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Isolation and characterization of bacterial strains with pyrene metabolic functions from cow dung and Terminalia catappa phylloplane
Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Original research paper
Isolation and characterization of bacterial strains with pyrene metabolic functions from cow dung and Terminalia catappa phylloplane Sunday A. Adebusoye n, Ogechukwu Okpalanozie, Nnedinma C. Nweke Department of Microbiology University of Lagos, Lagos, Nigeria
art ic l e i nf o
a b s t r a c t
Article history: Received 22 May 2015 Received in revised form 7 August 2015 Accepted 26 August 2015
Proteus vulgaris strain CPY1 and Pseudomonas aeruginosa strain LPY1 exhibiting competent pyrene metabolic functions isolated respectively from animal waste and phyllosphere were identified by analysis of their morphological, physiological and biochemical properties. A batch culture experiment with axenic cultures of both strains on 100 mg l 1 pyrene yielded over 88% degradation concomitant with over 3-orders-of-magnitude biomass production. A critical evaluation of the kinetic data showed that over 50% of the PAH was consumed in less than 9 days of cultivation, resulting in an approximate degradation rate of 0.255 mg l 1 h 1 compared with 0.147 mg l 1 h 1 determined for days 9–18. Overall, the degradative competence of both strains did not differ significantly (P o0.05) even though strain CPY1 appeared to utilize the substrate better. This study provides new insight into pyrene degradation, with the first evidence for the likely role of Proteus vulgaris – an enteric bacterium in pyrene metabolism. It further demonstrates that pyrene metabolic capability may be more widespread than previously believed and that such functionality may not be confined to contaminated systems and soil microorganisms. & 2015 Elsevier Ltd. All rights reserved.
Keywords: Pyrene HMW PAH Biodegradation Phyllosphere
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants comprising two or more fused aromatic rings. They are produced and released into the environment through natural and anthropogenic pyrolysis of organic material such as forest fires, automobile exhaust, coal-refining processes and activities of the petroleum industry. Owing to their genotoxicity, carcinogenicity, mutagenicity and recalcitrance to microbial attack (Ghosh et al. 2014; Moscoso et al. 2012; Cerniglia, 1992), most PAHs, particularly high molecular weight (HMW) PAHs, are of great environmental and public health concern. As a consequence, many of the HMW PAHs are listed as priority pollutants by USEPA (Keith and Telliard, 1979; Wang et al. 2000) and also by environmental protection agencies of most countries including Nigeria. Increasing number of aromatic rings and angularity confers greater thermodynamic stability and hydrophobicity to HMW PAHs, hence their persistence in the environment. Their hydrophobic property enables them to adsorb to organic-rich soils and n
Corresponding author. E-mail addresses: [email protected], [email protected] (S.A. Adebusoye).
sediments making them available for biological uptake. As a result, they have a high potential for biomagnification through trophic transfers, thus constituting a serious health threat to aquatic and terrestrial ecosystems. In addition, hydrophobicity is also a factor responsible for their non-bioavailability to microorganisms (Das and Mukherjee, 2007). Pyrene, a four-ring peri-condensed compound, is one of the most abundant HMW PAHs in environmental matrices that has demonstrated striking recalcitrance to microbial degradation. Nevertheless, in spite of its molecular stability and super-hydrophobicity, there have been some reports of microorganisms with remarkable pyrene metabolic functions. Mycobacterium vanbaalenii strain PYR-1, a bacterium originally isolated from estuarine sediment near an oil field, was the first organism reported to metabolize pyrene in a medium supplemented with low level of organic nutrients (Heitkamp and Cerniglia, 1988). Since this initial report, several other organisms, mostly Gram-positive actinomycetes, have been shown to degrade pyrene (Heitkamp et al., 1988; Mueller et al., 1990; Cerniglia, 1992; Wang et al., 2000; Vila et al., 2001; Silva et al., 2009; Kanaly and Harayama, 2010; Wen et al., 2011). The pathways for pyrene metabolism in these organisms unequivocally indicated that the PAH was metabolized through βketoadipic acid via protocatechuic acid (Kim et al., 2007; Kanaly and Harayama, 2010). Previously, reports of pyrene degradation by non-actinomycete bacteria have been scanty in literature.
http://dx.doi.org/10.1016/j.bcab.2015.08.015 1878-8181/& 2015 Elsevier Ltd. All rights reserved.
Please cite this article as: Adebusoye, S.A., et al., Isolation and characterization of bacterial strains with pyrene metabolic functions from cow dung and Terminalia catappa phylloplane. Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.08.015i
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S.A. Adebusoye et al. / Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
However, in the last fifteen years, new non-actinomycete bacteria that possess metabolic capabilities for pyrene degradation have been isolated from different contaminated systems. For instance, Paracoccus sp. strain Ophe1 utilized pyrene as a sole source of carbon and energy in addition to other HMW PAHs (Zhang et al., 2004). An enteric bacterium, Leclercia adecarboxylata strain PS4040 obtained from an oily sludge-contaminated soil, was shown to grow on pyrene resulting in nearly 60% degradation of 200 mg l 1 pyrene over an incubation period of 20 days (Sarma et al., 2004). In another study, Wang et al. (2008) reported that a deep sea bacterium identified as Cycloclasticus spirillensus strain P1 grew excellently well with pyrene. Obayori et al. (2008, 2009) also documented the isolation of pyrene-degrading Pseudomonas aeruginosa strains LP5 and LP6 as well as Pseudomonas sp. strain LP1 from contaminated soils in Lagos, Nigeria. With the exception of LP6, a poor degrader; the organisms, one of which was a biosurfactant producer, utilized over 66% of 100 mg l 1 pyrene in a 30-day incubation period. Currently, the metabolic functions of microorganisms are being challenged by unquantifiable amounts of xenobiotics indiscriminately released into the environment. The effectiveness of bioremediation technologies will largely be dependent on rigorous sourcing for competent microbial strains that will not only attack the target pollutant but also degrade potential co-contaminants within the vicinity of the environmental matrix. The main objective of the present study was to screen for microorganisms with pyrene metabolic capability from two unusual environmental sources, namely plant leave surfaces and animal fecal material. The standard protocol for isolating microorganisms with ability to degrade environmental pollutants is to source them from contaminated sites through repeated enrichment procedures. This process has been mostly successful for the isolation and characterization of pyrene degraders which mostly are actinomycete bacteria. It appears, therefore, that the gene pool for pyrene degradation, even though scarce, is somewhat restricted to Grampositive organisms. We reasoned that using alternative environmental sources, we might be able to isolate unusual microorganisms with unique metabolic capabilities. Interestingly, several workers have shown the abilities of some plants to induce metabolic capabilities in microorganisms including those for polychlorinated biphenyl (PCB) degradation (Gilbert and Crowley, 1997). Cow dung (CD) was recently shown to be rich in hydrocarbonoclastic organisms (Akinde and Obire, 2008; Agamuthu et al., 2013; Okoro et al., 2013). Field et al. (1993) established the association of lignin-degrading organisms with degradation of environmental pollutants. According to them, such organisms may be isolated from fecal materials obtained from animals that consume a woody plant diet. In another study, Juhasz and Naidu (2000) obtained multiple microorganisms from both contaminated and uncontaminated plant and animal fecal materials which were capable of degrading a wide range of pollutants such as PCBs, PAHs, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), and organochlorine pesticides. Previously too, we isolated several bacterial species belonging to different genera from the leave surfaces of Citrus sinensis, Mangifera indica, Cola acuminata, Musa sapiensis, Annona muricata, and Terminalia catappa (Ilori et al., 2006). These organisms grew very well on crude petroleum (CP) and petroleum derivatives. Although the bacteria were able to utilize the aliphatic components of the oil, none had the capacity to attack the aromatic fractions and PAHs and to the best of our knowledge, isolation of such organisms from phylloplanes is yet to be demonstrated for pyrene degradation.
