Isolation and characterization of a novel hydrocarbonoclastic and biosurfactant producing bacterial strain: Fictibacillus phosphorivorans RP3

Abstract

In this study, an indigenous novel hydrocarbonoclastic (kerosene and diesel degrading) and biosurfactant producing strain Fictibacillus phosphorivorans RP3 was identified. The characteristics of bacterial strain were ascertained through its unique morphological and biochemical attributes, 16S rRNA sequencing, and phylogenetic analysis. The degradation of hydrocarbons by F. phosphorivorans RP3 was observed at Day 7, Day 10 and Day 14 of the experimental duration. GC-FID chromatograms demonstrated a significant increase in hydrocarbon degradation (%) with progressing days (from 7 to 14). The bacterium exhibited capability to utilize and degrade n-hexadecane (used for primary screening) and petroleum hydrocarbons (kerosene and diesel; by ≥ 90%). With increase in the number of experimentation days, the optical density of the culture medium increased, whereas pH declined (became acidic) for both Kerosene and Diesel. Absence of resistance to routinely used antibiotics makes it an ideal candidate for future field application. The study is, thus, significant in view of toxicological implications of hydrocarbons and their degradation using environmentally safe techniques so as to maintain ecological and human health.

Introduction

Modern civilization is heavily dependent upon petrochemical industries and petroleum-based products towards achieving major developmental goals. Extreme and unique environments like north and south poles, deserts (both hot and cold), wetlands and deep sea are more susceptible to degradation in view of heavy industrial activities (Xu et al. 2018). These petrochemical industries have proved to be a bane rather than a boon over the years due to the excessive amount of unquantifiable environmental deterioration (Xue et al. 2015; Neris et al. 2019). With hydrocarbons serving as raw material in various industries such as petrochemical, refineries, pharmaceuticals, paints, synthetic polymers, etc. (Asghar et al. 2016) instances of spillage and untreated discharges are becoming more frequent, subsequently affecting environmental health and equilibrium. Biodegradation of hydrocarbons is an alternate process of detoxification and removal of pollutants. Of late, a new promising technology of utilizing bacteria and other microorganisms to mitigate the negative impacts and degrade such petroleum-based pollutants has gained popularity (Dvořák et al. 2017; Guerra et al. 2018). Although in contaminated sites natural bioremediation is often limited due to poor biodiversity of specialized microflora required for degrading petroleum-based hydrocarbons (Ron and Rosenberg 2014; Dvořák et al. 2017), but with the advancement in the field, it is fast becoming a forthcoming alternative to other remediation techniques (Guerra et al. 2018). Bioremediation is an intricately complex process, often depending upon nature and amount of hydrocarbon present in the contaminated sites (Das and Chandran 2011). Till date, strains from 79 genera of bacteria have been found to degrade petroleum products (Xu et al. 2018); for instance, Aeromonas, Acinetobacter, Achromobacter, Alcaligenes, Brevibacterium, Bacillus, Corynebacterium, Flavobacterium, Xanthomonas, Mycobacterium, Nocardia, Pseudomonas, Rhodococcus, etc. (Leahy and Colwell 1990; Plaza et al. 2008). Due to close proximity of toxic discharges and spills, the bacteria have evolved to be able to utilize petroleum products as a carbon source (Margesin et al. 2013; Ron and Rosenberg 2014; Xu et al. 2018). Bacteria have co-evolved with the toxic environments so as to develop various countermeasures against contaminants; for instance, improved adhesion property to enhance the extent of contact with the hydrocarbon compounds through surfactants (Inakollu et al. 2004; Kleindienst et al. 2015; Varjani 2017). Such bacteria can be screened, isolated, identified and utilized for bioremediation purposes (Krasowska and Sigler 2014). Indeed, not all bacteria are capable of degrading the complete fraction of petroleum hydrocarbons, as different indigenous strains contain different catalytic enzymes (Xu et al. 2018). As variable sets of microbes participate in the hydrocarbon biodegradation operation, there is a need to identify indigenous strains of bacteria with hydrocarbonoclastic properties.

