Biodegradation of Monoaromatic hydrocarbons Toluene and Xylene through native bacterial strain Bacillus subtilis RD20

doi:10.21203/rs.3.rs-1974378/v2

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Biodegradation of Monoaromatic hydrocarbons Toluene and Xylene through native bacterial strain Bacillus subtilis RD20

https://doi.org/10.21203/rs.3.rs-1974378/v2

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A bacterial strain designated as RD20 isolated from oil contaminated soil and identified as Bacillus subtilis through biochemical tests and 16S rDNA sequence analysis, have the ability to degrade toluene and xylene, the mono-aromatic hydrocarbons of petroleum contaminant. FTIR (Fourier Transform Infrared) analysis showed characteristic peaks, that reveals that the bacterial strain was able to degrade toluene and xylene efficiently within 7 days of incubation periods. Intermediate metabolites of toluene degradation in treated culture media were detected as benzeneacetic acid, acetic acid, 2-Propanol, 1,3-benzenediol, pivalic acid, terephthalic acid, hexanoic acid, pentane, propiolic acid, cyclopentane-1,2-diol, hexanedioic acid and propionic acid through GC-MS (Gas Chromatography and Mass Spectrometry). Whereas Xylene degradation products detected through GC-MS were 4,4-dimethyl-2-pentanol, propane, cyclopropane, hexane, acetic acid, benzyl alcohol, formic acid, propane, ethane, cyclobutane carboxylic acid, phenol, propanedioic acid, pentane, phthalic acid and succinic acid, respectively, which further confirms the biodegradation of the monoaromatic hydrocarbons. This bacteria is thus, suitable for application in bioremediation process in hydrocarbon contaminated sites.

Bacillus subtilis

Biodegradation

FT-IR

GC-MS

metabolites

Many volatile organic compounds (VOCs) are released to the environment through many industrial discharges, accidental spills and gasoline leakages which have resulted in environmental pollution and also groundwater contamination. Among the VOCs, aromatic VOCs are considered major environmental pollutants due to its high toxicity. Monoaromatic hydrocarbons such as toluene and xylene are used as industrial solvents and also they provide the starting materials for the production of pharmaceuticals, agrochemicals, polymers, explosives and many other everyday products (Smith 1990). Toluene and xylene are ubiquitous pollutants (Gibson and Subramanian, 1984) which affects all kinds of living organisms directly or indirectly. The removal of such VOCs from the environment is very much necessary so as to make the environment in which we live in sustainable for all living organisms.

Bioremediation is one such relatively efficient, economical, versatile and eco-friendly method that could successfully overcome these problems. Bioremediation uses microorganisms such as bacteria, fungi, etc to degrade or detoxify such compounds by means of microbial bio-geochemical processes, and returning the environment to its original condition. It is an emerging strategy for removing pollutants from the environment.

Hazards of the monoaromatics are very well documented but a thorough evaluation for the substitutes of the monoaromatics are yet to be explored. Many toluene and xylene degrading bacteria are isolated from diverse aerobic and anaerobic environments which has led to extensive studies on the metabolism of the degrading microorganisms. In this study, a strain Bacillus subtilis RD20 that could efficiently degrade toluene and xylene was isolated from the oil contaminated soil of Guwahati Refinery, Assam, India. Several intermediates of toluene and xylene are presented which shows ring cleavage of the monoaromatics and its transformation into less toxic intermediate metabolites.

Chemicals

All chemicals are of analytical grade, purchased from Himedia (India) and Sisco Research Laboratory Pvt. Ltd., Mumbai.

Enrichment and isolation

For isolation of toluene and xylene degrading bacteria, Bushnell Hass medium, BHM (MgSO₄.7H₂O 0.2g, CaCl₂.2H₂O 0.02g, KH2PO₄.2H₂O 1.0g, K₂HPO₄.2H₂O 1.0g, NH₄NO₃ 1.0g, FeCl3 0.05g per litre) was prepared, supplemented each with 1% toluene (v/v) and 1% xylene separately as sole carbon source. The aromatic hydrocarbons were subjected to membrane filtration before adding to the medium. Serial dilution plate technique (Johnson and Curl, 1972) was used to obtained bacterial isolates from oil contaminated soil. 10 serially diluted pre-treated soil suspensions were mixed with 15 ml of each selective isolation medium (BHM) with 1.5% agar in sterile petridishes. The plates were incubated at 35ºC for 7 days.