2. Materials and methods 2.1. Chemicals High purity (98%) pyrene, acetone and hexane were purchased from Sigma Aldrich (St. Louis, MO, USA). Escravos light crude petroleum was obtained from Chevron Nigeria Limited. All other chemicals were of analytical grade with high purity. 2.2. Sampling Fresh leaf samples of T. catappa (tropical almond) located behind the Faculty of Science, University of Lagos, Nigeria were collected and placed in a sterile, polyethylene bag. The plant leaves used for this study were those attached to the third and fourth nodes below the terminal bud so as to ensure comparable leaf ages. Fresh CD was collected from cow abattoir in Ikorodu, Lagos and placed in sterile screw-capped bottles. All samples were promptly transported to the laboratory for analysis. 2.3. Enumeration of bacterial populations Heterotrophic bacterial populations of both leaf and animal waste samples were analyzed by standard plate count techniques as previously described by Ilori et al. (2006) and Adebusoye et al. (2007). Populations of hydrocarbon utilizers were estimated on a mineral salts (MS) medium formulated by Kästner et al. (1994). The pH of the medium was adjusted to 7.2 and fortified with nystatin at 50 mg ml 1 to suppress fungal growth. Trace elements solution (1 ml l 1) described by Bauchop and Elsden (1960) was sterilized separately and added aseptically to the medium. Prior to sterilization, the medium was solidified by bacteriological agar and handled as described by Adebusoye et al. (2007). CP served as the sole carbon and energy source and was made available to the cultures through vapor-phase transfer. Unless otherwise stated, all incubations were performed at room temperature (27.072.0 °C) for 2–7 days. 2.4. Isolation of pyrene-degrading bacteria Bacteria able to degrade pyrene were isolated by use of a repeated enrichment technique as described previously (Adebusoye et al. 2007, 2008). Briefly, 2 g of ground dried CD were added into 250 ml Erlenmeyer flask containing 50 ml MS medium. In the case of phyllospheric microorganisms, a sterile cork borer was used to punch a whole sample of T. catappa leaf. The leaf discs were then placed in a flask with 50 ml MS medium. The flask was vortexed vigorously to dislodge the phyllospheric organisms. All flasks were supplemented with 100 mg l 1 pyrene and incubated with shaking for 30 days. After four successive transfers, aliquots of an appropriate dilution of the enriched cultures were inoculated onto nutrient agar as well as MS agar. The MS agar was subsequently coated with a thin film of pyrene and incubated for 10 days. The colonies that appeared were purified and screened for pyrene and CP utilization before taxonomic characterization using an API 20 E test system (bioMerieux Vitek, Hazelwood, MO, USA). 2.5. Evaluation of CP and pyrene biodegradation Hydrocarbon degradation was assayed by inoculating replicate 250 ml flasks containing 50 ml MS medium with fresh bacterial culture. Pyrene was added as a sole carbon and energy source at a concentration of 100 mg l 1 while CP was supplied at a concentration of 2.0% (v/v). Flasks inoculated with heat-killed cells served as controls. Biodegradation was monitored by determination of pH, total viable count (TVC) and residual hydrocarbon at
Please cite this article as: Adebusoye, S.A., et al., Isolation and characterization of bacterial strains with pyrene metabolic functions from cow dung and Terminalia catappa phylloplane. Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.08.015i
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3 days interval.. The data obtained were processed with Microsoft Excel 2013 and subjected to ANOVA using Prism version 6.01 (GraphPad Software, San Diego, CA, USA). 2.6. Analytical methods Residual hydrocarbon was extracted from the culture fluids with an equal volume of n-hexane (Adebusoye et al., 2007). Hexane extracts (1.0 ml) were analyzed with a Hewlett Packard 5890 Series II gas chromatograph (Hewlett Packard Co., Palo Alto, CA, USA) equipped with a flame ionization detector (FID) and 30 m long HP-5 column (internal diameter, 0.25 mm; film thickness,0.25 mm). The carrier gas was nitrogen. The injector and detector temperatures were maintained at 300 °C and 320 °C respectively. The column temperature was programmed to rise from 60 to 300 °C for 26 min. The GC was programmed at an initial temperature of 60 °C; this was held for 2 min, then ramped at 12 °C min 1 to 300 °C and held for 10 min.
