Assessment of the dibenzothiophene desulfurization potential of indigenously isolated bacterial consortium IQMJ-5: a different approach to safeguard the environment

Biodesulfurization is emerging as a valuable technology for the desulfurization of dibenzothiophene (DBT) and its alkylated substitutes, which are otherwise regarded as refractory to other physical and chemical desulfurizing techniques. The inability of the currently identified pure cultures and artificial microbial consortia due to lower desulfurization rate and product inhibition issues has compelled the researcher to look for an alternative solution. Thus, in the present study, an indigenously isolated microbial consortium was employed to tackle the desulfurization issue. Herein, we isolated several kinds of DBT desulfurizing natural microbial consortia from hydrocarbon-contaminated soil samples by conventional enrichment technique. The most effective desulfurizing microbial consortium was sequenced through illumine sequencing technique. Finally, the effect of the products of the desulfurizing pathway (such as 2-hydroxybiphenyl (2-HBP) and sulfate (SO4−2) was evaluated on the growth and desulfurization capability of the isolated consortium. The outcomes of Gibb’s assay analysis showed that six isolates followed the “4S” pathway and converted DBT to 2-HBP. Among the isolates, I5 showed maximum growth rate (1.078 g/L dry cell weight) and desulfurization activity (about 77% as indicated by HPLC analysis) and was considered for further in-depth experimentation. The analysis of 16S rRNA by high-throughput sequencing approach of the I5 isolate revealed five types of bacterial phyla including Proteobacteria, Bacteroidetes, Firmicutes, Patescibacteria, and Actinobacteria (in order of abundance). The isolate showed significant tolerance to the inhibitory effect of both 2-HBP and SO4−2 and maintained growth in the presence of even about 1.0 mM initial concentration of both products. This clearly suggests that the isolate can be an efficient candidate for future in-depth desulfurization studies of coal and other fossil fuels.