Screening of toluene and xylene tolerating bacteria

For screening the potentiality of the bacterial isolates to degrade toluene and xylene, 1 ml of each bacterium from mother culture prepared from each isolates was inoculated in 250ml Erlmeyer flask containing 100ml of sterilized BHM supplemented with 1% (v/v) toluene and xylene individually. All the cultures were incubated at 35˚C for 7 days. Growth of all the isolates in the enriched medium were estimated by optical density reading (O.D₆₀₀) at 0^th and 7^th day by UV-VIS Spectrophotometer.

The bacterial isolates capable of showing the maximum growth was selected and further studied for biodegradation of toluene and xylene respectively.

Characterization of bacterial isolates

The bacterial isolate was characterized based on the morphological and biochemical attributes using Cappuccino and Sherman (2014) and Bergey’s Manual of Systematic Bacteriology.

Morphological and microscopic attributes of the isolate was tested using Gram stain (Claus, 1992). Scanning Electron Microscopy (SEM) was used to study the morphological characteristics of the isolate. The biochemical traits studied for characterization of the isolate was: Indole test, Methyl Red test, Voges-Proskauer test, Catalase test, Oxidase test, Starch hydrolysis, Gelatin liquefiction, Citrate utilization, Carbohydrate fermentation test, Nitrate reduction test, Urease test, Casein hydrolysis and H₂S production test.

Identification of bacterial strain through 16SrDNA sequence analysis

DNA was isolated from the culture and its quality was evaluated on 1.0% Agarose Gel where a single band of high-molecular weight DNA was observed. Fragment of 16S rDNA gene was amplified by 27F and 1492R primers. The PCR amplicon observed was purified to remove contaminants. Forward and reverse DNA sequencing reaction of PCR amplicon was carried out with forward primer and reverse primer using BDT v3.1 Cycle sequencing kit on ABI 3730xl Genetic Analyzer. Consensus sequence of 16S rDNA gene was then generated from forward and reverse sequence data using aligner software.

Biodegradation study

Evaluation of biodegradation was done by inoculating the bacterial strain (CFU 3x10⁶) in 250 ml Erlenmeyer flasks containing 100ml Bushnell Hass broth with 1% toluene and 1% xylene separately as a sole carbon source. It was then incubated at 35^ºC for 7 days. Growth of the bacterial strain in the medium was monitored periodically by measuring the optical density at 600 nm in UV visible spectrophotometer (Agilent Carry-60 UV-visible spectrophotometer). Culture media inoculated with bacterial suspension without supplementation of hydrocarbon was treated as control.

Analytical procedures

Intermediate metabolites of toluene and xylene degradation were identified and quantified through FT-IR and GC-MS analysis. For this, 7 days old treated cultures were centrifuged at 15000 rpm for 15 minutes. The supernatant was extracted twice in 25 ml chloroform separately for each toluene and xylene treated cultures. It was then filtered through Whatman No.42 filter paper. The filtrate was then allowed to dry completely in a dessicator until further analysis.

For FT-IR analysis of the metabolites, a small amount of KBr was used to mix with the dried sample and from it, pellets of 0.26 mm thickness were made to analyze the FT-IR spectra of the intermediate metabolites. Culture broth supplemented with toluene and xylene without inoculation of bacteria which was treated as control was also centrifuged, extracted, dried and pellets with KBr were made for analysis.

As for GC-MS analysis, 3 ml chloroform was added in the completely dried extracts, mixed well and then transferred in a glass vile until analysis was done. For GC-MS analysis, the conditions were, carrier gas: He; EB-5MS column (30 m x 0.25 mm x 0.25 mm); injector temperature: 250ºC; initial oven temperature: 40ºC with 1 min hold time, 120ºC with 1 min hold time and then 280ºc with 5 min hold time; sample amount 1µl in splitless mode with column flow of 1.11 mL/min.

Isolation of the bacteria

A total of 21 bacterial isolates were obtained from the oil contaminated soil, of which four isolates exhibited maximum growth in the enriched medium. Furthermore, the isolate, labeled as RD20 showed best growth in the enrichment culture, incorporated with toluene and xylene. Therefore, the strain RD20 was selected for further biodegradation studies.