3. Results Preliminary investigation of the occurrence of hydrocarbonoclastic microorganisms in the animal waste sample as well as the phylloplane of T. catappa yielded a significantly high population of this physiological group of microorganisms, using CP as the carbon source. Not surprisingly, the proportion of these organisms in heterotrophic community was in the order of 20.8% and 26.1% for CD and T. catappa phylloplane respectively. These values are quite significant compared with less than 1% that would be expected from pristine environmental systems. This probably indicates the presence of hydrocarbon substrates in these samples which are needed to stimulate the proliferation of such huge population of organisms. The phylloplane and CD microflora enriched on MS medium supplemented with pyrene as the sole carbon and energy source were screened for their metabolic potential on the PAH following several successive transfers in the enrichment medium. Analysis of these microbial consortia on nutrient agar revealed multiple bacterial isolates exhibiting varying responses to pyrene and CP metabolism. However, when both enrichment cultures were screened on pyrene-coated MS agar plates, very few colonies were obtained with each surrounded by a large zone of clearance which unequivocally indicated pyrene utilization. Two of these colonies, one from each sample, were subsequently purified by a single subculturing on nutrient agar and characterized for further study. The isolates that were designated CPY1 and LPY1, degraded 1% (v/v) CP in less than 9 days and originated from CD and the phyllopshere of T. catappa respectively. Although typical colonies of strain CPY1 exhibited a characteristic swarming motility on nutrient agar, both strains were Gram-negative highly motile rods. Physiological and biochemical properties of the isolates were obtained using API 20E test kit system. These characteristics enabled putative identification of the organisms as Pseudomonas aeuruginosa strain LPY1 and Proteus vulgaris strain CPY1. Briefly, with the exception of xylose, rhamnose and sorbitol, all other carbohydrates supplied were utilized by strain LPY1 while CPY1 may be referred to as a non-sugar fermenter. Although both organisms were catalase, and Voges-Proskauer-positive, only strain LPY1 exhibited cytochrome oxidase activity and had the requisite enzymic machinery for nitrate reduction and citrate metabolism. On the other hand, strain CPY1 was an indole and H2S producer with urea and gelatin hydrolysis capabilities. Substrate profiling of the organisms revealed that they possess natural abilities to degrade a wide range of natural and xenobiotic organic pollutants. Biphenyl, benzoic acid, 4-chlorobenzoic acid,
Fig. 1. Growth curve (■), pH (●) changes and percent degradation in control (○) as well as experimental (□) flasks during aerobic degradation of crude petroleum by Proteus vulgaris strain CPY1 (a) and Pseudomonas aeruginosa strain LPY1 (b). The substrate was supplied at a concentration of 2.0 % (v/v). In the control flasks, CP was not utilized and minimal abiotic loss occurred. The experiment was repeated at least twice and data points represent the means ± standard deviation of two replicate flasks. Error bars for cell counts were eliminated to improve clarity and in the case of percent degradation for experimental, values were determined with reference to CP recovered from heat-killed controls.
dodecane, hexadecane, naphthalene, anthracene, phenanthrene and dibenzothiophene in addition to petroleum cuts were among the compounds utilized for growth. Both isolates showed visible signs of growth on these organics within 12 h of incubation with the exception of the PAHs and dibenzothiophene which took much longer. In contrast, apart from pyrene, growth was not sustainable on any of the HMW PAHs tested. Other compounds that failed to support growth include phenol, benzene and cyclohexane. When both strains were incubated with 2% (v/v) CP as a sole source of carbon and energy, growth commenced almost immediately without an observable lag period (Fig. 1). Utilization of the oil substrate resulted in over a 300-fold biomass increase concomitant with visual disappearance of the oil slick from the culture broth. This biomass increase occurred within the first 9 days of incubation which, not surprisingly, was a period of intense metabolic activity with nearly 60% depletion in the oil concentration. No significant change in the oil appearance was observed in the controls and no meaningful degradation occurred.
Please cite this article as: Adebusoye, S.A., et al., Isolation and characterization of bacterial strains with pyrene metabolic functions from cow dung and Terminalia catappa phylloplane. Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.08.015i
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S.A. Adebusoye et al. / Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Table 1 Fate of pyrene utilized by strains CPY1 and LPY1 under aerobic batch conditions. Isolate % Degradation Biomass yield
CPY1 LPY1
87.72 86.28
4 300-fold 4 300-fold
Rate of biodegradation (mg L 1 h 1) Days 0–9
Days 9–18
Days 0–18
0.259 0.253
0.147 0.147
0.203 0.20
Isolates were supplied with 100 mg l 1 pyrene and cultivated for 18 days. Degradation rate was calculated with the assumption that utilization per time point was constant throughout the cultivation period. All values presented are means of two replicate determinations.
respectively 13.72% and 12.87% of the initial concentration supplied. A critical evaluation of the kinetic data showed that approximately 32% of the substrate was utilized between days 9 and 18; this suggested an average degradation rate of 0.15 mg l 1 h 1. This value is nearly half of what was obtained during the first 9 days of the experiment which is not surprising according to the data summarized in Fig. 2. It is also noteworthy that biomass production during this period decreased dramatically, just as the pH of the culture media. In all incubations, strains CPY1 and LPY1 seem to possess similar metabolic capabilities irrespective of the choice of hydrocarbon substrate. This inference is further corroborated by the fact that the growth and degradative dynamics of both isolates produced no significant statistical differences (Po 0.05).
4. Discussion
Fig. 2. Growth curve (■), pH (●) changes and percent degradation in control (○) as well as experimental (□) flasks during aerobic degradation of pyrene by Proteus vulgaris strain CPY1 (a) and Pseudomonas aeruginosa strain LPY1 (b). The PAH was supplied at a concentration of 100 mg l 1. In the control flasks, pyrene was not utilized and minimal abiotic loss occurred. The experiment was repeated at least twice and data points represent the means ± standard deviation of two replicate flasks. Error bars for cell counts were eliminated to improve clarity and in the case of percent degradation for experimental, values were determined with reference to pyrene recovered from heat-killed controls.
