Metabolism of pyrene through phthalic acid pathway by enriched bacterial consortium composed of Pseudomonas, Burkholderia, and Rhodococcus (PBR)
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Polycyclic aromatic hydrocarbons (PAHs) are highly recalcitrant compounds due to their high hydrophobicity and tendency to partition in organic phase of soils. Pyrene is a high-molecular weight PAH, which has human health concerns. In the present study, a bacterial consortium, PBR, was developed from a long-term polluted site, viz., Amlakhadi, Ankleshwar, Gujarat, for effective degradation of pyrene. The consortium effectively metabolized pyrene as a sole source of carbon and energy. The consortium comprised three bacterial species, Pseudomonas sp. ASDP1, Burkholderia sp. ASDP2, and Rhodococcus sp. ASDP3. The maximum growth rate of consortium was 0.060/h and the maximum pyrene degradation rate was 16 mg/l/day. The organic and inorganic nutrients along with different surfactants did not affect pyrene degradation, but degradation rate moderately increased in the presence of sodium succinate. The significant characteristic of the consortium was that it possessed an ability to degrade six other hydrocarbons, both independently and simultaneously at 37 °C, in BHM (pH 7.0) under shaking conditions (150 rpm) and it showed resistance towards mercury at 10 mM concentration. Phthalic acid as one of the intermediates during pyrene degradation was detected through high-performance liquid chromatography (HPLC). The efficiency of consortium for pyrene degradation was validated in simulated microcosms’ study, which indicated that 99% of pyrene was metabolized by the consortium under ambient conditions.
KeywordsPyrene Sodium succinate Heavy metals Phthalic acid Bioremediation Enrichment technique
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous compounds present in the environment. Their origin in the environment may be attributed to extensive anthropogenic activities like industrialization, incomplete combustion of fossil fuels and oil spills or natural phenomena like wood fires, volcano eruptions and natural underwater oil spills (Juhasz and Naidu 2000; Atlas and Hazen 2011). PAHs are considered as priority pollutants by the US Environmental Protection Agency (USEPA). They persist in the environment for a long time because of the sequestration in sediment particles’ micropores and thus remain out of reach of microbial degradation. They are also concerned with human health because many PAHs are mutagenic, carcinogenic and teratogenic (Haritash and Kaushik 2009; Kumar et al. 2011).
Pyrene is high-molecular weight (HMW) PAH having very high hydrophobicity and because of its high octanol:water partition coefficient, there are relatively less reports of efficient degradation of this compound by microorganisms, though many efforts have been made by several investigators. In addition, few studies have reported on the utilization of these compounds as a sole carbon source (Bacosa and Inoue 2015; Ghosh et al. 2014; Ho et al. 2000; Wang et al. 2008). There are reports of degradation of pyrene by pure cultures and consortia developed from various origins like petroleum sludge, oil spill sites or other petroleum products contaminated sites. The isolated organisms may be from the Actinomycetes phylum (Mycobacterium sp.) or Proteobacteria (Pseudomonas sp., Burkholderia sp., etc.). But these are not very efficient degraders of HMW PAHs like pyrene. Bacosa et al. (2013) developed six consortia from sediment mainly consisting of different species of Pseudomonas and Burkholderia capable of degrading various PAHs. Availability of extra carbon or nitrogen sources may be limiting factor or the presence of toxic compounds may retard the degradation. So it is very useful to develop a versatile consortium that can degrade pyrene in the presence of other pollutants with their simultaneous degradation, since Amlakhadi canal received mixed pollutants from different surrounding industries (Patel et al. 2012a; Kathuria 2007).
The present study is focused on the development of efficient microbial consortium from the long-term polluted soil sediments of the Amlakhadi canal, Ankleshwar, which is the tributary of the Narmada River and polluted by extensive discharge of effluents from industries of the Ankleshwar Industrial Estate. Optimization of physicochemical parameters, effects of other additives and surfactants and effect of other hydrocarbons and heavy metals were other significant objectives. The most important objective of the study is a simultaneous degradation of mixture of PAHs (fluoranthene, pyrene, naphthalene, chrysene and phenanthrene) and related hydrocarbons (benzene, toluene and xylene) by developed consortium in simulated microcosms. Thus, study provides an important pace for the further bioremediation process like macrocosm study or reactor scale study.
Materials and methods
Media and chemicals
Bushnell-Haas broth (BHB), nutrient broth and nutrient agar were purchased from Himedia (Mumbai, India). All chemicals used in the study were of analytical and HPLC grade. Pyrene was purchased from Sigma-Aldrich® (Bellefonte, Pennsylvania, USA) with 99% purity.