Baddi (Himachal Pradesh, India) is an industrial hub with textile, pharmaceutical, petrochemical, and plastic manufacturing units. In the past, studies have reported isolation of indigenous bacterial strains for the degradation of polyvinyl chloride and textile dyes from the contaminated areas (Kaushik and Malik 2009; Sawhney and Bhojia 2011; Dhiman and Shirkot 2015; Kumar et al. 2017). Hydrocarbons are used as raw material in many of these industries (Asghar et al. 2016). In this context, it is pertinent to isolate and identify indigenous bacterial strain(s) capable of degrading petroleum hydrocarbons.

The present study focuses on the identification and isolation of microorganisms capable of producing biosurfactants and degrading differently structured petroleum hydrocarbons (diesel, n-hexadecane and kerosene). We selected three hydrocarbons, n-hexadecane (a medium-chain hydrocarbon), diesel, and kerosene (which are a mixture of mid-length hydrocarbons)—as model contaminants. Among them, n-hexadecane was employed in screening for hydrocarbon degradation, whereas degrading potential of strain for diesel and kerosene was investigated.

Materials and methods

Reagents

Diesel and kerosene were obtained from Sigma-Aldrich (St. Louis, USA), whereas n-hexadecane was procured from Hi-Media, Mumbai, India. The solvents used were of HPLC grade and procured from Spectrochem Pvt. Ltd., India. All other chemicals used in the study were procured from Sigma-Aldrich.

Sample collection, media and culture

Samples of industrial effluents were collected from three distinct locations in Baddi (Himachal Pradesh, India). The selected site has numerous manufacturing plants of pharmaceutical, textile, and steel companies; thus, generating a high volume of industrial effluent (containing hydrocarbons) that was easily available for collection. The following were the coordinates of sampling locations: Site 1, 30° 56′ 44.2932′′ N; 76° 46′ 59.1312′′ E; 406 m; Site 2, 30° 56′ 31.9632′′ N; 76° 46′ 50.1888′′ E; 390 m; and Site 3, 30° 55′ 11.2764′′ N; 76° 48′ 12.2364′′ E; 401 m asl. The collected samples were stored in dried and sterilized Duran bottles at 4 °C. These were analysed by liquid extraction (qualitatively and quantitatively) for the presence of major organic compounds using GC–MS (THERMO Trace 1300GC coupled with THERMO TSQ8000 Triple Quadrupole MS; Thermo Fisher, USA).

The biodegradation experiments were carried out in Minimal Salt Medium (MSM; pH 7.0 ± 0.2) composed of Na₂HPO₄ (33.90 g/L), NaCl (2.50 g/L), KH₂PO₄ (15 g/L), NH₄Cl (5 g/L), and 0.1% solution of trace elements containing the following: ZnSO₄.7H₂O (0.525 g/L), MnSO₄.4H₂O (0.2 g/L), CuSO₄.5H₂O (0.7 g/L), Na₂MoO₄.7H₂O (0.015 g/L), CoCl₂.6H₂O (0.2 g/L), H₃BO₄ (0.015 g/L) and NiSO₄.6H₂O (0.027 g/L). Carbon sources were added separately. Both the nutrient medium and MSM were autoclaved at 120 °C for 20 min before use.

To isolate bacterial colonies, 10 mL of effluent sample was shaken with 90 mL of autoclaved normal saline solution (NSS) for 30 min. The mixture was then diluted further (10–10,000 times) with autoclaved NSS and plated on nutrient agar plates. On the basis of distinct colour and morphology, bacterial colonies were isolated, purified, and streaked on nutrient agar slants. The streaked slants were kept at 4 °C till the next experiment. Cell-free supernatant (CFS) from nutrient broth (NB) was obtained by centrifugation of the culture in NB at 7400×g for 15 min. Cell free supernatant is advantageous as it is an open system and eliminates need of living cells for production of a particular protein of interest. As the supernatant retains a particular function (of interest) of the cell, it aids the efficient study of biochemical reactions and processes by avoiding complex interactions within processes (Albert et al. 2002).

Screening for biosurfactant production

A combination of methods was adopted to identify potential biosurfactant producers among the isolated bacteria. These included the following: Hemolytic assay and Oil spreading (Liu et al. 2012), Drop collapse and E₂₄ Emulsification index (Khan et al. 2018), and CTAB (Cetyl Trimethyl Ammonium Bromide) test (Siegmund and Wagner 1991).