Characterization of Bacterial strain:

The biochemical and morphological study revealed that, the isolate, RD20 was Gram positive, rod-shaped and motile. SEM analysis confirmed the short rod-shaped structure of the isolate (Fig 1 and 2). It showed positive test for Catalase, Oxidase, Voges-Proskauer, nitrate reduction and was able to ferment carbohydrates such as glucose, sucrose, lactose and fructose. It was able to hydrolysis starch and gelatin. However, the bacterial strain was variable for Galactose test. It showed negative test for Methyl Red, Indole, Citrate, Galactose, Lactose, hydrogen sulphide and Urease (Table 1).

Bacterial identification by 16S rDNA sequence analysis

PCR amplicon band of 1500 base pairs was observed after amplification of the fragment of 16S rDNA (Fig.2). The 16S rDNA gene sequence was used to carry out BLAST with the database of NCBI genebank database. Based on maximum identity score first twenty sequences were selected and aligned using multiple alignment software program Clustal W. Distance matrix was generated and the phylogenetic tree was constructed using MEGA 7 (Fig.3).

The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model (Kimura 1980). The bootstrap consensus tree inferred from 1000 replicates (Felsenstein 1985) is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches (Felsenstein 1985).

From the 16S rDNA sequence analysis, the isolated bacterial strain RD20 has been identified as Bacillus subtilis based on nucleotide homology and phylogenetic analysis. The sequence has been submitted to the Genebank of NCBI with accession no. ON994905 for global retrieval (https://www.ncbi.nlm.nih.gov/portal/utils/pageresolver.fcgi?log$=activity&recordid=62edea6d9aae413c1e3278de&absref=https://www.ncbi.nlm.nih.gov)

Biodegradation of Toluene and Xylene through Bacillus subtilis RD20

The growth profile of the bacterial strain Bacillus subtilis RD20 in enriched medium containing respective monoaromatic hydrocarbons as determined by the O.D reading of the culture medium at 600nm after 7 days of incubation is shown in the table 2 and 3. From the readings, it is observed that in 7 days, in both the treated cultures, the isolate RD20 exhibited maximum growth signifying utilization of the hydrocarbons. Also, there was gradual increase in turbidity in 7 days in both the culture that suggested degradation of hydrocarbons.

FT-IR analysis

The FT-IR analysis of toluene biodegradation was done for both treated and control. The characteristic peaks formed in both the spectra were observed and compared (Fig 4 and 5). As FT-IR analysis is a preliminary investigation to interpret the functional groups, in our study it was observed that the peaks found in control were absent in treated depicting degradation of the monoaromatic compound (table 4). In the FT-IR analysis of xylene biodegradation also it was noted that the peaks exhibited in the control were absent in treated suggesting the conversion of the monoaromatic into different intermediate metabolites (table 5).

GC-MS analysis of toluene and xylene degradation:

Intermediates detected during toluene biodegradation:

The important intermediate metabolites of toluene detected through GC-MS analysis reveals the presence of benzeneacetic acid, acetic acid, 2-Propanol, 1,3-benzenediol, pivalic acid, terephthalic acid, hexanoic acid, pentane, propiolic acid, cyclopentane-1,2-diol, hexanedioic acid, propionic acid and ethanol (table 6). The GC-MS chromatogram of toluene biodegradation is shown in Fig.8.

Intermediates detected during xylene biodegradation:

The main xylene degradative metabolites detected through GC-MS analysis are 4,4-dimethyl-2-pentanol, propane, cyclopropan, hexane, acetic acid, benzyl alcohol, formic acid, propane, ethane, cyclobutane carboxylic acid, phenol, propanedioic acid, pentane, phthalic acid and succinic acid (table 7). The GC-MS chromatogram of xylene biodegradation is shown in Fig.9.

