Gr ¼ green; Bl ¼ blue.b The first control contained no diesel (substrate), the third contained no cells.