For assessing biosurfactant producing ability of the bacterial isolates, they were grown on MSM plates with 1% n-hexadecane and 2% agar followed by incubation at 35 °C for 48 h. The isolates showing the maximum growth were selected for further experimentation.

Biodegradation of hydrocarbons

The method described by Mishra and Singh (2012) was followed with slight modifications. The bacterium was grown in NB for 24 h at 30 °C and 2 mL of this culture was transferred to 250 mL Erlenmeyer flasks (experimental and control flask). Three pairs of flasks were used for each hydrocarbon; one flask served as the experimental flask and the other as control flask. The control flask containing MSM with carbon source but without bacterial culture was kept to know the abiotic losses. All the flasks were transferred to an orbital shaker at 30 °C and 150 rpm for 7, 10, and 14 days. Residual substrate in the samples was extracted twice with 30 mL of dichloromethane. The extracts were evaporated till a final volume of 5 mL was achieved and analysed using Gas Chromatography (GC). All the experiments were done in triplicates. The degradation of hydrocarbon (%) was measured by using the following formula (Aydin and Icgen 2018):

$$\mathrm{Hydrocarbon}\mathrm{degradation}\left(\%\right)=\frac{(\mathrm{Hydrocarbon}\mathrm{in}\mathrm{Control}-\mathrm{Hydrocarbon}\mathrm{in}\mathrm{Inoculum})}{\mathrm{Hydrocarbon}\mathrm{in}\mathrm{Control}}\times 100$$

Hydrocarbon degradation rate was calculated by applying the Pseudo-first order kinetics (Behnajady et al. 2006; Rezaei and Habibi-Yangjeh 2013).

$$\ln \frac{[\mathrm{HC}]}{{[\mathrm{HC}]}_{\mathrm{o}}}=-kt$$

where k is rate constant of reaction; [HC] is concentration of Hydrocarbon (Kerosene and Diesel) in mol/L at any time; [HC]_o is initial concentration of Hydrocarbon (Kerosene and Diesel) in mol/L at any time; t is the time taken.

Identification and molecular characterization of bacterial isolate

The bacterial isolate yielding the best result was identified using morphological, biochemical, and molecular attributes. To identify the bacterial morphology, pure culture was subjected to Gram stain. The visible features, pigment production, motility, and characters of the colonies were recorded from growth on nutrient agar (NA) plates. Various basic biochemical tests such as catalase activity, indole, starch hydrolysis, gelatin liquification, and many more as outlined in Bergey’s manual of systematic bacteriology (Whitman et al. 2012) were performed to determine the biochemical properties of isolated bacterial strains.

The molecular characterization and identification of the selected isolate was undertaken using 16S rRNA sequencing. Polymerase chain reaction (PCR) was carried out and the isolate was sequenced using 27F 5′ (AGA GTT TGA TCM TGG CTC AG) 3′ as forward primer and 1492R 5′ (TAC GGY TAC CTT GTT ACG ACT T) 3′ as reverse primer, at Macrogen, South Korea. PCR operating conditions comprised of initial denaturation at 95 °C for 2 min, followed by 30 cycles each at 95 °C for 60 s, annealing at 58 °C, and extension at 72 °C for 60 s each. Dideoxy chain termination method was used to sequence both the strands of PCR products. The partially sequenced 16S rRNA gene fragment of 1495 base pairs was compared with the nucleotide sequence database in NCBI site using Basic Local Alignment Search Tool (BLAST) program (www.ncbi.nlm.nih.gov/BLAST/). Using Muscle programme, all the sequences were aligned with gene sequence of our isolate. In Mega-X software, using neighbour joining method, a phylogenetic tree was constructed (Pemmaraju et al. 2012; Shiri et al. 2014). The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The accession number for the bacterial isolate was obtained by depositing the 16S rRNA nucleotide sequence in the Genebank database of NCBI.