Development of microbial consortium PBR for degradation of test PAHs
Polluted sediment samples were collected from Amlakhadi canal, Ankleshwar, Gujarat. The microbial consortium for the degradation of pyrene was developed by the culture enrichment method. Ten grams of sediment samples inoculated in 200 ml BHB medium amended with 1000 ppm of pyrene (from a stock solution of 50,000 ppm, filter sterilized by 0.2 µm nylon filters) and incubated under shaking conditions (150 rpm) at 37 °C for nearly 15 days. After 15 days the content of the flask was centrifuged at 200×g for 3 min to discard the sample debris. Ten to fifteen milliliters supernatant was inoculated into the fresh BHB amended with 1000 ppm of pyrene and continued incubation under shaking conditions 37 °C for another 15 days.
After subsequent incubation of 15 days the content of flasks was again centrifuged at 11,000×g rpm for 5 min to harvest the cells. The cells were re-suspended in minimal quantity of sterile distilled water and inoculated in fresh BHB containing 1000 ppm of pyrene. Thus, after repeated sub-culturing of more than 25 times in minimal medium without any nutritional additives and pyrene provided as sole carbon source, consistent degradation of pyrene with stable growth of the consortium was observed. Further, the degradation of pyrene was observed on Bushnell-Haas agar medium spread with 50 µl of stock solution of pyrene. Different dilutions of the consortium were spread on the solid media spiked with pyrene. After incubation of 15 days at 37 °C, Bushnell-Haas agar plates were checked for degradation of pyrene by observing zone of clearance around the colonies. The grown organisms were purified by streaking and regular transfers on solid agar plates.
Characterization of microbial consortium
The enriched developed consortium was serially diluted and spread on Nutrient agar, Bushnell-Haas agar, Bushnell-Haas agar amended with 1000 ppm of pyrene and incubated for 1–8 days at various temperatures. Discrete colonies with distinctive morphology were further screened to pure cultures to enumerate the bacteria present in the consortium. Identification of bacterial isolates was performed using extracting genomic DNA from each pure culture using standard protocol of (Ausubel et al. 1997). 16S rRNA gene was amplified by eubacterial universal primers 8F and 1492R as described in (Desai and Madamwar 2007). The purified amplified product was sequenced using automated ABI 3500 Genetic Analyzer (Thermo Scientific, ABI, USA). Full length of 16S rRNA gene was analyzed by the BLAST tool at NCBI server to identify the bacteria.
Development of Inoculum, degradation conditions and preparation of samples for HPLC
Consortium PBR was grown in BHB amended with 1000 ppm of pyrene under shaking conditions (150 rpm) at 37 °C. Five percent (v/v) of this grown consortium was used as inoculum for further studies. The detailed method has been provided in the Supplementary Information.
The pyrene degradation experiments were conducted in BHM medium amended with 100 ppm of pyrene under shaking conditions of 150 rpm at 37 °C. The PAHs degradation efficiency of consortium PBR was studied by extracting the entire content (100 ml) of the flask and its degraded products in 20 ml of dichloromethane by incubating the mixture under shaking condition at 150 rpm for 1 h followed by separation of aqueous and organic phases under static condition. The 500 µl of separated organic phase (i.e. dichloromethane) collected in fresh microfuge and the solvent was evaporated under vacuum using SpeedVac (Thermo Electron Corporation, Waltham, MA) and the dried content was re-suspended in 1 ml 70% acetonitrile. This prepared sample also diluted by 70% acetonitrile as the pyrene concentration will be in range of 10 ± 5 ppm to compare it with standard of 10 ppm pyrene. HPLC analysis was performed using Prominence LC system (Shimadzu, Japan), on Pursuit 3 PAH C18 reverse phase column (100 mm × 4.6 mm, 3 µm) (Agilent, USA), under ambient conditions, with acetonitrile:water (70:30, v/v) as eluent and isocratic flow rate of 1 ml/min. The standards and degraded products were detected at 254 nm with a Photo Diode Array Detector.
Study of auxiliary nutrient and environmental parameters
For the effective degradation of pyrene by consortium PBR, several parameters were studied and optimized.
Effect of supplementary co-substrates
For enhancing the degradation potential of consortium PBR, BHM was supplemented with peptone, yeast extract, sodium succinate, ammonium nitrate (0.1%, w/v) or glucose (2.0%, w/v) and intermediates such as phthalic acid and salicylic acid (20 g/L) along with 100 ppm of pyrene. Uninoculated media with the respective co-substrates amended with 100 ppm of pyrene were served as abiotic controls. Another set of control experiments was performed with only BHB amended with 100 ppm of pyrene, inoculated with 5% of consortium PBR. All experiments were conducted under shaking conditions of 150 rpm at 37 °C, pH of 7.0 (unless specified) in triplicates.