This observation readily suggests that the extensive hydrocarbon degradation was a function of microbial metabolic activities and not due to physical, chemical or other abiotic components. Incubation beyond 12 days yielded no significant cell increase. In fact, the microbial populations in both flasks took a downward trend (Fig. 1). In addition to substrate depletion, accumulation of toxic acidic products as indicated by a pH reduction of the culture fluids from 7.2 to 6.18 must have played a significant role. When pyrene (100 mg l 1) was supplied as the growth substrate, both organisms grew exponentially in a similar fashion to that observed for CP. Within 9 days of propagation, which also corresponded to periods of rapid biomass production over 50% of the PAH was consumed (see Fig. 2). This metabolic activity translated to an approximate degradation rate of 0.255 mg l 1 h 1 assuming a constant rate of pyrene uptake. After the first 9 days of incubation, degradation of pyrene slowed considerably (Table 1). The amounts of the substrate recovered from the culture fluids of strains LPY1 and CPY1 by the end of the 18- day cultivation were
Microorganisms with specific metabolic capabilities are routinely sourced from soils contaminated with the target compounds, especially when it is believed that prior exposure may, enhance the metabolic potentials of the organisms. In this study, however, bacterial strains with CP and pyrene metabolic potentials were sourced from unusual environmental samples. Prior exposure to pyrene or CP was, therefore, not a necessity for demonstration of such metabolic competence. Worthy of note is the huge collection of hydrocarbonoclastic organisms (26%) from these matrices. Our previous study on the microflora of T. catappa leaf surfaces indicated a similar trend of over 18% hydrocarbon degraders compared with the total microbial community (Ilori et al., 2006). Reports emanating from several investigations have shown that hydrocarbon utilizers often constitute less than 1% of the total microbial community found in pristine systems (Atlas, 1981; Leahy and Colwell, 1990; Van Hamme et al., 2003). According to Atlas (1981), probable sensitive index of environmental exposure to hydrocarbons and by extension, petroleum pollution are population densities of hydrocarbonoclastic organisms and their proportions within the microbial community. Therefore, the high density of hydrocarbonoclastic organisms found in both the phyllosphere and CD may be an indication of prior exposure of these organisms to readily utilizable hydrocarbons. Conversely, since the leaves used for this investigation were obtained from sites with no known history of petroleum hydrocarbon contamination, such hydrocarbons, if any, are most likely to be of biogenic origin. The sources of phyllospheric microorganisms are controversial, some of which are transient, nevertheless, and could have originated from dust, air currents and splashes (Ilori et al., 2006). Their establishment on the phylloplane could have been aided by the abundance of nutrients including exudates or waxes from the cuticle (Rao, 1977). Plant surfaces and interior are important habitats for microorganisms. Some of these organisms actively utilize compounds exuded from leaves of which some
Please cite this article as: Adebusoye, S.A., et al., Isolation and characterization of bacterial strains with pyrene metabolic functions from cow dung and Terminalia catappa phylloplane. Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.08.015i
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have been analysed to be actually hydrocarbons (Swaminathan and Kochlar, 1989). According to Schlegel (1995), some plants synthesize waxes which cover leaf surfaces. Such waxes are mixtures of essential oils, cyclohexane and alkanes which could be alternative carbon and energy sources for both endo/epiphytic microorganisms. The persistency, recalcitrancy and relative dearth of catabolic phenotypes in the environment have renewed interest in the biodegradation of HMW PAHs. As a consequence, many microorganisms, the majority of which are actinomycetes, have been characterized and unequivocally shown to degrade these compounds and use them as a sole source of carbon and energy. Many of these isolates have been used to identify biochemical pathways involved in the catabolism of PAHs they degrade (Kanaly and Harayama, 2000, 2010). However, such metabolic phenomena are not commonly encountered in enteric organisms. The successful isolation of Proteus vulgaris strain CPY1 and its ability to effectively metabolize pyrene is an uncommon feature. Strain CPY1 is a member of enterobactericaea mainly regarded as inhabitants of the intestinal tracts of humans and animals. The ability to degrade pyrene therefore, appears to be an unusual property since this phenomenon is not only associated with soil bacteria but mostly confined to members of the phylum actinobacteria. Sarma et al. (2004) first demonstrated pyrene metabolic functionality in an enteric bacterium, Leclercia adecarboxylata strain PS4040. The authors recorded a 61.5% reduction in pyrene concentration over a period of 20 days in flasks seeded with the organism. In another report, we documented exceptional PCB metabolic capabilities in an Enterobacter sp. strain SA-2 (Adebusoye et al., 2008). Unlike CPY1, strains PS4040 and SA-2 were obtained from soils with a long history of petroleum hydrocarbon and PCB contaminations respectively. Thus prior exposure must have imposed selective pressure and enhancement of their acquisition of degradative ability. In any case, irrespective of the origin of isolation, these reports show a microbial shift in degradation of pyrene and other xenobiotics and that such a property may no longer be an exclusive preserve of soil organisms. The metabolic competence of our isolates is comparable or relatively superior to those previously reported to grow on pyrene. Evaluation of the data summarized in Table 1 and Fig. 2 show that nearly 88% of the substrate was consumed to over the 18-day cultivation even though biomass recovery was somewhat low. Lin and Cai (2008) developed a microbial consortium which achieved a 92.18% removal of 50 mg l 1 pyrene after 21 days of incubation. However, Bacillus cereus strain Py5 and B. megaterium strain Py6 isolated from the consortium were observed to consume respectively 65.8% and 33.7% of a similar substrate concentration. In an earlier study undertaken by Heitkamp and collaborators (1988), strain PYR1 which is now regarded as a model bacterium for HMW PAH degradation could only utilize 52.4% of the pyrene supplied at an initial concentration of 0.5 mg l 1. It must be mentioned that no metabolism occurred until the MS medium was heavily fortified with yeast extract, bacteriological peptone and soluble starch. Bacterial strains LP1 and LP5 also degraded 100 mg l 1 pyrene by nearly 68% during a 30 day incubation (Obayori et al., 2008). The degradation rates recorded for these organisms were in the order of 0.082–0.111 mg l 1 h 1 which are much lower than 0.203 mg l 1 h 1 determined for strains CPY1 and LPY1. In a parallel study where the growth medium was further fortified with corn steep liquor, Obayori et al. (2010) reported an increase of 13.09–37.05% in pyrene utilization within an incubation period of 21 days. Unfortunately, growth of the organisms lagged significantly for 72 h. During the course of this investigation, we could not assay for metabolic products due to limited instrumentation; neither was there any attempt to determine the pathway of pyrene
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metabolism. Although this will be the focus of subsequent investigations, findings from Kanaly and Harayama (2010), Kim et al. (2005), Heitkamp et al. (1988) and Liang et al. (2006) have implicated production of several acidic intermediate products including phenanthrene-4,5-dicarboxylic acid, 4-phenanthroic acid, 1-hydroxy-2-naphthoic acid, 6,6’-dihydroxy-2,2’-biphenyl dicarboxylic acid, phthalic acid and those of the lower protocatechuic acid pathway. Accumulation of these products may be the reason that the change in pH corresponded to the change in pyrene concentration (Fig. 2). A review of the data presented in this paper shows that both strains CPY1 and LPY1 shared common metabolic properties. The isolation of a pseudomonad is not unexpected since it is a metabolically versatile organism readily encountered in most environmental systems and demonstrated to degrade various organic pollutants. The unusual observation is not only in the fact that an enteric strain CPY1 could grow with pyrene but also in the fact that it appeared to degrade the PAH better than LPY1. This study, therefore, provides new insight into pyrene degradation with the first evidence for the likely role of Proteus vulgaris in pyrene metabolism. It further demonstrates that pyrene metabolic capability may be more widespread than previously believed and that such functionality may not be confined to contaminated systems and soil microorganisms.