Determination of hydrocarbon degradation using GC-FID

The biodegradation of 1% diesel and kerosene on various days (7, 10, and 14) was measured by GC (GC-2014, Shimadzu Corporation, Japan) fitted with a DB5 column (5% diphenyl/95% dimethyl polysiloxane; 30 m × 0.25 mm × 0.25 µm, i.d.), and connected to Flame Ionization Detector (FID). Nitrogen (N₂) was used as carrier gas at a flow rate of 30 mL min^‒1. One microlitre of the sample was injected into the instrument. The injector and detector temperatures were set at 250 °C and 280 °C, respectively. Initially, the oven temperature was held at 100 °C for 3 min and then programmed to 300 °C at the rate of 10 °C min^‒1, and held at 300 °C for 10 min. No internal standards were used (Zhang et al. 2012).

Relationship of culture time with pH and growth (as optical density)

To determine changes in growth of bacteria and pH of the medium during degradation, the Erlenmeyer flasks containing the bacterial colonies and their controls were incubated for 14 days at 30 °C and 150 rpm. Samples were drawn in every two days. Absorbance was measured at 600 nm using spectrophotometer (UV-1800, Shimadzu, Japan) and pH was measured using EcoScan pH meter (Eutech Instruments, Singapore).

Antibiotic sensitivity testing

The susceptibility of bacterial isolate to commonly used antibiotics was determined through disc diffusion method of Kirby-Bauer to rule out the presence of any inherent or acquired resistance which might be transmitted horizontally to the pathogenic bacteria in the environment. The antibiotic discs were placed on NA plates spread with the bacterium to be tested and incubated for 48 h. After incubation, the zone of clearance was measured (Bauer et al. 1966; Mishra and Singh 2012).

Statistical analysis

All experiments were performed in triplicate and the results were obtained as an average of the three sets with their standard error. Non-linear regression (third-order polynomial) was used for the assessment of variations in pH and optical density along with days of incubation. All the graphical representations were performed using Sigma Plot software ver. 8.0.

Results and discussion

Characterization of hydrocarbons in industrial effluents and screening of bacteria for biosurfactant producing properties

The collected samples have different type of organic compounds such as cyclopentaneundecanoic acid, n-hexadecane, octadecanol, 9-octadecenoic acid, etc. (Table 1). In the past, studies have reported the isolation of indigenous bacterial strains for the degradation of polyvinyl chloride and textile dyes from the contaminated areas in Baddi, Himahcal Pradesh, India (Kaushik and Malik 2009; Sawhney and Bhojia 2011; Dhiman and Shirkot 2015; Kumar et al. 2017).

Table 1.

Organic compounds identified in effluents collected from Baddi, Himachal Pradesh, using GC–MS

Sample	Name of the compound	Molecular formula
Site 1	3-(benzylthio)acrylic acid methyl ester	C11H12O2S
	Cyclopentaneundecanoic acid	C17H32O2
	Hexanedioic acid, Dioctyl ester	C22H42O4
Site 2	9,9-Dimethoxybicyclo[3.3.1]nona-2,4-dione	C11H16O4
	Docosene	C22H44
	Octadecanol	C18H38O
Site 3	2-Bromotetradecane	C14H29Br
	n-Hexadecane	C16H34
	9-Octadecenoic acid	C18H34O2

The presence of hydrocarbons suggests that the bacterial colonies isolated from the contaminated samples may have an inherent capability to survive in this toxic environment and utilize them as carbon source. All the bacteria were streaked on NA plates and around 30 bacterial colonies were isolated as pure culture on the basis of distinct morphology. Individual screening of these isolates for biosurfactant production led to the selection of 18 bacterial colonies that yielded promising results in multiple screening methods. Only ten out of these 18 strains (55.5%) showed growth on model contaminant, i.e. 1% n-hexadecane, which indicated their potential to degrade hydrocarbons. Out of these 10 strains, one strain (identified as the RP3) was selected for further studies as it showed the most impressive growth (Fig. 1). The selected strain showed positive results for hemolytic assay and drop collapse test, and negative for CTAB test (Fig. 1a, b, e, f). It created a clear zone of 25 mm diameter in oil displacement test and showed emulsification activity of 55% (Fig. 1c, d, g). Drop collapse method, which can be employed with the oil spreading technique, was used to screen and quantify biosurfactant production (Youssef et al. 2004). On comparative analysis of the screening results, it was confirmed that the selected strain was an active biosurfactant producer. The bacterial properties of surface tension reduction and formation of a stable emulsion are rarely associated (Balan et al. 2017); hence, we used methods for screening both emulsification and surface tension reduction potential. As per our knowledge, no study till date has reported biosurfactant production by F. phosphorivorans RP3; making our study the first one to report so.