Introduction
Coal is an abundant and rich energy source of fossil fuels origin, that has been exploited in several industries. The primary use of coal is in electricity generation, fulfilling about 39% of global electricity demand (Vega et al. 2019). In addition, it is also used in several other industries and for household heating (Marks and Callaghan 2018). However, the combustion of an excessive volume of sulfur-containing coal is responsible for an increased discharge of harmful or toxic gases, especially SO 2 (Xu et al. 2019). Such sulfurcontaining toxic gases after release can cause health issues, acid rain, and other environmental problems including air pollution (Khan et al. 2022a).
To achieve sustainable growth in a civilized society, it is not a wise decision to burn fossil fuels and get industrial development while causing harm to the environment (Ghosh et al. 2015). Therefore, a level of sulfur lesser than 15 ppm has been demanded by environmental regulations (Mohebali and Ball 2016). To achieve such a goal, researchers all over the world are trying to reduce the fraction of sulfur in coal and other fossil fuels before it is utilized for different industrial purposes (Maass et al. 2015).
The most common method of eliminating sulfur from fossil fuels is hydrodesulfurization (HDS). During HDS, the sulfur in fossil fuels is reduced to hydrogen sulfide (H 2 S) in the presence of hydrogen gas, a metal catalyst like NiMo/ Al 2 O 3 or CoMo/Al 2 O 3, and at an elevated level of temperature (ranging from 200 to 425 ℃) and pressure (150-250 psi) (Soleimani et al. 2007). HDS remains successful in removing a large amount of inorganic sulfur and a small amount of organic sulfur from fossil fuels. However, this process finds it difficult to remove sulfur from recalcitrant heterocyclic sulfur-containing organic compounds such as dibenzothiophene (DBT) and its alkylated derivatives which constitute about 70% of the organic sulfur in fossil fuels (Martinez et al. 2015). Some other disadvantages of the process include the involvement of costly catalysts, the requirement of an extensive amount of hydrogen gas, and taking place at an elevated level of temperature and pressure conditions, ultimately making the process energy-intensive (Sadare et al. 2017). To achieve full-scale fossil fuels desulfurization, it is essential to have a method that must be cost-effective and capable of eliminating sulfur from recalcitrant compounds under ambient operating conditions (Etemadifar et al. 2014). Biodesulfurization (BDS) is a complementary and alternative technique to HDS. This process has the capability of removing sulfur from recalcitrant heterocyclic sulfur-containing organic compounds without damaging the carbon skeleton of the parent compounds (Magdy et al. 2015). The other advantages of the process are lower energy consumption, a lesser amount of sulfur emission, and the minimum generation of unwanted byproducts, mainly attributed to the capability of the biocatalyst (microbes or enzymes) (Khan et al. 2022b). DBT is used as a model sulfur-containing compound due to which the search for an efficient microbial strain has been continued (Al-Jailawi et al. 2015).
To obtain sulfur from DBT and its alkylated derivatives for energy purposes as well as for maintaining growth, bacteria have followed different types of biochemical pathways. The two most important pathways identified in different bacteria are the Kodama and the "4S" pathways (Mohebali et al. 2007;Bordoloi et al. 2016). The Kodama pathway is also called a degradative or ring-destructive pathway because in this pathway the bond between two carbon atoms is broken (Silva et al. 2018). The "4S" pathway is also called a sulfurspecific pathway in the sense, that the sulfur atom in DBT is released as sulfite while the carbon skeleton of DBT remains unchanged, and results in retaining the calorific value of fossil fuels.
Most of the earlier work on the desulfurization of polyaromatic sulfur heterocycles (PASH) was performed with either pure microbial culture or microbial consortium constructed artificially (Ghazali et al. 2004). However, pure culture or artificial microbial consortia are not the actual representers of the ongoing activities of microbes in the environment because the relationship between the new combined species is changed in an artificial microbial consortium (González et al. 2011). Similarly, pure culture is also assumed to be incapable of metabolizing different compounds in a mixture. In comparison, mixed microbial consortia with a broader substrate range, have the benefits of co-metabolism and synergic effect performing biodegradation in a way of commensalism and co-oxidation (Gojgic-Cvijovic et al. 2012).
Further, mixed microbial consortia have the advantage of containing different types of metabolic capabilities that can enhance the rate of BDS. Such bacterial consortia frequently grow in highly contaminated regions, where they are facing intense environmental conditions. This can further enhance their capability of degrading and tolerating different types of recalcitrant substances (Krishna and Philip 2008). Thus, such characteristics of combined and cooperative interaction of natural microbial consortia must be examined for an enhanced rate of desulfurization of DBT and fossil fuels (Ismail et al. 2016).
Based on the above-mentioned facts, the purpose of the study explained herein was to isolate and characterize an efficient DBT desulfurizing microbial consortium from hydrocarbon-contaminated soil samples by the traditional enrichment technique. In addition, the impact of the product of DBT desulfurization (2-HBP and SO 4 −2 ) on the sulfur metabolizing activities of the isolated consortium was also investigated.

Media and growth conditions
Sulfur-deficient minimal salt medium (MSM) was used for the isolation and enrichment of desulfurizing microorganisms. (Ashutosh et al. 2011). The stock solutions of each component of the MSM medium per liter of distilled water were separately prepared in such a way as phosphate buffer (86.6 g of Na 2 HPO 4 and 53 g of KH 2 PO 4 ), 53.491 g of NH 4 Cl, 203.3 g of MgCl 2 .6H 2 O, 44.1 g of CaCl 2 .2H 2 O, 5 g of yeast extract, and 180.15 g of anhydrous glucose. One liter of working media was prepared from stock solutions by adding 50 mL phosphate buffer, 30 mL glucose, 10 mL NH 4 Cl, 1 mL each of MgCl 2 .6H 2 O, CaCl 2 .2H 2 O, and yeast extract. A final concentration of 0.3 mM DBT from a stock of 100 mM (0.72g dissolved in 40 mL ethanol without sterilization) was added as the primary sulfur source. The final pH of the medium was adjusted to 7.2 with 1M solutions of either NaOH or H 2 SO 4 . The media were sterilized by autoclaving at 121 ℃ and 15 psi pressure for 15 min.