The present study deals with the degradation of mono-aromatic hydrocarbons; toluene and xylene by Bacillus subtilis RD20. Microbial enzymes transform aromatic compounds into ortho or para dihydroxy-phenol derivatives, followed by ring cleavage to produce aliphatic acids (Dutton and Evans, 1969). Muccee et al. (2019) detected cyclohexene, cyclohexane, cyclohexanone and cyclohexanol in the culture of Bacillus paralicheniformis strain KJ-16 and Brevibacillus agri strain NBRC 15538 as biodegradable metabolites of toluene. In our study we have detected benzene and phenol as initial metabolites of toluene. The metabolite benzene is produced through addition of a methyl group in 2, 3 or 4 position of toluene whereas hydroxylation of benzene at 1 and 2 or 1 and 3 positions further converted into o-phenol or p-phenol. Further conversion of phenol, generates certain low molecular weight aliphatic acids such as, acetic acid, propionic acid and oxalic acid through ring cleavage which are the intermediates formed in TCA cycle, also reported by a number of authors (Shields et al.1989; El-Naas et al, 2014). The intermediate metabolites such as Cyclopentane, Cyclohexane, Cyclopropanone, Cyclopentanol and Cyclopentane-1,2-diol, were also detected in GC-MS study; however, the metabolite cyclohexene as reported by Dutton and Evans (1969) was not detected in our study. The enzyme involved in this degradation pathway were cyclohexane monoxygenase, cyclohexanol dehydrogenase and cyclohexanone monoxygenase (Muccee et al., 2019). Ring cleavage is considered as major criteria for biodegradation of aromatic compound (Smith, 1990). Toluene, a mono-alkyl benzene is biodegraded through both ring cleavage methylation and hydroxylation. Oxidation of the methyl group of toluene leads to the formation of Benzoic acid which shows normal biodegradation routes of toluene (Smith, 1990). Benzoic acid (1,3-Benzenediol), an oxidation product of phthalic acid and benezeneacetic acid has also been detected in our study. Conversion of the aromatic hydrocarbon into catechol followed by catechol ring cleavage has been reported by many authors (Ranya et al, 2008; Yu et al 2001; Zhang et al, 2013; Parales et al, 2008). Though catechol, an intermidiate metabolite of toluene biodegradation was not detected in our study, however, carboxylic acids such as Pivalic acid, propionic acids, pentanoic acids, hexanoic acids and hexanedioic acids were obtained. This might be due to further degradation of catecholic products by ring cleavage. The reaction products can be easily converted to tricarboxylic acid cycle (TCA cycle) intermediates, which are further broken down into CO₂ and water (Madigan et al. 2000; Rittmann & McCarty 2001).

Xylene, a di-alkyl benzene is biodegraded by initial oxidation of one of the methyl groups to the corresponding methyl benzylalcohols, tolualdehydes, toluic acids and methyl catechol (Davey & Gibson 1974). Benzenemethanol (benzylalcohol) has been detected in our study which is due to the oxidation of one methyl group on xylene (Assinder and Williams, 1990). The metabolites detected in this study such as 1,2,4-Benzenetricarboxylic acid, 1,3-Cyclopentanedicarboxylic acid and Cyclobutane carboxylic acid also suggested further oxidation of methylbenzyl alcohol. Phenol and Benzene are also reported in GC-MS analysis indicating the initial step of degradation of xylene. Catechol as major intermediate during degradation of xylene has been reported by many authors (Assinder and Williams, 1990; Choi et al., 2012). Subsequently catechol intermediates are mineralized via various pathways through aromatic ring cleavage. (Ramos et al. 1987; Mazzeo et al. 2010). Intermediate compounds like Phthalic acid along with Propanedioic acid (malonic acid), 2-Propanone and Butanone were also detected in this study. Aliphatic compounds like ethane, propane and pentane are another metabolites also reported in our study that suggest the conversion of toxic aromatic compound to less toxic metabolites. Organic acids of low molecular weight compounds such as succinic acid, formic acid and acetic acid as detected in our study are the catabolic intermediate of xylene degradation pathway via Krebs cycle (Gibson et al.,1974; Barbieri et al.,1992; Choi et al.,2012; Brammuci et al., 2002; Wang et al.,2015).

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Table. 1 Biochemical characterization of the isolate RD20

Sl. Nos.	Biochemical Test	RD20
1	Gram Staining	+ve
2	Catalase	+ve
3	Oxidase	-ve
4	IMViC Test
	i) Indole production test	-ve
	ii) Methyl-Red	-ve
	iii) Voger -Proskauer test	+ve
	iv) Citrate Utilization test	+ve
5	Carbohydrate Fermentation test - (1% w/v)
	i) Glucose	+ve(acid)
	ii) Fructose	+ve
	iii) Sucrose	+ve(acid)
	iv) Lactose	+ve(gas)
	v) Galactose	variable
6	Nitrate Reduction test	+ve
7	Urease test	-ve
8	Casein Hydrolysis test	+ve
9	Starch Hydrolysis test	+ve
10	Gelatin liquification test	+ve
11	H2S Production test	-ve