Effect of environmental conditions
Different environmental factors viz. pH (5.0–9.0), temperature (30–50 °C), dissolved oxygen concentration with respect to shaking speed [0 (static), 50, 100 and 150 rpm], initial pyrene concentrations (100–4000 ppm), presence of different hydrocarbons [fluoranthene, phenanthrene and naphthalene (100 ppm)] other aromatic compounds [benzene, toluene and xylene (0.1%, w/v)], heavy metals at different concentrations [mercury (Hg), lead (Pb), chromium (Cr), cadmium (Cd) and zinc (Zn) at 1, 5 and 10 mM concentrations] surfactants CTAB (Cetyl-trimethyl ammonium bromide), SDS (sodium dodecyl sulfate), Tween-80 and Triton-X-100 (0.02% w/v and v/v) were studied to observed the variable effect on pyrene degradation by the consortium PBR.
The µ max, K S and q max were obtained from the exponential growth phase and degradation rate (while studying the initial substrate concentration) using the Monod equation as described in Ghosh et al. (2014) and Okpokwasili and Nweke (2005).
The effect on indigenous microflora and the ability of the consortium on pyrene degradation during microcosm studies
Pristine, non-sterile soil amended with 100 ppm pyrene, 100 ppm fluoranthene, 500 ppm naphthalene, 250 ppm phenanthrene and 5 ppm chrysene and consortium PBR
Pristine, non-sterile soil amended with 100 ppm pyrene and consortium PBR
Pristine, non-sterile soil amended with 100 ppm pyrene, to determine the ability of indigenous microflora for pyrene degradation
Polluted, non-sterile soil amended with 100 ppm pyrene, 100 ppm fluoranthene, 500 ppm naphthalene, 250 ppm phenanthrene and 5 ppm chrysene and consortium PBR
Polluted, non-sterile soil amended with 100 ppm pyrene, and consortium PBR
Polluted, non-sterile soil amended with 100 ppm pyrene, to determine the ability of indigenous microflora for pyrene degradation
Pristine, sterile soil amended with 100 ppm pyrene, 100 ppm fluoranthene, 500 ppm naphthalene, 250 ppm phenanthrene and 5 ppm chrysene and consortium PBR
Pristine, sterile soil amended with 100 ppm pyrene and consortium PBR
Pristine, sterile soil amended with 100 ppm pyrene, to determine abiotic loss of pyrene
Polluted, sterile soil amended with 100 ppm pyrene, 100 ppm fluoranthene, 500 ppm naphthalene, 250 ppm phenanthrene and 5 ppm chrysene and consortium PBR
Polluted, sterile soil amended with 100 ppm pyrene, and consortium PBR
Polluted, sterile soil amended with 100 ppm pyrene, to determine abiotic loss
Results and discussion
Pyrene is a high-molecular weight polycyclic aromatic hydrocarbon having low water solubility (0.12–0.18 mg/L) which, therefore, tends to sequester in the sediments. Hence, there are relatively very less reports on efficient microbial degradation of pyrene. The present study demonstrated the development of consortium PBR (and its characterization) for the degradation of pyrene from the polluted sediments of Amlakhadi canal, Ankleshwar, Gujarat. The physicochemical parameters of Amlakhadi are shown in Table S1. From Table S1 it is clear that there is the presence of heavy metals in the sediments of the Amlakhadi canal. More than 1200 industrial units are manufacturing petroleum products, chemicals, pesticides, pharmaceuticals, bulk drugs, engineering, plastic and many other products and effluents from these industries are released into the Amlakhadi canal. The continual release of these effluents (for the last four decades) containing compounds of xenobiotic origin, have acclimatize the inhabitant microorganisms which have evolved the necessary resilient mechanisms for their metabolism. Hence it was a pertinent site to obtain the enriched bacterial community, having the inherent capacity to metabolize PAHs.
Development of bacterial consortium PBR and bacterial identification
Growth rate and effect of substrate concentration
It is imperative to determine the load of a pollutant that can be degraded by defined number of microorganisms. In a polluted environment, there is a range of concentrations of different pollutants and microorganisms have different sensitivities towards the various concentrations of pollutants (Leahy and Colwell 1990; Boopathy 2000). In kinetic terms, there is always a threshold concentration below which the pollutants cannot be detected by particular numbers of organisms and above a particular concentration of pollutant; it will prove toxic to microorganisms (Doick et al. 2005; Okpokwasili and Nweke 2005 ). Moreover, it was generally observed that in certain confined range, as the concentration of pollutant increases, the rate of degradation of that pollutant also increases. This can be determined by kinetic parameters viz. specific growth rate (µ), specific degradation rate (q) and half saturation rate constant (k).