Fig. 1.

Preliminary screening for biosurfactant production by bacterium Fictibacillus phosphorivorans RP3. a Positive result for hemolytic assay; b Negative result on CTAB test; c Control of Oil spreading technique; d Oil displacement shown by the isolate; e Beaded drop of control in drop collapse method; f Collapsed drop showing positive result for biosurfactant production; g The stable emulsion after 24 h showing positive result for biosurfactant production

Biodegradation of diesel and kerosene

The strain RP3 was found to be hydrocarbonoclastic and a potential degrader of both the petroleum hydrocarbons in MSM. The percent degradation was calculated by the peak intensity of hydrocarbons on various days of incubation. Martin-Sanchez et al. (2018) suggested that GC-FID can be used for fuel quantification as an improved method for detecting hydrocarbon degradation since it provides additional valuable information on the alteration of fuel component patterns. RP3 strain was able to degrade 37% of diesel (Table 2) and 74% of kerosene (Table 3) in first 7 days of incubation. It was observed that rate of degradation (day⁻¹) for diesel increased till 14 days of incubation (Table 2), whereas for kerosene it increased upto 10 days of incubation followed by a decline (Table 3). The degradation of diesel and kerosene reached 95% (Table 2) and 96% (Table 3), respectively, after incubation of 14 days. Previous studies have demonstrated the degradation of n-hexadecane by Pseudomonas aeruginosa strains NY3 and ATCC 2007 (Zhong et al. 2016; Nie et al. 2017) isolated from marine sediments (García-Cruz et al. 2018). Similarly, Rhodococcus sp. T1 isolated from oil-contaminated site degraded 2% n-hexadecane by 90.81% after 5 days, when single substrate was used; however, the use of dual substrate reduced the degradation of n-hexadecane to 87.55% (Jia et al. 2019). A consortium of six bacteria, namely Serratia marcescens C11S1, S. marcescens C7S3A, Citrobacter freundii CCC4DS3, Raoultella ornithinolytica C5S3, Stenotrophomonas maltophilia CCC10S1, and St. pavanii C5S3FN showed 97.9% diesel degradation; whereas strain C7S3A alone showed 96% diesel degradation (Morales-Guzmán et al. 2017). Dixit et al. (2018) reported 65% and 80% degradation of diesel by Bacillus cereus 3E and B. subtilis 4F strains, respectively, over a period of 15 days. Mojarad et al. (2016) demonstrated that Enterobacter cloacae, E. hormaechei and Pseudomonas stutzeri degraded 5% kerosene by 67.43%, 48.48%, and 65.48%, respectively, within 7 days.

Table 2.

The rate of degradation (k) of diesel (organic pollutants) calculated using Pseudo-first order kinetics

Days	Peak Intensity		Degradation %		Rate of degradation (k) day−1
	Control	Diesel	Control	Diesel	Control	Diesel
0	900,000	900,000	0	0	0	0
7	900,000	566,726	0	37	0	0.0660
10	897,827	195,906	0.24	78.2	0.0008	0.3540
14	857,952	42,266	4.6	95.3	0.0113	0.3834

Table 3.

The rate of degradation (k) of kerosene (organic pollutants) calculated using Pseudo-first order kinetics

Days	Peak Intensity		Degradation %		Rate of degradation (k) day−1
	Control	Kerosene	Control	Kerosene	Control	Kerosene
0	1,734,368	1,917,651	0	0	0	0
7	1,720,700	496,108	0.78	74.1	0.0011	0.1931
10	1,525,581	117,531	12.0	93.8	0.0401	0.4800
14	1,307,851	76,499	24.5	96.0	0.0384	0.1073

Identification of bacterial strain

Morphological identification

The RP3 strain grew as circular, smooth, convex, opaque, shiny colonies; which were orange in colour with entire margin on nutrient agar. The bacterium was non-motile, rod-shaped, and Gram-positive. It was able to grow between 15 and 35 °C (optimum temperature 25‒30 °C) and pH 6‒11 (optimum pH 7–8) when the growth was supported by 1% NaCl. Till date, very few studies have been performed on F. phosphorivorans, and thus there is no record/information available regarding pathogenicity.