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Please cite this article as: Adebusoye, S.A., et al., Isolation and characterization of bacterial strains with pyrene metabolic functions from cow dung and Terminalia catappa phylloplane. Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.08.015i
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Please cite this article as: Adebusoye, S.A., et al., Isolation and characterization of bacterial strains with pyrene metabolic functions from cow dung and Terminalia catappa phylloplane. Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.08.015i
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Original research paper
Isolation and characterization of bacterial strains with pyrene metabolic functions from cow dung and Terminalia catappa phylloplane Sunday A. Adebusoye n, Ogechukwu Okpalanozie, Nnedinma C. Nweke Department of Microbiology University of Lagos, Lagos, Nigeria
art ic l e i nf o
a b s t r a c t
Article history: Received 22 May 2015 Received in revised form 7 August 2015 Accepted 26 August 2015
Proteus vulgaris strain CPY1 and Pseudomonas aeruginosa strain LPY1 exhibiting competent pyrene metabolic functions isolated respectively from animal waste and phyllosphere were identified by analysis of their morphological, physiological and biochemical properties. A batch culture experiment with axenic cultures of both strains on 100 mg l 1 pyrene yielded over 88% degradation concomitant with over 3-orders-of-magnitude biomass production. A critical evaluation of the kinetic data showed that over 50% of the PAH was consumed in less than 9 days of cultivation, resulting in an approximate degradation rate of 0.255 mg l 1 h 1 compared with 0.147 mg l 1 h 1 determined for days 9–18. Overall, the degradative competence of both strains did not differ significantly (P o0.05) even though strain CPY1 appeared to utilize the substrate better. This study provides new insight into pyrene degradation, with the first evidence for the likely role of Proteus vulgaris – an enteric bacterium in pyrene metabolism. It further demonstrates that pyrene metabolic capability may be more widespread than previously believed and that such functionality may not be confined to contaminated systems and soil microorganisms. & 2015 Elsevier Ltd. All rights reserved.
Keywords: Pyrene HMW PAH Biodegradation Phyllosphere
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants comprising two or more fused aromatic rings. They are produced and released into the environment through natural and anthropogenic pyrolysis of organic material such as forest fires, automobile exhaust, coal-refining processes and activities of the petroleum industry. Owing to their genotoxicity, carcinogenicity, mutagenicity and recalcitrance to microbial attack (Ghosh et al. 2014; Moscoso et al. 2012; Cerniglia, 1992), most PAHs, particularly high molecular weight (HMW) PAHs, are of great environmental and public health concern. As a consequence, many of the HMW PAHs are listed as priority pollutants by USEPA (Keith and Telliard, 1979; Wang et al. 2000) and also by environmental protection agencies of most countries including Nigeria. Increasing number of aromatic rings and angularity confers greater thermodynamic stability and hydrophobicity to HMW PAHs, hence their persistence in the environment. Their hydrophobic property enables them to adsorb to organic-rich soils and n
Corresponding author. E-mail addresses: [email protected], [email protected] (S.A. Adebusoye).
sediments making them available for biological uptake. As a result, they have a high potential for biomagnification through trophic transfers, thus constituting a serious health threat to aquatic and terrestrial ecosystems. In addition, hydrophobicity is also a factor responsible for their non-bioavailability to microorganisms (Das and Mukherjee, 2007). Pyrene, a four-ring peri-condensed compound, is one of the most abundant HMW PAHs in environmental matrices that has demonstrated striking recalcitrance to microbial degradation. Nevertheless, in spite of its molecular stability and super-hydrophobicity, there have been some reports of microorganisms with remarkable pyrene metabolic functions. Mycobacterium vanbaalenii strain PYR-1, a bacterium originally isolated from estuarine sediment near an oil field, was the first organism reported to metabolize pyrene in a medium supplemented with low level of organic nutrients (Heitkamp and Cerniglia, 1988). Since this initial report, several other organisms, mostly Gram-positive actinomycetes, have been shown to degrade pyrene (Heitkamp et al., 1988; Mueller et al., 1990; Cerniglia, 1992; Wang et al., 2000; Vila et al., 2001; Silva et al., 2009; Kanaly and Harayama, 2010; Wen et al., 2011). The pathways for pyrene metabolism in these organisms unequivocally indicated that the PAH was metabolized through βketoadipic acid via protocatechuic acid (Kim et al., 2007; Kanaly and Harayama, 2010). Previously, reports of pyrene degradation by non-actinomycete bacteria have been scanty in literature.
