Morphological, biochemical and molecular identification of petroleum hydrocarbons biodegradation bacteria isolated from oil polluted soil in Dhahran, Saud Arabia.

Accumulation of petroleum hydrocarbon residual considered a major environmental problem in the kingdom of Saudi Arabia cause of intensive efforts for oil detecting. Until now, In situ biodegradation considered the most effective method for petroleum hydrocarbon residual biodegradation. The aim of this study is isolation and identification biodegradable capability bacteria from contaminated sites in Khurais oil field, Dhahran, Saud Arabia via Different morphological and biochemical and molecular methods. Furthermore, degradation level in contaminated liquid medium and soil were evaluated. Three bacterial strains were selected from petroleum-contaminated soils of Khurais oil field depending on their capacity to grow in the existence of hydrocarbon components and identified according to morphological, biochemical. Interestingly, 16S rDNA sequencing fingerprinting results confirmed our bacterial identification as Bacillus subtilis, Pseudomonas aeruginosa and Bacillus cereu. Phyllogenetic tree was constructed and genetic similarity was calculated according to alignments results. Biodegradation patterns for different three isolates were reflected varied degradation ability for three isolates regarding incubation time. Different features were studied for three biodegrading bacterial strains and identified as Bacillus subtilis, Pseudomonas aeruginosa and Bacillus cereus. Remarkable degradation rate % patterns for hydrocarbons residual were recorded for all three isolates with varied.

Introduction

Petroleum (crude) oil are compounded of thousands compounds mixture. 50–98% of crude oil is Petroleum hydrocarbons which considered a major component depending on the source of the oil. Different microorganisms could be applied for Petroleum hydrocarbons biodegradation. However, bacteria considered important biodegrable microorganisms which play a critical role in hydrocarbon degradation (Udgire et al., 2015).

One of the initiative efficient, economical and environmental treatment mechanisms for petroleum biodegradation is In situ biodegradation through degrade petroleum and other hydrocarbons from culture via widely distributed microorganisms and applied for varied hydrocarbon-contaminated soils and waters (Margesin and Schinner, 1997, Whyte et al., 1997). Thus, continuous evaluation for biodegradation rate considered a critical needed for different biodegradable microorganisms (Alquati et al., 2005). Petroleum hydrocarbons biodegradable illumination depends on the indigenous microorganisms to transform or mineralize the organic contaminants (Fig. 1). Many different factors of contaminated soil characterize influence petroleum hydrocarbons bacterial biodegradation such as pH, electrical conductivity, total nitrogen and heavy metal which are important indicators of soil quality, fertility and productivity. Eight hydrocarbon degrading bacteria were specifically detected as Alcaligen sp, Bacillus sp, Chromobacterium sp, Corynebacterium sp, Pseudomonas sp, Aeromonas sp, Serratia sp, and Flavobacterium sp. (Gayathiri et al., 2017).

Fig. 1

Detectable conditions for Hydrocarbon degradation rates in soil, fresh water and marine environments.

Recently, many advanced molecular culture-dependent techniques (like library clone, TGGE/DGGE, LH-PCR, RISA, RT-Q-PCR, FISH, RAPD and RFLP) were developed and considered a helpful tool for isolation and identification new bacterial strains with degradation capabilities (Stancu, 2018).

Powerful points for Molecular especially, rDNA-dependent methods to identify microorganisms are rapidity, precisely and reliability analysis of microbial cultures comparing with traditional, biochemical culture-dependent techniques, thus molecular technique has great potential for bacterial identification in new era. 16S rRNA specific molecular marker has been constructed to identify specific bacterial genes (Wang et al., 1996, Wheeler et al., 1996) which dedicated to two methods. Firstly, designed to amplify a wide spectrum of bacterial sequences (Teske et al., 1996, Marchesi et al., 1998).

This investigation was carried out to identify isolated microbial strains from oil-contaminated soils through extensive study consisting of morphological, biochemical and molecular fingerprinting method. Furthermore, hydrocarbons biodegradation capabilities were evaluated for identified isolates through estimated degradation rate % during incubation time.

Materials and methods

Soil samples collection

In this investigation, approximately 10 g of oil-contaminated soil samples were collected from various locations of Khurais oil field (Fig. 2) with an area of 2890 km2, 250 km southwest of Dhahran and 150 km east-northeast of Riyadh, 25.0715°N, 48.0556°E), Saudi Arabia in 2018. After taking out 5 cm of perfunctory soil were collected to a depth of 20 cm. Sterile polyethene bags were used to samples reserving and stored under −20 C. One gram of soil was liquefied in ten ml of double distilled water to prepare soil suspensions.