Isolation, screening, and enrichment of microorganisms
Different types of microbial isolates, capable of using DBT as a sulfur source were isolated from the hydrocarbon-contaminated soil samples. About 17 of the subsurface samples of soil were collected from various oil filling stations and mechanical workshops in different areas of Chaman (5 in total labeled as C1 to C5) and Islamabad (12 in total and labeled as I1-I12) Pakistan. Soil samples (One gram each) were inoculated in 250 mL flasks containing 100 mL of sterilized MSM medium. The suspensions were continuously stirred at 30 ℃ in a rotary shaker (Model 14000, IRMECO GmbH & co, Germany) with 100 r/min agitation speed. After 7 days, 1mL from incubated suspension was added as inoculum in fresh MSM containing DBT and incubated at the conditions mentioned above. Inoculation was repeated in a fresh MSM medium, until obtaining efficient microbial isolates capable of desulfurizing DBT, as indicated by the appearance of blue color after conducting Gibb's assay. The remaining culture media of the isolates exhibiting desulfurization activity were harvested by centrifugation at 10000 r/ min for 10 min. The pallets were washed twice with 0.1 M phosphate buffer and preserved in 50% glycerol for further use.

DBT desulfurization and screening of suitable microbial consortium
A shake flask experiment was conducted for screening and identifying the most efficient microbial isolate. The efficient isolates were screened by separately inoculating 0.5 mL of each of the glycerol-preserved cultures into 100 mL MSM medium and incubated at the conditions explained above. During the process of incubation, aliquots of suspension were regularly collected at about 24-h intervals for about 7 days to measure the desulfurization activity and growth rate.

DNA isolation and PCR amplification
The Cetyl trimethyl ammonium bromide (CTAB) method was employed for the isolation of whole metagenomic DNA and was confirmed by running on 1% agarose gel. The 16S rRNA genes of the isolated DNA were PCR amplified with bacterial universal 1492R reverse (TAC GGY TAC CTT GTT ACG ACTT) and 27F forward primers (AGA GTT TGATCMTGG CTC AG) (Mao et al. 2012). The bacterial primers were used based on visual observation of the solid and liquid cultures. The composition of the 20 µL PCR blend was such that: each primer concentration (1.32 µM), DNA polymerase (0.5 U), deoxynucleoside triphosphate (0.2 µM), PCR buffer (5 µL), total DNA (1 µL), and rest of the volume was fulfilled by PCR water. The reaction conditions of PCR were initial denaturation step at 94 ℃ for 10 min (extended due to utilization of whole bacterial DNA as templates) with subsequent 35 cycles of denaturation for 1 min at 94 ℃, annealing for 1 min at 60 ℃, an extension step for 2 min at 72 ℃, and a concluding extension at 72 ℃ for 10 min was performed.

Statistical and bioinformatics analysis
Metagenomic 16S rRNA sequencing datasets were submitted under accession number KFIT00000000 to NCBI DDBJ/EMBL/GenBank (Yergeau et al. 2012). Based on the metagenomic analysis, the consortium was named bacterial consortium IQMJ-5. Qiime 2 v 2021.4 software was used for processing raw paired-end data that was imported from the original DNA segments. Initially, pair-end reads for all the samples were linked with the fastq-join technique having a lower possible permissible overlap of 120 bp and 15% higher possible permissible variation in the overlap. Next, reads that contain over three successive base calls with a Phred score <20 were cut, reads having unclear base calls were thrown away and various sample reads were marked with sample identifiers using the VSEARCH instrument and linked into an individual FASTA file. The reference database for 16S rRNA was SILVA (https:// www. arb-silva. de/ downl oad/ archi ve/ qiime). The classifier was identified on the 97% resemble operational taxonomic units (OTUs) using reference databases like q2-feature classifier and Naïve Bayes classifier plugin. The diversity (composition) of the bacterial community was measured with alpha metrics (Shannon diversity index). Bacterial communities were presented at the phylum and genus levels with the help of bar charts. Alpha diversity was detected by performing Shannon Diversity Index at both the phylum and genus levels.

Effect of final products on the growth and desulfurization activity
To determine the inhibitory effect of the final products of the desulfurization reaction (such as 2-HBP and SO 4 −2 ), MSM was supplemented with an increasing amount of 2-HBP (ranging from 0.1 to 1 mM) and sodium sulfate (ranging from 0.1 to 1 mM). A control containing only 0.3 mM of DBT dissolved in the culture medium was run in parallel.