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Please cite this article as: Adebusoye, S.A., et al., Isolation and characterization of bacterial strains with pyrene metabolic functions from cow dung and Terminalia catappa phylloplane. Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.08.015i
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However, in the last fifteen years, new non-actinomycete bacteria that possess metabolic capabilities for pyrene degradation have been isolated from different contaminated systems. For instance, Paracoccus sp. strain Ophe1 utilized pyrene as a sole source of carbon and energy in addition to other HMW PAHs (Zhang et al., 2004). An enteric bacterium, Leclercia adecarboxylata strain PS4040 obtained from an oily sludge-contaminated soil, was shown to grow on pyrene resulting in nearly 60% degradation of 200 mg l 1 pyrene over an incubation period of 20 days (Sarma et al., 2004). In another study, Wang et al. (2008) reported that a deep sea bacterium identified as Cycloclasticus spirillensus strain P1 grew excellently well with pyrene. Obayori et al. (2008, 2009) also documented the isolation of pyrene-degrading Pseudomonas aeruginosa strains LP5 and LP6 as well as Pseudomonas sp. strain LP1 from contaminated soils in Lagos, Nigeria. With the exception of LP6, a poor degrader; the organisms, one of which was a biosurfactant producer, utilized over 66% of 100 mg l 1 pyrene in a 30-day incubation period. Currently, the metabolic functions of microorganisms are being challenged by unquantifiable amounts of xenobiotics indiscriminately released into the environment. The effectiveness of bioremediation technologies will largely be dependent on rigorous sourcing for competent microbial strains that will not only attack the target pollutant but also degrade potential co-contaminants within the vicinity of the environmental matrix. The main objective of the present study was to screen for microorganisms with pyrene metabolic capability from two unusual environmental sources, namely plant leave surfaces and animal fecal material. The standard protocol for isolating microorganisms with ability to degrade environmental pollutants is to source them from contaminated sites through repeated enrichment procedures. This process has been mostly successful for the isolation and characterization of pyrene degraders which mostly are actinomycete bacteria. It appears, therefore, that the gene pool for pyrene degradation, even though scarce, is somewhat restricted to Grampositive organisms. We reasoned that using alternative environmental sources, we might be able to isolate unusual microorganisms with unique metabolic capabilities. Interestingly, several workers have shown the abilities of some plants to induce metabolic capabilities in microorganisms including those for polychlorinated biphenyl (PCB) degradation (Gilbert and Crowley, 1997). Cow dung (CD) was recently shown to be rich in hydrocarbonoclastic organisms (Akinde and Obire, 2008; Agamuthu et al., 2013; Okoro et al., 2013). Field et al. (1993) established the association of lignin-degrading organisms with degradation of environmental pollutants. According to them, such organisms may be isolated from fecal materials obtained from animals that consume a woody plant diet. In another study, Juhasz and Naidu (2000) obtained multiple microorganisms from both contaminated and uncontaminated plant and animal fecal materials which were capable of degrading a wide range of pollutants such as PCBs, PAHs, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), and organochlorine pesticides. Previously too, we isolated several bacterial species belonging to different genera from the leave surfaces of Citrus sinensis, Mangifera indica, Cola acuminata, Musa sapiensis, Annona muricata, and Terminalia catappa (Ilori et al., 2006). These organisms grew very well on crude petroleum (CP) and petroleum derivatives. Although the bacteria were able to utilize the aliphatic components of the oil, none had the capacity to attack the aromatic fractions and PAHs and to the best of our knowledge, isolation of such organisms from phylloplanes is yet to be demonstrated for pyrene degradation.
2. Materials and methods 2.1. Chemicals High purity (98%) pyrene, acetone and hexane were purchased from Sigma Aldrich (St. Louis, MO, USA). Escravos light crude petroleum was obtained from Chevron Nigeria Limited. All other chemicals were of analytical grade with high purity. 2.2. Sampling Fresh leaf samples of T. catappa (tropical almond) located behind the Faculty of Science, University of Lagos, Nigeria were collected and placed in a sterile, polyethylene bag. The plant leaves used for this study were those attached to the third and fourth nodes below the terminal bud so as to ensure comparable leaf ages. Fresh CD was collected from cow abattoir in Ikorodu, Lagos and placed in sterile screw-capped bottles. All samples were promptly transported to the laboratory for analysis. 2.3. Enumeration of bacterial populations Heterotrophic bacterial populations of both leaf and animal waste samples were analyzed by standard plate count techniques as previously described by Ilori et al. (2006) and Adebusoye et al. (2007). Populations of hydrocarbon utilizers were estimated on a mineral salts (MS) medium formulated by Kästner et al. (1994). The pH of the medium was adjusted to 7.2 and fortified with nystatin at 50 mg ml 1 to suppress fungal growth. Trace elements solution (1 ml l 1) described by Bauchop and Elsden (1960) was sterilized separately and added aseptically to the medium. Prior to sterilization, the medium was solidified by bacteriological agar and handled as described by Adebusoye et al. (2007). CP served as the sole carbon and energy source and was made available to the cultures through vapor-phase transfer. Unless otherwise stated, all incubations were performed at room temperature (27.072.0 °C) for 2–7 days. 2.4. Isolation of pyrene-degrading bacteria Bacteria able to degrade pyrene were isolated by use of a repeated enrichment technique as described previously (Adebusoye et al. 2007, 2008). Briefly, 2 g of ground dried CD were added into 250 ml Erlenmeyer flask containing 50 ml MS medium. In the case of phyllospheric microorganisms, a sterile cork borer was used to punch a whole sample of T. catappa leaf. The leaf discs were then placed in a flask with 50 ml MS medium. The flask was vortexed vigorously to dislodge the phyllospheric organisms. All flasks were supplemented with 100 mg l 1 pyrene and incubated with shaking for 30 days. After four successive transfers, aliquots of an appropriate dilution of the enriched cultures were inoculated onto nutrient agar as well as MS agar. The MS agar was subsequently coated with a thin film of pyrene and incubated for 10 days. The colonies that appeared were purified and screened for pyrene and CP utilization before taxonomic characterization using an API 20 E test system (bioMerieux Vitek, Hazelwood, MO, USA). 2.5. Evaluation of CP and pyrene biodegradation Hydrocarbon degradation was assayed by inoculating replicate 250 ml flasks containing 50 ml MS medium with fresh bacterial culture. Pyrene was added as a sole carbon and energy source at a concentration of 100 mg l 1 while CP was supplied at a concentration of 2.0% (v/v). Flasks inoculated with heat-killed cells served as controls. Biodegradation was monitored by determination of pH, total viable count (TVC) and residual hydrocarbon at
Please cite this article as: Adebusoye, S.A., et al., Isolation and characterization of bacterial strains with pyrene metabolic functions from cow dung and Terminalia catappa phylloplane. Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.08.015i
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3 days interval.. The data obtained were processed with Microsoft Excel 2013 and subjected to ANOVA using Prism version 6.01 (GraphPad Software, San Diego, CA, USA). 2.6. Analytical methods Residual hydrocarbon was extracted from the culture fluids with an equal volume of n-hexane (Adebusoye et al., 2007). Hexane extracts (1.0 ml) were analyzed with a Hewlett Packard 5890 Series II gas chromatograph (Hewlett Packard Co., Palo Alto, CA, USA) equipped with a flame ionization detector (FID) and 30 m long HP-5 column (internal diameter, 0.25 mm; film thickness,0.25 mm). The carrier gas was nitrogen. The injector and detector temperatures were maintained at 300 °C and 320 °C respectively. The column temperature was programmed to rise from 60 to 300 °C for 26 min. The GC was programmed at an initial temperature of 60 °C; this was held for 2 min, then ramped at 12 °C min 1 to 300 °C and held for 10 min.
