Bacteria and fungi mediated degradation of poly aromatic hydrocarbons and effect of surfactant Tween-80

ABSTRACT Microorganisms (17 bacteria and 3 fungi) isolated from crude oil-contaminated soils were evaluated for degradation of five polyaromatic hydrocarbons (PAHs), both in the absence (-T80) and the presence of surfactant Tween-80 (+T80) at a 10 mg L−1 concentration of each PAH. Results of the heat map and Tukey HSD revealed that highest PAH degradation was observed by Kocuria rosea in +T80 treatment. Degradation (%) of individual PAH (-T80, +T80) was naphthalene: 96.6, 98.9; fluorene: 93.8, 95.1; phenanthrene: 58.1, 70.9; anthracene: 19.9, 32.5 and pyrene: 13.8, 54.7, respectively. Tukey HSD further revealed that all three fungi Trichoderma atroviride, Aspergillus nidulans and Aspergillus sydowii showed almost similar degradation of selected PAH. Based on degradation ability, consortia of K. rosea and A. sydowii were used for PAH degradation at 10, 50 and 100 mg L−1 in the presence of Tween-80. Results suggested that PAH degradation slowed down with the increase in the concentration. PAH degradation products phthalic acid and anthrone or phenanthrene-9,10 oxide or 9-phenanthrol were identified as a product of degradation. Bacterial strain Arthrobacter pascens, Pseudomonas aeruginosa, Bacillus megaterium, Bacillus sp., Bacillus niacini and Bacillus siamensis showed high fluorescence diacetate (FDA) hydrolase activity, whilst A. pascens, K. rosea, Pseudomonas sp. and B. niacini showed a significant soluble protein content.


Introduction
Polycyclic aromatic hydrocarbons (PAHs), a major cause of environmental concern, are derived from either pyrogenic or petrogenic sources in the environment.Pyrogenic PAHs are gradually created due to incomplete combustion of carbon-based fuel including wood, coal or dung, whereas petrogenic PAHs are generated due to refining petroleum products as well as during production and use of plastic consumer products [1,2].PAHs are aromatic recalcitrant chemicals belonging to three different classes based on their ring structures.PAHs with 2-3, 4 and 5-6 rings are named low molecular weight (LMW), medium molecular weight (MMW) and high molecular weight (HMW) PAHs, respectively.They readily bind to sediments and are highly recalcitrant and persistent in the nature due to higher hydrophobicity and became ubiquitous contaminants in the environment, leading to bioaccumulation and biomagnification in biological systems [3,4].Fadzil et al. [5] reported that the concentrations of PAHs in Indian soils were 6.7 μg g −1 (agricultural land), 9.3 μg g −1 (residential area), 12.9 μg g −1 (along roadside) and 13.7 μg g −1 (industrial region).Devi et al. [6] reported that the concentrations of polyaromatic hydrocarbons were found to be varying from 15.3 to 4762 ng g −1 (mean 458 ng g −1 ) in surface soil of the Indian Himalayan region.These PAHs are carcinogenic, teratogenic and mutagenic in nature, ultimately posing serious threat to the environment and human.Therefore, USEPA has listed 16 PAHs as priority pollutants [7].
Low-molecular weight (LMW) PAHs like naphthalene, phenanthrene and anthracene are widely present in the environment and designated as signature compounds to detect PAH contamination.Due to the presence of bay and K regions in phenanthrene (simplest PAHs), it is also used as a model substrate for studies on the metabolism of carcinogenic PAHs [8].Sleight et al. [2] used acenaphthene, anthracene, fluorene and phenanthrene for degradation and analysis studies due to the variation in the ring structure.Among different PAHs, pyrene and phenanthrene are highly associated with industrial processes and are most widely transported PAHs in the hydrosphere [9].Physicochemical properties of naphthalene, fluorene, phenanthrene, anthracene and pyrene (Table 1) suggest that due to the hydrophobic nature and high water-octanol partition coefficient (log K ow ) of PAHs, the amphiphilic surface active agents (anionic, non-ionic, cationic or zwitter ionic surfactants) are employed to enhance solubilisation of hydrocarbons in degradation experiments [7,10].
Despite several methods for degradation of PAHs (combustion, photolysis, landfill and ultrasonic decomposition), biodegradation has attracted attention of researchers due to its economic and environmentally friendly nature [9,11].Some microbes utilise PAHs as the source of carbon and energy due to their adaptability as well as metabolic capabilities, finally converting them into carbon dioxide and water or non-toxic compounds.Researchers have exploited potential of bacteria and fungi for degrading PAHs.Obayori et al. [3] studied degradation of pyrene, fluoranthene and dibenzothiophene at 100 mg L −1 by Proteus mirabilis and after 30 days, 87.92, 93 and 97.25% degradation was observed, respectively.Hadibarata and Kristanti [12] reported that 95% fluorene (10 mg L −1 ) was degraded by Armillaria sp.F022 within 30 days.Bacterial fungal co-culture has been reported for 46-91% degradation of phenanthrene and pyrene (200 mg L −1 ) within 46 days [13].Other studies showed that mixed microbial culture in the anaerobic-anoxic-aerobic sequential bioreactor operated in the fedbatch condition resulted in 71% degradation of naphthalene (500 mg L −1 ) and 36% anthracene (100 mg L −1 ) within 16 days [14].Wang et al. [15] reported 63, 49 and 69% degradation of pyrene (10 mg L −1 ) by Trichoderma sp., Aspergillus Niger and Fusarium sp., respectively.White rot fungus, Ganoderma lucidum, was found to be more efficient (99.65 and 99.58%) for degradation of phenanthrene and pyrene (20 mg L −1 ) [16].Degradation of PAHs is limited due to low aqueous solubility; therefore, surfactants have been used to enhance their solubility.Tween-80 is a hydrophilic non-ionic surfactant and is widely used due to its nontoxic nature [17].The present investigation uses microbes (bacteria and fungus) isolated from an oily sludge-contaminated soil for PAH degradation in the presence and the absence of surfactant Tween-80.The effect of the PAH concentration was investigated using the selected bacterium and fungus.PAH degradation products were identified using LC-MS/MS.Microbial parameters like soluble protein content and fluorescein diacetate hydrolase were determined during the degradation process.