Table 2: Growth (OD₆₀₀) of the bacterial isolate RD20 in Bushnell Hass Broth supplemented with 100µl toluene

Time (Hrs)	100µl toluene treated with RD20	Control 2 (Media + RD20)
24	0.017	0.002
48	0.051	0.002
72	0.091	0.002
96	0.193	0.002
120	0.286	0.002
144	0.499	0.002
168	0.562	0.002

Table 3: Growth (OD₆₀₀) of the bacterial isolate RD20 in Bushnell Hass Broth supplemented with 100µl xylene

Time (Hrs)	100µl xylene treated with RD20	Control 2 (Media + RD20)
24	0.015	0.002
48	0.087	0.002
72	0.155	0.002
96	0.237	0.002
120	0.352	0.002
144	0.478	0.002
168	0.519	0.002

Table 4. Analysis of the peaks shown by FTIR for toluene treated with RD20

Control (Toluene)		RD20 Treated (Toluene)
Wavelength (cm^-1)	Assignment of peaks	Wavelength (cm^-1)	Assignment of peaks
3026	C-H stretch (aromatic)	2925	C-H stretch
2920	C-H stretch (alkyl)	2847	O-H stretch (partial obscure C-H stretching bands)
1604	C=C stretch (aromatic)	1659	C=O stretch
1495	C-C stretch in the aromatic ring	1461	C-O-H bending

Table 5. Analysis of the peaks shown by FTIR for xylene treated with RD20

Control (Xylene)		RD20 Treated (Xylene)
Wavelength (cm^-1)	Assignment of peaks	Wavelength (cm^-1)	Assignment of peaks
3063.68	=C-H stretch (aromatic)	3451	O-H (H-bonded), usually broad
2965.15	C-H stretch (alkyl)	2920	C-H stretch
1604.96	C=C (aromatic)	2850	C-H bands
1453.65	C-C stretch (in ring)	1733	C=O (saturated aldehyde)
1030.47	C-H bends (in plane)	1650	C=C stretch
903.63	C-H bend (out of plane)	1464	CH₂ bending
-	-	914	C-H (Out of plane bending)

Table 6. Intermediate metabolites detected during GC-MS analysis of toluene biodegradation through Bacillus subtilis RD20

Retention time	Metabolites detected
7.099	Benzeneacetic acid (toluic acid)
7.670, 11.552, 20.606, 28.637, 42.155	Acetic acid
7.985	Butanoic acid
8.022	Ethane
10.153, 15.140, 27.449	2-Propanol
10.290	Cyclopentane
16.647	3-Pentenoic acid
17.523	1,3-Benzenediol
21.240	Pivalic acid
21.721	Cyclopentane
22.900	4-Dodecene
24.610	Hexadecan-1-one
26.309, 26.395	Phenol
27.250	Benzene
27.689	Acetic acid
28.513	Octane
28.780	Terephthalic acid
29.655	Hexanoic acid
31.275	Pentane
32.462	Cyclopentanol
32.530, 32.202	Oxalic acid
33.328	1,6-Hexanediol
34.520, 36.500, 48.801	Phthalic acid
34.950	Pentanoic acid
35.060	2-Propenoic acid
36.066	Cyclopropanone
40.707	Cyclopentane-1,2-diol
40.925	Cyclohexane
43.905	Hexanedioic acid
44.451	Propionic acid
45.804	Ethanol
46.958	Pentane
50.670	Propane

Table 7. Intermediate metabolites detected during GC-MS analysis of Xylene biodegradation through Bacillus subtilis RD20

Retention time	Metabolites detected
5.319	4,4-Dimethyl-2-pentanol
5.620, 15.460, 15.795	Propane
7.412	Cyclopropane
10.745	Acetic acid
12.749, 14.923, 15.154, 17.221	Benzenemethanol (Benzyl alcohol)
13.305	2-Propanone
15.396	Formic acid
16.515	Ethane
16.746	Cyclobutanecarboxylic acid
19.935	Benzene
20.751	Phenol
26.826	Butanone
38.110	Propanedioic acid
41.807	1,3-Cyclopentane carboxylic acid
43.707	Pentane
46.948	Phthalic acid
50.421	1,2,4-Benzenetricarboxylic acid
54.770	Succinic acid

No competing interests reported.

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Biodegradation of Monoaromatic hydrocarbons Toluene and Xylene through native bacterial strain Bacillus subtilis RD20

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