Analytical determination
The growth of the isolated microbial consortia was measured by optical density (OD) at 600 nm with Analytik Jena UV visible spectrophotometer (SPECORD 200 PLUS, Germany). The concentration of the dry cell weight (DCW) was determined from a standard curve made between DCW and obtained OD readings. Triplicate runs were performed for each experiment. In addition, the phenolic product (2-HBP) of the DBT desulfurization pathway was detected by performing Gibb's assay (Oldfield et al. 1997). Gibb's reagent (2,6 -dichloroquinone-4-chloroimide) reacts with 2-HBP and forms a blue-color complex. In short, culture media of 1.5 mL was taken in a 2 mL Eppendorf tube and centrifuged for 10 min at 10000 rpm. The pH of the supernatant was adjusted to 8 with the addition of 33 μL of 1M Na 2 CO 3 . 10 µL of Gibb's reagent (50 mg/L in absolute ethanol) were inoculated into the alkaline supernatant and incubated at room temperature for 30 min. Following incubation, the positive reaction developed blue color and was examined spectrophotometrically at A610 nm. The result obtained was compared with the standard curve made with authentic 2-HBP. Similarly, DBT and 2-HBP in the reaction mixture were further identified and quantified by High-Performance Liquid Chromatography (HPLC) (Agilent, USA) equipped with a C18 column. The mobile phase consisted of acetonitrile and water (60/40) with a flow rate of 1mL/min. The injection volume was kept at 40 μL and peaks were detected with a UV detector at 280 nm. Briefly, 20 mL of eight-dayold culture media were centrifuged, and the supernatant was acidified to pH 2.2 with 4M HCL. The acidified supernatant was extracted twice with an equal volume of ethyl acetate. The top layer containing acidic metabolites was concentrated by a rotary evaporator and the residual moisture was removed by the addition of 1 g/5mL Na 2 SO 4 and was next filtered through a 0.2 μm syringe filter. The substrate and product identification and quantification were carried out by matching their retention time and peak length with that of purified reference standards of DBT and 2-HBP.

Isolation and screening of desulfurization microorganisms
All the results explained in the result section were operationally defined. The standard Gibb's assay revealed that six (6) of the isolated microbial consortia (Fig. 1) showed desulfurization of DBT into 2-HBP or other phenolic compounds, which was evident from the appearance of the blue color in the reaction mixture. The isolates were repeatedly tested in MSM media containing DBT and ensured desulfurization efficiency.

Selection of the most efficient desulfurizing isolate
The results in Fig. 1a revealed that isolate I5 exhibited 1.078 g/L DCW, which was the highest relative to the other isolates. Similarly, the produced 2-HBP (0.2093 mM) by isolate I5 was also the highest (Fig. 1b). Therefore, based on the obtained results, isolate I5 was chosen for further in-depth characterization and desulfurization experimentation.
The capabilities of 6 microbial isolates using DBT as the only bioavailable sulfur source, were further screened by HPLC analysis as shown in Fig. 2. The results of the analysis proved that all the microbial isolates were capable of desulfurizing DBT to some extent. However, the maximum DBT desulfurization was detected for the I5 isolate, which converted about 77.68% DBT to 2-HBP.  (1051 in number and 1%) and Actinobacteria (512 in number and 0.65%). The Shannon diversity index has shown a value of 1.1519 which indicated that the consortium was highly diverse at the phylum level (Table 1).

Characterization of the isolate on the molecular basis
Two of the phyla such as Bacteroidetes and Patescibacteria are yet not reported for any kind of desulfurizing activity. At the genus level (Fig. 3 right), the phylogenetic relatives (based on the increasing number) in the sample were such that: Empedobacter, Raoultella, Stenotrophomonas, Enterobacter, Bacillus, Pseudomonas, Uncultured cyanobacterium,  Ochrobactrum, Allorhizobium, Achromobacter, Uncultured sphingobium, Rhodococcus, Microbacterium, and other genera. Seven of the genera (followed by a star in Table 2) are not yet reported for any kind of desulfurization activity. The Shannon diversity index indicated that the consortium is less diverse at the genus level.