Biochemical identification

The RP3 strain was positive for Citrate, Glucose, Starch and Sucrose utilization tests and negative for Catalase, Gelatin, Indole, Methyl red and Voges-Proskauer tests. It did not show any utilization of Adonitol, Arabinose, Lactose, Mannitol, Rhamnose and Sorbitol. These morphological and biochemical characteristics of RP3 were found to be similar to those reported for the Fictibacillus genus (Glaeser et al. 2013).

Molecular identification and accession number

Using BLAST, the sequence of selected RP3 strain was examined. On the basis of 16S rRNA sequencing, the most potent isolate was identified as Fictibacillus phosphorivorans RP3. The strain belongs to the family Bacillaceae and phylum Firmicutes. The 1495 long gene sequence generated by the forward and reverse primers was submitted to NCBI GeneBank database and the accession number MG263748 was successfully obtained.

Phylogenetic analysis

The 16S rRNA gene sequence of Fictibacillus phosphorivorans RP3 was amplified to know the phylogenetic position of isolated bacterium. Muscle alignment and the neighbour joining method of MEGA-X software were used to study evolutionary relationships. It showed 100% genetic similarity of the isolated strain with F. phosphorivorans strain Ca7 (NR_118455) and 99.86% identification with F. halophilus strain AS8 (NR_149289) (Fig. 2). However, there is not much of literature available on the degradation potential of the genus Fictibacillus. Nevertheless, a recent study has reported Fictibacillus sp YS-26 as a potential microbe for the biodegradation of lignocellulosic agricultural residues, which can improve carbon metabolic properties and functional diversity of the rhizosphere microbial community (Chen et al. 2020). A closely related genus Bacillus (Glaeser et al. 2013) has been exploited tremendously for bioremediation (Pemmaraju et al. 2012; Ayed et al. 2015; Dixit et al. 2018; Banerjee et al. 2019). Previous study on Proteobacteria, Firmicutes, and Bacteroidetes phyla present in petroleum-contaminated soil revealed significant biodegradation activity (Shahi et al. 2016). Al-Dhabaan (2019) reported that B. cereus had highest degradation rate of petroleum hydrocarbon, i.e. 68%, after 21 days of incubation but it decreased after 28 days of incubation. In another study, a new strain (B. cereus CPOU13) caused 84.23% degradation of anthracene in 13 days of incubation (Poornachander et al. 2016). Das et al. (2017) demonstrated that B. cereus strain JMG-01 caused 98% degradation of anthracene within 21 days. Marchut-Mikolajczyk et al. (2020) demonstrated that endophytic B. cereus EN18 showed better efficiency of degradation when supplemented with lipase from Rhizomucor miehei and was able to biodegrade up to 40% of aromatic hydrocarbon (oil) within 14 days of incubation.

Fig. 2.

Evolutionary relationships of isolated bacteria Fictibacillus phosphorivorans RP3 with reference closest NCBI strains based on 16S rRNA gene sequence. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree with the sum of branch length = 0.10697072 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The analysis involved 16 nucleotide sequences. All ambiguous positions were removed for each sequence pair. There were a total of 1572 positions in the final dataset. Evolutionary analyses were conducted in MEGA X. Bootstrap values more than 50 are given. The collection centre of all the type strains is NCBI

Determination of hydrocarbon degradation using GC-FID

The identified strain (F. phosphorivorans RP3) exhibited almost similar trend of degradation for both the carbon sources as observed on GC (Fig. 3). The percent degradation of the hydrocarbons was calculated by the peak intensity on various days of incubation. It was observed that with an increase in the incubation time, the concentration of the carbon sources decreased although after a period of time the rate of degradation also declined, which could be attributed to the observed trends of changes in pH and optical density (OD) (discussed later). Martin-Sanchez et al. (2018) suggested that GC-FID can be used for fuel quantification as an improved method for detecting hydrocarbon degradation since it provides additional valuable information on the alteration of fuel component patterns.