3. Results Preliminary investigation of the occurrence of hydrocarbonoclastic microorganisms in the animal waste sample as well as the phylloplane of T. catappa yielded a significantly high population of this physiological group of microorganisms, using CP as the carbon source. Not surprisingly, the proportion of these organisms in heterotrophic community was in the order of 20.8% and 26.1% for CD and T. catappa phylloplane respectively. These values are quite significant compared with less than 1% that would be expected from pristine environmental systems. This probably indicates the presence of hydrocarbon substrates in these samples which are needed to stimulate the proliferation of such huge population of organisms. The phylloplane and CD microflora enriched on MS medium supplemented with pyrene as the sole carbon and energy source were screened for their metabolic potential on the PAH following several successive transfers in the enrichment medium. Analysis of these microbial consortia on nutrient agar revealed multiple bacterial isolates exhibiting varying responses to pyrene and CP metabolism. However, when both enrichment cultures were screened on pyrene-coated MS agar plates, very few colonies were obtained with each surrounded by a large zone of clearance which unequivocally indicated pyrene utilization. Two of these colonies, one from each sample, were subsequently purified by a single subculturing on nutrient agar and characterized for further study. The isolates that were designated CPY1 and LPY1, degraded 1% (v/v) CP in less than 9 days and originated from CD and the phyllopshere of T. catappa respectively. Although typical colonies of strain CPY1 exhibited a characteristic swarming motility on nutrient agar, both strains were Gram-negative highly motile rods. Physiological and biochemical properties of the isolates were obtained using API 20E test kit system. These characteristics enabled putative identification of the organisms as Pseudomonas aeuruginosa strain LPY1 and Proteus vulgaris strain CPY1. Briefly, with the exception of xylose, rhamnose and sorbitol, all other carbohydrates supplied were utilized by strain LPY1 while CPY1 may be referred to as a non-sugar fermenter. Although both organisms were catalase, and Voges-Proskauer-positive, only strain LPY1 exhibited cytochrome oxidase activity and had the requisite enzymic machinery for nitrate reduction and citrate metabolism. On the other hand, strain CPY1 was an indole and H2S producer with urea and gelatin hydrolysis capabilities. Substrate profiling of the organisms revealed that they possess natural abilities to degrade a wide range of natural and xenobiotic organic pollutants. Biphenyl, benzoic acid, 4-chlorobenzoic acid,
Fig. 1. Growth curve (■), pH (●) changes and percent degradation in control (○) as well as experimental (□) flasks during aerobic degradation of crude petroleum by Proteus vulgaris strain CPY1 (a) and Pseudomonas aeruginosa strain LPY1 (b). The substrate was supplied at a concentration of 2.0 % (v/v). In the control flasks, CP was not utilized and minimal abiotic loss occurred. The experiment was repeated at least twice and data points represent the means ± standard deviation of two replicate flasks. Error bars for cell counts were eliminated to improve clarity and in the case of percent degradation for experimental, values were determined with reference to CP recovered from heat-killed controls.
dodecane, hexadecane, naphthalene, anthracene, phenanthrene and dibenzothiophene in addition to petroleum cuts were among the compounds utilized for growth. Both isolates showed visible signs of growth on these organics within 12 h of incubation with the exception of the PAHs and dibenzothiophene which took much longer. In contrast, apart from pyrene, growth was not sustainable on any of the HMW PAHs tested. Other compounds that failed to support growth include phenol, benzene and cyclohexane. When both strains were incubated with 2% (v/v) CP as a sole source of carbon and energy, growth commenced almost immediately without an observable lag period (Fig. 1). Utilization of the oil substrate resulted in over a 300-fold biomass increase concomitant with visual disappearance of the oil slick from the culture broth. This biomass increase occurred within the first 9 days of incubation which, not surprisingly, was a period of intense metabolic activity with nearly 60% depletion in the oil concentration. No significant change in the oil appearance was observed in the controls and no meaningful degradation occurred.
Please cite this article as: Adebusoye, S.A., et al., Isolation and characterization of bacterial strains with pyrene metabolic functions from cow dung and Terminalia catappa phylloplane. Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.08.015i
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S.A. Adebusoye et al. / Biocatalysis and Agricultural Biotechnology ∎ (∎∎∎∎) ∎∎∎–∎∎∎ Table 1 Fate of pyrene utilized by strains CPY1 and LPY1 under aerobic batch conditions. Isolate % Degradation Biomass yield
CPY1 LPY1
87.72 86.28
4 300-fold 4 300-fold
Rate of biodegradation (mg L 1 h 1) Days 0–9
Days 9–18
Days 0–18
0.259 0.253
0.147 0.147
0.203 0.20
Isolates were supplied with 100 mg l 1 pyrene and cultivated for 18 days. Degradation rate was calculated with the assumption that utilization per time point was constant throughout the cultivation period. All values presented are means of two replicate determinations.
respectively 13.72% and 12.87% of the initial concentration supplied. A critical evaluation of the kinetic data showed that approximately 32% of the substrate was utilized between days 9 and 18; this suggested an average degradation rate of 0.15 mg l 1 h 1. This value is nearly half of what was obtained during the first 9 days of the experiment which is not surprising according to the data summarized in Fig. 2. It is also noteworthy that biomass production during this period decreased dramatically, just as the pH of the culture media. In all incubations, strains CPY1 and LPY1 seem to possess similar metabolic capabilities irrespective of the choice of hydrocarbon substrate. This inference is further corroborated by the fact that the growth and degradative dynamics of both isolates produced no significant statistical differences (Po 0.05).
4. Discussion
Fig. 2. Growth curve (■), pH (●) changes and percent degradation in control (○) as well as experimental (□) flasks during aerobic degradation of pyrene by Proteus vulgaris strain CPY1 (a) and Pseudomonas aeruginosa strain LPY1 (b). The PAH was supplied at a concentration of 100 mg l 1. In the control flasks, pyrene was not utilized and minimal abiotic loss occurred. The experiment was repeated at least twice and data points represent the means ± standard deviation of two replicate flasks. Error bars for cell counts were eliminated to improve clarity and in the case of percent degradation for experimental, values were determined with reference to pyrene recovered from heat-killed controls.