Effect of initial concentration of 2-HBP
The results in Fig. 4 showed the effect of various concentrations of 2-HBP on the growth of the isolated bacterial consortium IQMJ-5. A gradual decrease in the cell growth of the bacterial consortium was observed with an increase in 2-HBP concentration. The percentage reduction of growth with the increasing initial concentration of 2-HBP in relation to control was found to be 97.35%, 75.76%, 54.55%, and 11.36% at 0.1 mM, 0.2 mM, 0.3 mM, and 1.0 mM 2-HBP, respectively.

Effect of initial concentration of SO 4 −2
The effect of an exogenous source of SO 4 −2 on both the growth and desulfurization rate of the IQMJ-5 consortium has been shown in Fig. 5. The results indicated that the consortium showed a concentration-dependent increase in growth with the addition of SO 4 −2 (Fig. 5a). On the other hand, the desulfurization activity was decreased when the concentration of SO 4 −2 was increased (Fig. 5b). The activity progressively decreased with the increase in SO 4 −2 concentration, though not completely retarded even at about 1 mM initial concentration of SO 4 −2 . Discussion BDS has been emerging as an efficient approach for removing unwanted sulfur contents from heterocyclic sulfurcontaining compounds. The process recently has gained ample scientific and technological attention owing to its environmental significance, particularly the reduction in SO 2 emission. An important criterion for the application of the desulfurization process in the coal industry has been the appropriate bacterial candidates with efficient metabolic activities.
In the present study, different types of natural microbial consortia were obtained from hydrocarbon-contaminated soil samples by employing the culture enrichment techniques using DBT as the only bioavailable source of sulfur. Six of the naturally isolated microbial consortia exhibited blue color after Gibb's assay analysis. This result confirmed the desulfurization of DBT by the "4S" route, an indication of the conservation of the carbon chain. Previously, Akhtar et al. (2009) obtained about 110 bacterial isolates after screening 17 different types of samples. Among them, only a single isolate, identified as Rhodococcus spp. Eu-32 has shown desulfurization activity. Nassar et al. (2013) isolated about 28 desulfurizing strains from an artificially contaminated Coke sample with a high sulfur content of about 3.8%. The highest desulfurization rate of about 60% was shown by an isolate later identified as Brevibacillu invocatus C19.
Gibb's assay is considered a non-reliable technique because it sometimes gives false-negative outcomes resulting from the production of phenolic compounds other than 2-HBP and the detoxification and transformation of 2-HBP by microbial action. Papizadeh et al. (2011) showed that the false-negative results of Gibb's assay were caused by the detoxification and methylation of 2-HBP. Thus, the results of Gibb's assay were further confirmed by a secondary screening technique called HPLC. The results of the HPLC analysis confirmed the desulfurization capabilities of the consortia after comparing their retention time with that of standards. Among the six types of natural microbial consortia, the desulfurization performed by the I5 consortium was comparatively better than others both in terms of concentration and time taken. The consortium was named IQMJ-5 based on sample collection locality, institution, department, name of researcher, and sequence of samples collected.
Previously, Papizadeh et al. (2017) obtained about 64% desulfurization of 0.8 mM DBT with the bacterial isolate Enterobacter sp. NISOC-03. Raheb et al. (2010) conducted Gibb's assay only for the detection of BDS capability of the recombinant Pseudomonas aeruginosa ATCC9027 strain. Ashutosh et al. (2011) detected about 60% desulfurization of DBT with the bacterium Lysinibacillus sphaericus DMT7 strain after incubating for 15 days. Al-Jailawi et al. (2015) have used Pseudomonas aeruginosa strains (M9, M19, S25) in their desulfurizing studies. All the strains exhibited a decreased desulfurization activity when the concentration of DBT was increased. In yet another study with the thermophilic bacterial strain Klebsiella sp. 13T, about 22-53% of desulfurization activity was obtained (Bhatia and Sharma 2012).
However, consortium IQMJ-5 in the present study exhibited about 77.68% (in HPLC results) production rate of 2-HBP, indicating an increased rate of desulfurization in terms of both concentration and time taken in relation to the above studies. The increased desulfurization activities can be attributed to the synergistic effect among members of the consortium. This aspect is probably associated with the better adoption of the consortia toward higher concentrations of DBT. Since this bacterial consortium was isolated from the hydrocarbon-contaminated soil sample, therefore, it has established pathways for the degradation of complex hydrocarbons like DBT under ambient environmental conditions. Due to the better adoption of this consortium to complex hydrocarbons, it showed better growth and desulfurization activity as compared to the already available reports.