Fig. 2

Khurais oil field, Dhahran, Saudi Arabia.

Bacterial isolation from contaminated oil soil

To isolate crude oil degrading bacteria, Enrichment technique was used via added 5 g of oil-contaminated soil to 500 ml of mineral salts media in sterilized Erlenmeyer flasks. Sole carbon source was applied as 2000 µl of crude oil and incubated with shaking for 7 days at room temperature. Then, ten-fold serial dilutions were performed for enrichment sample suspension and one millilitre of each dilution was poured into oil agar plates to isolate crude oil utilizing bacteria and incubated at 30 °C for 3–7 days. Sub-culturing repeated for selected pure colonies on oil agar plates and transferring on Nutrient agar slants for morphological, biochemical and molecular identification.

Bacterial isolates identification and characterization

Morphological characterization

Colour, shape, transparency and margin were examined and recorded as colony morphological characteristics according to Cheesbrough (1991). Microscopic features were recorded for all isolates via Gram stain protocol.

Biochemical characterization

Oxidase test

Tested bacterial colony was smeared on the filter paper previously saturated with freshly prepared oxidase reagent. Positive oxidase test was recorded as the development of a blue-purple colour within 10 s (Cheesbrough, 1991).

Catalase test

Gas bubbles detecting within 10 s after added purified bacterial culture to 5 ml of hydrogen peroxide solution, considered as a positive catalase test (Cheesbrough, 1991).

Urease test

Slanted two millilitres of urea medium which placed in bijou bottles applied for the incubated bacterial colony at room temperature. Red-pink colour in the medium was considered as a positive test for urease induction (Cheesbrough, 1991).

Indole test

Appearance of bright red and yellow color which composed after added 0.5 ml of Kovac's reagent to incubated bacterial culture at 35 °C for 24 h on SIM media indicated a positive and negative results respectively (Cheesbrough, 1991).

Simmons Citrate test

Simmons Citrate test was performed via inculcate Simmons Citrate Agar plates (TSBA, Himedia) surface with bacterial cultures then, incubated at 37 °C up to 48 h. changing media colour from green to bright blue indicate positive reaction.

Methyl red (MR) test

After adding methyl red indicator solution (TSBA, Himedia) to inoculated culturing media and incubation at 35 °C for up to 4 days, changing color to red indicate MR test positive- appearance of tested bacteria (Color Atlas and Textbook of Diagnostic Microbiology, 2016).

Gelatin hydrolysis

Nutrient gelatin stab method was applied according to Edison et al. (2012). Heavy inoculums of a test bacterium inoculated into tubes containing nutrient gelatin, gelatin liquefaction is the positive results for bacterial gelatin hydrolysis.

Molecular fingerprinting as a confirmatory identification technique

E.Z.N.A.®Bacterial DNA Kit (Omega Bio-Tek, D3350-01, USA) was applied to extract total bacterial genomic DNA. Universal 16S rDNA primers (27 F, 5-AGAGtttGAtcAtGGctcAG-3 and 1492 R, 5-tAcGG ttAccttGttAcGActt-3) were applied for bacterial identification through PCR technique with targeting fragment size 1400 bp. Thermal cycler conditions was designed as 94 °C for 5 min, 3 cycles at 94 °C for 45 s, 57 °C for 30 s, 72 °C for 120 s; 3 cycles at 94 °C for 45 s, 56 °C for 30 s, 72 °C for 120 s; 3 cycles at 94 °C for 45 s, 55 °C for 30 s, 72 °C for 120 s; 26 cycles at 94 °C for 45 s, 53 °C for 30 s, 72 °C for 120 s; and a final step at 72 °C for 5 min (Linderman, 1998). 1.5% of Agarose gel was applied for detecting amplified amplicon of 16S rDNA fragment which eluted (E.Z.N.A.®Gel Extraction Kit, Omega Bio-Tek D2500-01, USA), sequenced and alignment through comparing with other sequences from gene bank database using Blast (www.ncbi.nlm.nih.gov/Blast) for bacterial identification (Stach et al., 2001).

Biodegradation studies

Degradation in liquid medium

Broth (LB) liquid medium containing 1% crude oil was applied for culturing and incubating three investigated isolated at 37C. Degradation rate % was evaluated spectrophotometrically according to the described method by Odu (1972).