Isolation of microbes using the enrichment technique
The bacteria and fungi from the crude oil-contaminated soil were isolated using serial dilution methods.Crude oil-enriched contaminated soil (1 g), as the carbon source, was added in Afterwards, 1 mL of culture was serially diluted up to 10 9 fold and 100 µL of each dilution was plated on 0.1% oil-enriched MSM agar plates.The bacteria and fungi grown on MSM agar were further purified and maintained on nutrient agar and potato dextrose agar, respectively.

Molecular identification
The bacteria were identified by 16S rDNA gene sequencing (17), whilst fungal strains were identified by sequencing of the rDNA gene cluster, consisting of ITS-1, the 5.8S rDNA and ITS-2, which was amplified with primers homologous to conserved sequences within the small subunit (SSU) rDNA gene.Genomic DNA was isolated from selected strains using Zymo Research Fungal/Bacterial DNA MicroPrep TM following the manufacturer's standard protocol [19].Bacterial 16S rRNA gene of approximately 1.5 kb internal region was amplified using the universal primer set of forward primer pA (5ʹ GA GTT TGA TCC TGG CTC AG 3ʹ) and reverse primer pH (5ʹ AAG GAG GTG ATC CAG CCG CA 3ʹ).The fungal rRNA gene cluster, consisting of ITS-1, the 5.8S rDNA and ITS-2, was amplified with primers homologous to conserved sequences within the small subunit (SSU) rRNA gene.The ITS primers used were ITS-1 (5ʹ TCC GTA GGT GAA CCT GCG G 3ʹ) and ITS-4 (5ʹ TCC TCC GCT TAT TGA TAT GC 3ʹ) to amplify the fragment of approximately 550 bp.The partial sequences of the isolates were compared with sequences available in the National Center for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov)through BLAST search to identify the nearest taxa and were submitted to NCBI GenBank and the accession number was obtained.

Screening of microbes for PAH degradation
Isolated bacteria (17) [20].One millilitre of PAH mixture containing 250 mg L −1 of each in acetonitrile was added to 100 mL of sterilised conical flasks under aseptic conditions and solvent was allowed to evaporate in laminar flow.Afterwards, 25 mL of sterile RMM, without Tween-80 (-T80) and with 0.1% Tween-80 (+T80) was added to each conical flask.Both sets of media were inoculated with bacterial (10% v/v culture, OD~1.0) and fungal cultures (5 discs) individually.Uninoculated samples served as control.All treatments were maintained in triplicate.Bacterial samples were incubated at 27°C in a rotary incubator maintained at 120 rpm for 7 days, whereas fungal samples were at the static position.On the sampling day (7 th for bacteria-inoculated medium and 10 th day for fungiinoculated medium), flasks from each treatment, in triplicate, were removed for PAH extraction and enzymatic parameters.The effect of the PAH concentration on degradation was studied using K. rosea AK4 strain and A. sydowii AK20 strain, individually and in mixture at 10, 50 and 100 mg L −1 concentrations using Reese minimal media containing 0.1% Tween-80.Studies were performed as mentioned in the previous section for 10 days.