Thus, the results provided the significant implications about the substrate sensitivity of the consortium PBR, which prompted us to conduct further experimentation at 100 ppm of pyrene. It was observed that, the efficiency of xenobiotic degradation by microorganisms mostly dependent on the environmental conditions and different parameters needs to be studied precisely so as it can be applied further for large scale actual onsite or reactor scale remediation process. Hence, the effect of each significant factor for PAHs degradation was studied to enhance the degradation potential of the consortium PBR.
Effect of temperature, pH and oxygen concentration
pH is another important factor in the biochemical reactions carried out by enzymes that catalyze the reactions. Figure 3b demonstrates the effect of pH on the degradation of pyrene by PBR. It suggested that for better/efficient degradation of pyrene, lower pH was more effective. At pH 6.0, 70% of 100 ppm of pyrene was degraded within 7 days, while the maximum degradation (97%) was observed at pH 7.0. At higher pH 9.0 nearly 1.8-fold decrease in degradation rate was observed. In various earlier studies it was observed that setting of pH to near neutrality has proven beneficial for the bioremediation of gasoline-contaminated soil, oily sludge in the soil and degradation of octadecane, naphthalene, and other PAHs, e.g. pyrene (Leahy and Colwell 1990; Verstraete et al. 1976; Patrick and DeLaune 1977; Hambrick et al. 1980; Dibble and Bartha 1979). In a recent work, Ravanipour et al. (2015) proved that pH 6.8 was better for the development and maintenance of bacterial consortia for the phenanthrene degradation in artificially contaminated soil. Pure culture study of degradation of pyrene by Rhodococcus sp. UW1 also indicated that, maximum degradation of pyrene and highest activity of dioxygenase system was found at pH 7.1 and 7.2, respectively (Walter et al. 1991).
Nevertheless, PAHs with 2–3 rings are observed to be degraded under anaerobic conditions at very slow rates, but there are very rare observations for the degradation of PAHs with more than three rings (Johnson et al. 2005). Proportional utilization of PAH carbon to natural organic carbon is 3 orders of magnitude higher during cooler months when water temperatures are low and dissolved oxygen (DO) percent saturation is higher and infusion of cooler, well-oxygenated water to the water column overlying contaminated sediments during summer stimulates PAH metabolism (Boyd et al. 2005). In the context of these observations, it was obvious that degradation of pyrene, which possesses four ring clusters, requires high dissolved oxygen concentration and in turn higher shaking conditions.
Effect of organic carbon sources, intermediates and surfactants
In our earlier studies, similar observations were made, where naphthalene degradation by Pseudomonas sp. HOB1 was not enhanced by addition of external surfactants (Pathak et al. 2009). In another study, Patel et al. (2012) also observed that external supplementation of surfactants did not enhance the degradation of phenanthrene by Pseudoxanthomonas sp. DMVP2. The anionic and cationic surfactants SDS and CTAB, respectively, decreased the phenanthrene degradation by consortium ASP (Patel and Madamwar 2013). These results additionally suggested that the strains involved in PAHs degradation might be producing bio-surfactant and further supplementation of surfactant may have negative effect on PAHs degradation (Pathak et al. 2009).
Effect of inoculum size
Effect of other hydrocarbons on pyrene degradation and simultaneous degradation of different PAHs
It is the most important and positive aspect of the developed consortium because during further studies like microcosm or reactor level the consortium must grow in presence of mixture of compounds. The decrease in the rate of degradation of pyrene in the presence of other hydrocarbons may be due to the toxicity of aromatic hydrocarbons like chrysene and fluoranthene. The toxicity is believed to be due to cell membrane disruption by aromatic hydrocarbons (Jacques et al. 2005). Moreover, due to structural similarity of many hydrocarbons, the bacterial oxygenases may utilize more than one hydrocarbon and co-metabolize different substrates, however, at significantly low rates (Ascon-Cabrera and Lebeault 1993). The observed results are very similar with this notion because degradation of pyrene occurs in the presence of other hydrocarbons but at very low rates relative to control experiment where no other hydrocarbons are added.