Fig. 3.

GC-FID chromatogram of residual hydrocarbons extracted after 7 and 14 days from flasks supplemented with 1% diesel (a–c) and 1% kerosene (d–f) with respect to control, when incubated with bacterium Fictibacillus phosphorivorans RP3

Relationship of culture time with pH and optical density (OD)

The bacterial strain identified in present study was isolated from industrial effluent; hence, it survived and adapted to the hydrocarbon content in culture medium. We further explored whether F. phosphorivorans RP3 strain has potential to degrade other hydrocarbon sources such as diesel and kerosene. For diesel and kerosene, pH declined non-linearly and became acidic, whereas OD increased non-linearly with increasing days (Fig. 4). Initially, pH of the medium was neutral, which was the optimum for enzyme activity (Cases and de Lorenzo 2005). A reduction in pH in the second week of experiment might have led to a reduction in the rate of hydrocarbon degradation (Shen et al. 2015). Although, a reduced availability of carbon sources upon degradation can also lead to the same effect. The first 3 days of incubation did not yield any significant decrease in the OD or pH of the medium, which could be explained by the lag phase of bacteria (Bertrand 2019). As the bacteria entered log phase, the utilization of carbon sources by the microbes significantly increased, as observed by an increase in OD with a concomitant reduction in pH between 3rd and 10th day of incubation, which can be explained by the acidic nature of metabolic products. The degradation rate was the maximum around 10th day (Tables 2, 3). However, the highest bacterial cell increase was obtained on 10th day of kerosene utilization as the carbon source. With further incubation, OD of the culture decreased and pH became stable (Fig. 4). This can be attributed to the fact that decrease in the rate of degradation beyond 10th day could have already moved the bacterial growth into death phase in both the cases. Another observation was that the change in pH between both the carbon sources over the entire period of investigation (14 days) was almost the same (Fig. 4). It suggested that the structure of the carbon sources did not affect the rate of degradation or the ability of bacteria to degrade carbon source, as observed by Adebusoye et al. (2007).

Fig. 4.

Variations in optical density (OD) and pH with number of days for diesel (a, b) and kerosene (c, d) incubated with bacterium Fictibacillus phosphorivorans RP3, analysed by non-linear regression (third-order polynomial)

Antibiotic susceptibility tests

Kirby-Bauer disc diffusion method demonstrated that the bacterium F. phosphorivorans RP3 is sensitive to most of the commonly used antibiotics, with the highest sensitivity to gentamycin (30 µg disc^‒1), for which a 23-mm zone of clearance was observed (Fig. 5). These results show that drug resistance factor is absent in the bacterium; therefore, its employment in bioremediation programmes is unlikely to spread drug resistance to the indigenous pathogens.

Fig. 5.

The susceptibility profile of isolated bacteria Fictibacillus phosphorivorans RP3 to various antibiotics determined using disk-diffusion method. The values in parenthesis along each antibiotic indicates the concentration in µg disc^‒1

Conclusion and future outlook

High ecological significance is associated with bioremediation using indigenous microorganisms/bacteria. Fictibacillus phosphorivorans RP3 is one such novel bacterial strain identified through the course of this study which was hydrocarbonoclastic in nature. Owing to highly efficient hydrocarbonoclastic nature of this bacterial strain, it can be used for large-scale bioremediation of petroleum hydrocarbon pollutants, with field application and experimentation being the next logical step in this direction. The study is significant in view of toxicological implications of hydrocarbons and their degradation using environmentally safe techniques so as to maintain ecological and human health. Combining other molecular approaches (metabolomics, metagenomics, transcriptomics, etc.) will allow for a better and realistic understanding of mechanism of biodegradation so as to enhance the biodegradation capacity.

Author contributions

RP and BK formulated the hypothesis, RP carried out work of the study; RP and BK prepared the first draft of the manuscript; RP, PS and SR analysed and edited the manuscript; HPS, DRB and RKK supervised and reviewed the work; HPS, DRB and RKK proofread the manuscript.

Compliance with ethical standards

Conflict of interest

Authors declare that they have no conflict of interest in the publication.