Extraction and analysis of PAHs
The PAHs from samples were extracted using dichloromethane following the method reported by Sharma et al. [21].Briefly, the sample (25 mL) was diluted with saline (15% NaCl) and extracted using dichloromethane (50 mL, thrice).Organic fractions were pooled together and dried over anhydrous Na 2 SO 4 and solvent was concentrated on a rotary vacuum evaporator.Finally, residues were redissolved in acetonitrile and samples were filtered through 0.22 μm membrane filter prior to analysis using high-performance liquid chromatography (HPLC).A Hewlett Packard HPLC instrument (series 1100) equipped with a degasser, a quaternary pump and a photodiode array detector connected with a rheodyne injection system was used in the study.The conditions for analysis included the following: RP-18 (PAH) column 25 cm x 5μ (Merck), acetonitirle:water (70:30) as the mobile phase at a flow rate of 0.5 mL min −1 at wavelengths of 220 nm for naphthalene, 210 nm for fluorene, and 246 nm for phenanthrene, anthracene and pyrene.The limit of detection (LOD) and limit of quantification (LOQ) for PAHs were naphthalene, 0.05 and 0.2 μg mL −1 ; fluorene, 0.08 and 0.5 μg mL −1 ; phenanthrene, 0.1 and 0.5 μg mL −1 ; anthracene, 0.08 and 0.5 μg mL −1 and pyrene, 0.1 and 0.5 μg mL −1 .The chromatogram of the mixture of PAHs, control sample (-T80, +T80) and degradation by K. rosea (-T80, +T80) on the 7 th day are depicted in Supplementary Figures S1-3.

Identification of metabolites using HPLC-MS/MS
Extracted samples of K. rosea bacteria (AK4) and A. sydowii fungus (AK20) were dissolved in acetonitrile and injected in the UPLC-MS/MS system (8030).Samples were scanned for mass values (m/z) ranging from 70 to 320 in ESI (±) mode.The condition for the LC-MS/MS system was as follows: the temperature of the column, 40°C; flow rate, 0.2 ml min −1 ; injection volume, 2 µL; run time, 30 min; DL temperature, 250°C, heat block temperature, 400°C; nebulising gas flow, 3 L min −1 and drying gas flow, 15 L min −1 .The column used in the stationary phase was Agilent Eclipse plus C18 (3 × 100 mm; 3.5 µm) and the mobile phase was the mixture of water: methanol (80:20, 5 mM ammonium formate) and water:methanol (10:90, 5 mM ammonium formate) with a gradient of 45-100%.

Biochemical assay
The soluble protein content was estimated using the alkaline copper reagent (ACR) and Folin Ciocalteu reagent (FCR) as per the procedure developed by Lowry et al. [22].The fluorescein diacetate (FDA) hydrolase activity was analysed as per the method described by Green et al. [23].

Statistical analysis
The data were subjected to two-way ANOVA taking the two different sources of variability, viz., bacteria/fungi type and surfactant using SAS 9.4 software.Tukey's HSD test was performed for multiple comparisons among treatment effects and interactions at the 5% level of significance.The treatments, which got the same letter grouping, are at par and the treatment pairs getting different letter grouping are significantly different.Besides, a heat map has been prepared using an R package 'heatmap' [24] using R-studio [25] to visualise the homogeneity among different bacteria in the presence/ absence of surfactant and also amongst various compounds.The hierarchical clustering approach was used to prepare the dendrogram in the heat map based on the similarities calculated through the Euclidean distance.All other analyses were performed using Microsoft Excel 2007.

Identification of microbes
Seventeen bacteria and three fungi were isolated from the enrichment culture.Based on the results of partial 16S rDNA and 18S rDNA sequences and their NCBI BLAST analyses, the bacterial and fungal strains were identified and accession numbers were obtained from NCBI (Supplementary Table 1).The phylogenetic analysis constructed by using MEGAX and all the bacterial and fungal strains were showing approximately 99-100% similarity with their close relatives.Most of the PAHs utilising bacteria belong to genus Bacillus, Pseudomonas, Kocuria, Arthrobacter and Lysinibacillus (Figure 1), where fungi were identified as Aspergillus and Trichoderma (Figure 2).