The products of the DBT desulfurizing "4S" pathway such as 2-HBP and SO 4 −2 are reported to have an inhibitory effect on the growth of BDS microorganisms (Ansari et al. 2007). 2-HBP is commonly regarded as a biocide and its presence in the medium has an inhibitory effect on the growth and so on desulfurizing activities ). In the present work, a similar kind of inhibitory effect was observed in the growth of the consortium IQMJ-5, as reported in the past with that of the Rhodococcus erythropolis IGTS8 strain (Chen et al. 2008). However, contrary to the aforementioned studies, the consortium retained growth even at about 1 mM concentration of 2-HBP. Previously, Chen et al. (2019) incubated Gordonia sp. SC-10 in a sulfurfree medium supplemented with different 2-HBP concentrations experience complete hindrance of growth at about 0.5 mM 2-HBP. In another study, Magdy et al. (2015) provided different concentrations (from 0 to 1.0 mM) of 2-HBP to novel bacterial species such as Rhodococcus sp. SA11, Stenotrophomonas sp. SA21, and Rhodococcus sp. SA31 and experienced complete retardation of desulfurization activity at about 1.0 mM concentration. Akhtar et al. (2018) have inoculated Gordonia sp. HS126-4N in MG medium containing an initial concentration of 2-HBP ranging from 0.1 to 1.0 mM. The bacterium showed comparatively better growth at a lower concentration of 2-HBP, but the growth was entirely absent in the medium supplemented with 1.0 mM 2-HBP concentration.
To the best of our knowledge, there is no previous report regarding the stability of bacterial strains toward such a high concentration of 2-HBP. The bacterial consortium used in the present study showed significant growth at 1 mM concentration, which indicates their better adaptation and metabolic fitness for the BDS of 2-BHP. In addition, in the presence of complex hydrocarbon substrates, microorganisms develop very effective membrane transportation systems for a wide variety of hydrocarbon substances. Therefore, it can be suggested that the complex substrates enable bacteria to manipulate their membrane transport system for the effective transport of 2-HBP. However, a detailed investigation in this area would be helpful for better membrane adaptation and triggered metabolic efficiencies of our used bacterial isolate.
Like 2-HBP, the other product of the "4S" pathway such as SO 4 −2 also has an inhibitory effect on the desulfurization activity of the isolates (a characteristic of the "4S" pathway) (Alves et al. 2005). In this study, the presence of an exogenous source of inorganic SO 4 −2 has resulted in the increased growth of the bacterial consortium. On the other hand, the presence of the SO 4 −2 source triggered a concentration-dependent reduction in the desulfurization activity of the consortium. However, the bacterial consortium showed some desulfurization activity even at about 1.0 mM initial concentration of Na 2 SO 4 although at a very low level. Prayuenyong (2001) experienced a similar behavior of growth and desulfurization activity when the medium was supplemented with different concentrations of SO 4 −2 . Bhatia and Sharma (2010) also experienced inhibition of growth and 2-HBP production of the isolate Pantoea agglomerans D23W3 in the medium supplemented with an external source of SO 4 −2 .

Conclusion
In the present research work, bacterial consortium IQMJ-5 capable of performing desulfurization of DBT was isolated and characterized. The consortium IQMJ-5 carried out about 77.68% of the desulfurization of DBT into 2-HBP as revealed by the results of the HPLC chromatogram. The appearance of 2-HBP in the results of HPLC analysis and Gibb's assay confirmed that the I5 isolate has followed the "4S" pathway of desulfurization. This confirmed the cleavage of the bond between carbon and sulfur atom which is an indication of the preservation of the fuel calorific value. Furthermore, the IQMJ-5 consortium showed growth and desulfurization activity even when the medium was supplemented with a 1.0 mM initial concentration of 2-HBP and Na 2 SO 4 . Such capabilities may have resulted from the cometabolic and synergetic approaches of different species in the consortium. Therefore, it is assumed that in the future, the consortium IQMJ-5 could be an efficient candidate for enhancing the quality of coal and other fossil fuels.