Degradation of contaminated soil

For obtaining soil sample with 1% concentration of crude oil, five grams of drying, sieving and sterilizing polluted soil, was mixed with contaminated oil solution and 1% inoculum was added and cultured at 30 °C for a month. Oil degradation rate was evaluated weighting method weekly and germ-free soil was considered as the control.

Results and discussion

Strain isolation and identification

Three different oil degrader’s bacteria were selected and isolated from contaminated soil samples from Khurais oil field, 250 km southwest of Dhahran and 150 km east-northeast of Riyadh, Saudi Arabia and initially labelled as A, B and C. Fig. 3 and Table 1 illustrated morphological characteristics for isolates. Isolate A was distinguished with rod, Creamy white, Circular flat and entire margin. By contrary, B isolates characterized by cylindrical rods size, white, Irregular large form, negative gram stain and undulate margin. Rods size, Creamy white, Circular flat, entire margin and positive gram stain features characterize isolate C.

Fig. 3

Morphological characters and gram stain for three bacterial strains A, B and C isolated from Khurais oil, Dhahran, Saudi Arabia.

Table 1

Morphological colony features for three bacterial isolates.

Isolates	size	Gram stain	Color	Form	Margin
A	Rods	+	Creamy white	Circular flat	Entire
B	Cylindrical rods	−	white	Irregular large	Undulate
C	Rods	+	Creamy white	Circular flat	Entire

Based on biochemical assays (as shown in Table 2) and previous morphological examination, three targeted isolates reflected different biochemical features. Positive for Oxidase test and negative for Catalase, Indole and Urease tests identified isolate A as Bacillus sp. On the other hand, positive for Oxidase and Catalase tests and negative for Indole and Urease tests refer to Pseudomonas sp. negative oxidase, Indole and Urease tests and positive Catalase test remarked third isolate as Bacillus cereus. Performed morphological colony characteristics examination (Gram stain, mobility and motility, shape and color) and biochemical tests (catalase, urease, oxidase activities, nitrate reduction, Idol production, acid/gas production from carbohydrates and fermentation of sugars) to identify our Hydrocarbons degradation isolates were considered a traditionally identification methods (Ventosa et al., 1982, Smibert and Krieg, 1994, Claus and Berkeley, 1986, Udgire et al., 2015).

Table 2

Three bacterial isolates Biochemical tests results.

Isolates	Oxidase test	Catalase test	Simmons Citrate test	Indole test	Methyl Red (MR) test	Urease test	Gelatin Hydrolysis
Bacillus subtilis(A)	Variable	(+)	(+)	(−)	(−)	(−)	(+)
Pseudomonas aeruginosa(B)	(+)	(+)	(+)	(−)	(−)	(−)	(+)
Bacillus cereus(C)	(−)	(+)	(+)	(−)	(−)	(−)	(−)

16S rDNA fingerprinting method which performed to identify hydrocarbons degradation bacteria is based on highly conserved regions, which help in the analysis. Almost 1.4 Kb of targeted amplicons were amplified, eluted and sequenced (Fig. 4). Regarding morphological, biochemical and molecular features for three obtained hydrocarbon-degrading isolates (as shown by Table 3), strain A was identified as Bacillus subtilis with 99% of homology percentage and B strain was identified as Pseudomonas aeruginosa with 100% and of homology percentage. Third isolate (C) was identified as Bacillus cereus with 99% of homology percentage. As shown by a Fig. 5, Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences was constructed for our obtaining three bacterial isolates based on 16S rDNA sequence alignments results. The highly genetic similarity was detected among our bacterial isolates (A), (B) and (C) which identified as Bacillus subtilis, Pseudomonas aeruginosa and Bacillus cereus and different strains for Bacillus subtilis, Pseudomonas aeruginosa and Bacillus cereus which indicated our identification results. Our obtaining results for applied 16S rRNA sequences for hydrocarbonoclastic bacteria (HCB) was in accordance of. They compared the limited capability of PCR-based technique such as cell lysis biases ability, a variation of rRNA copy number or different templates amplification efficiency with PCR-based studies which considered as an important procedure to classify microbial diversity. Based on previous findings, 16S rRNA genes considered an effective method to get an association between taxonomic identification with degrading hydrocarbons capability. More support was added to our sequencing identification data based on findings of Więckowicz (2009) via clearing efficiency of a sequencing technique for bacterial identification through sequences alignments lower than 3%. More light was added to our findings by Subathra et al. (2013) through applied both biochemically and phylogenetically methods to identify 3 crude oil biodegradation bacteria isolates as Bacillus subtilis I1, Pseudomonas aeruginosa I5 and Pseudomonas putida I8. Furthermore, our obtaining results for applying biochemical tests and 16S rRNA sequences to identify Petroleum-degrading bacteria were in agreement of Godini et al. (2018). They identified Brevibacillus sp., Microbacterium oxydans, Staphylococcus arlettae, Staphylococcus warneri, Methylobacterium persicinum, and Achromobacter xylosoxidans as degrading bacteria in the light of biochemical and molecular identification results.