Degradation of PAHs by bacteria
Results of the PAH degradation study by 17 bacterial strains after the 7 th day incubation (Figure 3, Table 2) demonstrated treatment effects and interactions for different compounds, namely naphthalene, fluorene, phenanthrene, anthracene, pyrene and Tween-80 were noted as p < 0.001.Amongst the PAHs studied, maximum degradation was observed for naphthalene followed by fluorene, phenanthrene, pyrene and anthracene Thus, naphthalene was degraded at a faster rate by the isolated bacteria, whilst anthracene was the most recalcitrant PAH.Degradation of PAHs in -T80 treatments by different microbes varied in the range of 59.5-100% (naphthalene), 53.9-95% (fluorene), 30.1-62.4% (phenanthrene), 6.9-26.9%(anthracene) and 9.6-26.8%(pyrene).PAH-degrading ability of bacteria can be positively correlated with their aqueous solubility, suggesting that availability of PAHs to bacteria limits their degradation.This was further confirmed from the results of the PAH degradation in the presence of surfactant Tween-80 (+T80).that K. rosea showed 55.4% degradation of naphthalene.Surfactant increases the solubility of pollutants in liquid media and contributes to a higher mixing level and physiology of cell surface uptake [28].Reports suggest that non-ionic surfactants were destructed into small moieties and increased the bioavailability of PAHs to bacteri0061, resulting in a higher level of degradation during the transformation process (Hadibarata and Kristanti 2014).Al-Farraj et al. [29] observed that addition of Tween-80 enhanced pyrene- degrading ability of Hortaea sp.B15, where 88% of pyrene (100 mg L −1 ) was degraded within 25 days.Hadibarata and Kristanti [12] reported a significant increase in PAH degradation by Polyporus sp.S133 and Armillaria sp F22 and concluded that Tween-80 was better surfactant than Tween-20 and Tetradecyl trimethyl ammonium bromide (TDTMA).
Previous studies reported degradation of 99.67 phenanthrene by Burkholderia fungorum FM-2 [9], 24-32% fluorene by P. aeruginosa [30] and 41% pyrene by Pseudomonas aeruginosa san ai after 72 h [31].Our study suggested that degradation of PAHs varied amongst microbes used and can be attributed to their genetic potential for utilising them as a preference substrate as a source of carbon for energy and solubility/availability of PAH.Data are expressed as mean values.Different superscripts for the same parameter represent significant differences (p < 0.001).SE, B, F and S represent the standard error of mean, bacteria, fungus, surfactant and their interaction, respectively, for each compound

Degradation of PAHs by fungus
Three fungal strains, viz., T. atroviride, A. nidulans and A. sydowii, isolated from the enrichment culture, were evaluated for PAH degradation (Table 2).Results of the PAH degradation study after 10 th day incubation demonstrated that percent degradation of naphthalene and pyrene was noted as non-significant (p > 0.05).However, degradation of fluorene, phenanthrene and anthracene was found to be significant (p < 0.05).The percent degradation of anthracene amongst different fungi (T.atroviride, A. nidulans and A. sydowii) was found to be significant as compared to other compounds (p < 0.05).The influence of Tween-80 was observed to be significant for degradation of fluorene, phenanthrene and anthracene (p < 0.05).However, interaction of fungus and Tween-80 was found to be nonsignificant for all compounds (p > 0.05).Results suggested that compared to bacteria, fungi were more effective in degrading PAHs.Similar to PAH degradation potential of bacteria, fungi in -T80 treatment exhibited maximum degradation of naphthalene (99.8-100%), followed by fluorene (79.3-80.5%),phenanthrene (56.1-57.9%),pyrene (45-46.2%)and anthracene (27.9-29.5%)(Figure 4) .Tween-80 showed a significant degradation increase for all three fungi in phenanthrene (64.2-66.6%)and anthracene (30.6-32.1%)degradation, whilst no effect was observed on naphthalene (99.8-99.9%),fluorene (81.4-82.1%)and pyrene (45.3-46.6%)degradation.Earlier reports suggested that Aspergillus sp. and Trichoderma sp.showed ability to degrade some of PAHs individually [4,15,32,33], whilst fungi used in the present study were able to degrade these PAHs in mixture; however, efficiency varied for individual PAH.