Effect of heavy metals
The study also revealed another significant observation that the consortium was more resistant towards mercury, i.e. at higher concentration (10 mM) nearly 30% pyrene was degraded after seven days, which was less than 10 mM for another four heavy metals under similar conditions. Moreover, Figure S1 shows the relationship between percent growth retardation by different concentrations of heavy metals which was measured as described by Pepi et al. 2008. EC50 values represent the 50% reduction in the growth of the consortium at the different concentrations of heavy metals. For the consortium of pyrene degradation the EC50 for Pb2+, Hg2+, Cr2+, Zn2+ and Cd2+ is 6, 5, 6.9, 5.2 and 5.4 mM, respectively. The high tolerance to heavy metals may be due to production of bio-surfactant by the consortium (Sandrin et al. 2000) or precipitation of heavy metals by phosphate and sulfate of the medium (Patel et al. 2012b; Hughes and Poole 1991). At higher concentrations lead, mercury and cadmium were precipitated and therefore may not retard the degradation of test PAHs.
Degradation profile of pyrene and stoichiometry
The stoichiometric relation between pyrene and phthalic acid was established and the results indicated that, from 0.4 mM of pyrene, 0.003 mM of phthalic acid was produced through synergistic metabolism of consortial species. That is, the stoichiometric ratio of nearly 1:100 between pyrene and phthalic acid can be postulated for 100 ppm of initial pyrene concentration and can be correlated from HPLC chromatogram of degraded products of pyrene (Fig. 11a, c). Therefore, the results evidently suggested that phthalic acid is not a dead end product of bacterial consortial metabolism, because the ratio of pyrene:phthalic acid was at much lower side. If it could have been a dead end product, the expected ratio would have higher value. Moreover, from Fig. 11c, the HPLC chromatogram of pyrene degradation revealed the emergence of other peaks at different retention time, along with major peak of phthalic acid. The difference between peaks of native pyrene molecule and phthalic acid was comparatively higher which clearly suggested that phthalic acid was not accumulated in the medium and effectively being metabolized by consortium PBR.
This is an important observation for the complete mineralization of pyrene, as Krishnan et al. (2004) observed the accumulation of phthalic acid while monitoring the degradation of phenanthrene by Pseudomonas sp. strain PP2, which have similar structural properties with pyrene. This implied the importance of consortium, where two or more microorganisms synergistically degrade and metabolize the xenobiotic compounds which is recalcitrant for complete degradation by single organism.
The microcosm studies provide an insightful observation about the competence of the consortium PBR for pyrene and other PAHs degradation under soil system and in the presence of the native microflora of polluted and pristine soils. Results from Table 1 evidently suggested that indigenous microflora of the polluted and pristine soils can degrade 39% of the pyrene without augmentation of consortium PBR whereas augmentation increases the degradation of pyrene in polluted and pristine soil to 99%. This indicates the ability of developed consortium to work exclusively in presence of components of pristine soil as well as polluted indigenous which reveal the high efficiency of PBR consortium and reveal positive effect of augmentation of PBR consortium. Also the abiotic loss of PAH is negligible in presence of sterile polluted and pristine soil. It can be noted here that in sterile polluted and pristine soils the degradation occurs at 75 and 77%, respectively, as compared to 99% degradation in both non-sterile polluted and pristine soils, which indicates the aid of indigenous microflora for the degradation by PBR consortium. Thus, indigenous microflora also works in cooperation with consortium to increase the degradation rate of pyrene. Also the degradation of pyrene was higher in the presence of non-sterile polluted samples as compared to sterile polluted samples. The λmax for pyrene in 70% ACN was 340 nm and for degradation calculation the O.D. at this wavelength was taken into consideration. But due to the cyclic structure and multiple benzene rings, pyrene has a complex absorption spectrum in the UV region that is from 200 to 400 nm. Thus, consortium PBR is highly efficient in degrading pyrene in microcosm and may be applicable for the macrocosm and reactor scale study.
The study revealed the effectiveness of developed consortium PBR for pyrene degradation, where it was metabolized as a sole source of carbon and energy through the phthalic acid pathway. The competence of the consortium was revealed by the observations that it individually and simultaneously degraded six different hydrocarbons other than pyrene without supplementing any additional nutrient in BHM. Owing to the growth of consortium and degradation of pyrene in presence of other PAHs and heavy metals, low requirements like nutritional additives and surfactants and successful working in simulated microcosms, the developed consortium is indeed an efficient consortium and can be further used for macrocosm and reactor scale studies.
The authors acknowledge Department of Biotechnology (DBT), Ministry of Science and Technology, New Delhi, India, for the financial support in form of Centre of Excellence and Innovations in Biotechnology (CEIB) (BT/01/CEIB/09/V/05).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest in the publication.
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