Fig. 4

Amplified product of 16S rDNA for three bacterial isolates (A), (B), (C) and negative control (N).

Table 3

16S rDNA sequencing data of the isolated strains A and B from contaminated soils in Khurais oil field; Saudi Arabia.

Strain number	Total length (bp)	Gene bank accession no.	Identification	Identity %
A	1440	KC197028.1	Bacillus subtilis	99
B	1417	MG818964.1	Pseudomonas aeruginosa	100
C	1500	KR071870.1	Bacillus cereus	99

Fig. 5

Phyllogenetic tree for three degradation of petroleum hydrocarbons bacterial isolates based on 16S rDNA sequence.

Evaluation of hydrocarbons degradation rate %

Different patterns were monitored, recorded and detected for Hydrocarbons degradation rate in liquid media and soil for Bacillus subtilis, Pseudomonas aeruginosa and Bacillus cereu, as shown by Fig. 6, Fig. 7, Fig. 8 and Table 4. Generally, degradation rate in polluted soil was superior compared with liquid media. Hydrocarbons degradation rate of Bacillus subtilis was increasing gradually by incubation time. Degradation rate % was 20, 38, 50, 52 and 25, 30, 55, 70 after 7, 14, 21 and 28 days of Hydrocarbons incubation with liquid media and contaminated soil respectively. Interestingly, incubation Hydrocarbons in liquid media for 14 days was superior for biodegradation rate % comparing with Hydrocarbons incubation for contaminated soil. Pseudomonas aeruginosa remarked with low Hydrocarbons degradation rate % for 14 and 21 incubation days comparing with Hydrocarbons degradation rate of Bacillus subtilis. Unique Hydrocarbons degradation rate distinguished Bacillus cereus with highest degradation rate % after 21dayes of incubation and sudden decrease after 28 days of incubation. Our findings of compared residual crude oil quantitatively with control sample were reflected varied biodegradation ability for different bacterial genera for varied incubation time (Mirdamadian et al., 2010).

Fig. 6

Degradation dendogram of petroleum hydrocarbons of 7, 14, 21 and 24 days for Bacillus subtilis in liquid medium and in polluted soil.

Fig. 7

Degradation dendogram of petroleum hydrocarbons of 7, 14, 21 and 24 days for Pseudomonas aeruginosa in liquid medium and in polluted soil.

Fig. 8

Degradation dendogram of petroleum hydrocarbons of 7, 14, 21 and 24 days for Bacillus cereus in liquid medium and in polluted soil.

Table 4

Degradation % of petroleum hydrocarbons of isolates Bacillus subtilis, Pseudomonas aeruginosa and Bacillus cereus in liquid medium and in polluted soil after 7, 14, 21 and 28 days of incubation.

Incubation days	Degrative samples	Degradation %
		Bacillus subtilis	Pseudomonas aeruginosa	Bacillus cereus
7	Liquid medium	20	15	19
	Polluted soil	25	16	25
14	Liquid medium	38	20	20
	Polluted soil	30	32	27
21	Liquid medium	50	30	55
	Polluted soil	55	38	68
28	Liquid medium	52	56	46
	Polluted soil	70	68	50

Conclusion

Three bacterial strains isolate from the contaminated soil of Khurais oil field Dhahran, Saudi Arabia were isolated and characterized for hydrocarbons degradation capability. Bacterial identification was carried out though morphological and biochemical methods and confirmed via

16S rDNA sequence fingerprinting method as Bacillus subtilis, Pseudomonas aeruginosa and Bacillus cereus. Distinguishable capability for hydrocarbons degradation was evaluated for three isolates which reflected varied degradation rate % patterns.