Effect of the concentration and microbial co-inoculation on degradation
Based on the results obtained in previous studies, bacterium K. rosea AK4 and fungus A. sydowii AK20, which exhibited maximum capability to degrade PAHs, were evaluated in combination, for PAH degradation (Table 3).The treatment effects and interactions for different compounds, namely, naphthalene, fluorene, phenanthrene, anthracene and pyrene degradation, by microbial consortia and concentration (10, 50 and 100 mg L −1 ) were noted as p < 0.001.Results after 10-day incubation suggested that except naphthalene, degradation of remaining PAHs slowed down with the increase in their concentration from 10 to 100 mg L −1 .No effect of bacterial and fungal co-inoculation was observed on phenanthrene degradation at higher concentrations.However, anthracene (10 mg L −1 ) degradation was significantly increased when both microbes were used together (49.3 a ) than when individual microbes were used (34.6 b , 32.8 bc ).Degradation of fluorene (95.6 a ) and pyrene (57.1 a ) in co-inoculated medium was at par with K. rosea AK4 (alone) although it was significantly higher than degradation by A. sydowii AK20 alone.The result showed that microbial consortia can be effectively used for 10 mg L −1 for degradation of PAH.However, for other PAHs, results were at par, whether used alone or in combination.

Identification of PAH metabolites
The PAH metabolites produced during degradation were identified using LC-MS/MS using extract of K. rosea.Based on the total ion chromatogram (TIC), peaks at 2.165 min in ESI(+) mode and 19.643 min in ESI(-) mode were obtained.Based on the molecular ion peak (m/ z), these two metabolites were identified as phthalic acid (166.14) and anthrone or phenanthrene-9,10 oxide or 9-phenanthrol (m/z:~194.23),respectively.Earlier, phthalic acid was detected as a common metabolite during degradation of phenanthrene [34], anthracene [4] and fluorene [31].Fu et al. [35] and Ye et al. [4] reported the formation of anthrone or phenanthrene-9,10 oxide or 9-phenanthrol (m/z:~194.23)during degradation of phenanthrene and anthracene.Based on probable degradative products (Figure 5

Conclusion
Seventeen bacterial and three fungal strains were isolated from the crude oilcontaminated soil and were identified using standard molecular characterisation techniques.Amongst the isolated microbes, K. rosea and A. sydowii were found to be best bacterium and fungus for naphthalene, fluorene, phenanthrene, anthracene and pyrene degradation.Tween-80 affected PAH degradation by selected microorganisms and the effect was more for anthracene and pyrene degradation.Consortium of the best bacteria and fungi showed significant enhancement of phenanthrene and anthracene degradation at the 10 µg mL −1 concentration.Bacteria/fungi identified in the present study can be exploited for PAH remediation in the environment.

Figure 1 .
Figure 1.Phylogenetic tree of crude oil degrading bacteria isolated from the oil sludge-contaminated soil.

Figure 2 .
Figure 2. Phylogenetic tree of crude oil degrading fungi isolated from the oil sludge-contaminated soil.

Figure 5 .
Figure 5. Possible intermediates identified during biodegradation of PAH (a) and probable mechanism derived based on probable identified degradative products (b) in the Reese minimal medium (RMM).

Table 1 .
Physico-chemical properties and some relevant information of PAHs enlisted as priority pollutants by US EPA.
a Toxic equivalent factor (TEF) relative toBenzo[a]pyrene (Chang et al., 2014).b International Agency for Research on Cancer (IARC) Classification Monographs Volume 1-111 updated on 18 February 2015 (1, carcinogenic to humans; 2A, probably carcinogenic to humans; 2B, possibly carcinogenic to humans; 3, not classifiable as carcinogenic to humans; n. c., not classified).c Environment Protection Agency (EPA) carcinogenic classification: A, human carcinogenic; B1 and B2: probable human carcinogenic; C, possible human carcinogenic; D, not Classifiable as to human carcinogenicity; E, evidence of non-carcinogenicity for humans

Table 2 .
Interaction analysis of percent degradation of PAHs by bacteria(7 th

Table 3 .
Interaction analysis of percent degradation of PAHs by bacteria and fungi after 10 days with Tween-80 in the Reese minimal medium (RMM).

Table 4 .
Interaction analysis of enzymatic activity by bacteria (7 th day) and fungus on 10 th day, without (-T80) and with Tween-80 (+T80) in the Reese minimal medium (RMM).Pairwise comparison of soluble protein and fluorescein diacetate hydrolase activity with different bacteria and fungi; SE, B, S and F represent the standard error, bacteria, surfactant and fungi.Statistical analysis was performed by Tukey's HSD test