This observation readily suggests that the extensive hydrocarbon degradation was a function of microbial metabolic activities and not due to physical, chemical or other abiotic components. Incubation beyond 12 days yielded no significant cell increase. In fact, the microbial populations in both flasks took a downward trend (Fig. 1). In addition to substrate depletion, accumulation of toxic acidic products as indicated by a pH reduction of the culture fluids from 7.2 to 6.18 must have played a significant role. When pyrene (100 mg l 1) was supplied as the growth substrate, both organisms grew exponentially in a similar fashion to that observed for CP. Within 9 days of propagation, which also corresponded to periods of rapid biomass production over 50% of the PAH was consumed (see Fig. 2). This metabolic activity translated to an approximate degradation rate of 0.255 mg l 1 h 1 assuming a constant rate of pyrene uptake. After the first 9 days of incubation, degradation of pyrene slowed considerably (Table 1). The amounts of the substrate recovered from the culture fluids of strains LPY1 and CPY1 by the end of the 18- day cultivation were
Microorganisms with specific metabolic capabilities are routinely sourced from soils contaminated with the target compounds, especially when it is believed that prior exposure may, enhance the metabolic potentials of the organisms. In this study, however, bacterial strains with CP and pyrene metabolic potentials were sourced from unusual environmental samples. Prior exposure to pyrene or CP was, therefore, not a necessity for demonstration of such metabolic competence. Worthy of note is the huge collection of hydrocarbonoclastic organisms (26%) from these matrices. Our previous study on the microflora of T. catappa leaf surfaces indicated a similar trend of over 18% hydrocarbon degraders compared with the total microbial community (Ilori et al., 2006). Reports emanating from several investigations have shown that hydrocarbon utilizers often constitute less than 1% of the total microbial community found in pristine systems (Atlas, 1981; Leahy and Colwell, 1990; Van Hamme et al., 2003). According to Atlas (1981), probable sensitive index of environmental exposure to hydrocarbons and by extension, petroleum pollution are population densities of hydrocarbonoclastic organisms and their proportions within the microbial community. Therefore, the high density of hydrocarbonoclastic organisms found in both the phyllosphere and CD may be an indication of prior exposure of these organisms to readily utilizable hydrocarbons. Conversely, since the leaves used for this investigation were obtained from sites with no known history of petroleum hydrocarbon contamination, such hydrocarbons, if any, are most likely to be of biogenic origin. The sources of phyllospheric microorganisms are controversial, some of which are transient, nevertheless, and could have originated from dust, air currents and splashes (Ilori et al., 2006). Their establishment on the phylloplane could have been aided by the abundance of nutrients including exudates or waxes from the cuticle (Rao, 1977). Plant surfaces and interior are important habitats for microorganisms. Some of these organisms actively utilize compounds exuded from leaves of which some
Please cite this article as: Adebusoye, S.A., et al., Isolation and characterization of bacterial strains with pyrene metabolic functions from cow dung and Terminalia catappa phylloplane. Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.08.015i
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have been analysed to be actually hydrocarbons (Swaminathan and Kochlar, 1989). According to Schlegel (1995), some plants synthesize waxes which cover leaf surfaces. Such waxes are mixtures of essential oils, cyclohexane and alkanes which could be alternative carbon and energy sources for both endo/epiphytic microorganisms. The persistency, recalcitrancy and relative dearth of catabolic phenotypes in the environment have renewed interest in the biodegradation of HMW PAHs. As a consequence, many microorganisms, the majority of which are actinomycetes, have been characterized and unequivocally shown to degrade these compounds and use them as a sole source of carbon and energy. Many of these isolates have been used to identify biochemical pathways involved in the catabolism of PAHs they degrade (Kanaly and Harayama, 2000, 2010). However, such metabolic phenomena are not commonly encountered in enteric organisms. The successful isolation of Proteus vulgaris strain CPY1 and its ability to effectively metabolize pyrene is an uncommon feature. Strain CPY1 is a member of enterobactericaea mainly regarded as inhabitants of the intestinal tracts of humans and animals. The ability to degrade pyrene therefore, appears to be an unusual property since this phenomenon is not only associated with soil bacteria but mostly confined to members of the phylum actinobacteria. Sarma et al. (2004) first demonstrated pyrene metabolic functionality in an enteric bacterium, Leclercia adecarboxylata strain PS4040. The authors recorded a 61.5% reduction in pyrene concentration over a period of 20 days in flasks seeded with the organism. In another report, we documented exceptional PCB metabolic capabilities in an Enterobacter sp. strain SA-2 (Adebusoye et al., 2008). Unlike CPY1, strains PS4040 and SA-2 were obtained from soils with a long history of petroleum hydrocarbon and PCB contaminations respectively. Thus prior exposure must have imposed selective pressure and enhancement of their acquisition of degradative ability. In any case, irrespective of the origin of isolation, these reports show a microbial shift in degradation of pyrene and other xenobiotics and that such a property may no longer be an exclusive preserve of soil organisms. The metabolic competence of our isolates is comparable or relatively superior to those previously reported to grow on pyrene. Evaluation of the data summarized in Table 1 and Fig. 2 show that nearly 88% of the substrate was consumed to over the 18-day cultivation even though biomass recovery was somewhat low. Lin and Cai (2008) developed a microbial consortium which achieved a 92.18% removal of 50 mg l 1 pyrene after 21 days of incubation. However, Bacillus cereus strain Py5 and B. megaterium strain Py6 isolated from the consortium were observed to consume respectively 65.8% and 33.7% of a similar substrate concentration. In an earlier study undertaken by Heitkamp and collaborators (1988), strain PYR1 which is now regarded as a model bacterium for HMW PAH degradation could only utilize 52.4% of the pyrene supplied at an initial concentration of 0.5 mg l 1. It must be mentioned that no metabolism occurred until the MS medium was heavily fortified with yeast extract, bacteriological peptone and soluble starch. Bacterial strains LP1 and LP5 also degraded 100 mg l 1 pyrene by nearly 68% during a 30 day incubation (Obayori et al., 2008). The degradation rates recorded for these organisms were in the order of 0.082–0.111 mg l 1 h 1 which are much lower than 0.203 mg l 1 h 1 determined for strains CPY1 and LPY1. In a parallel study where the growth medium was further fortified with corn steep liquor, Obayori et al. (2010) reported an increase of 13.09–37.05% in pyrene utilization within an incubation period of 21 days. Unfortunately, growth of the organisms lagged significantly for 72 h. During the course of this investigation, we could not assay for metabolic products due to limited instrumentation; neither was there any attempt to determine the pathway of pyrene
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metabolism. Although this will be the focus of subsequent investigations, findings from Kanaly and Harayama (2010), Kim et al. (2005), Heitkamp et al. (1988) and Liang et al. (2006) have implicated production of several acidic intermediate products including phenanthrene-4,5-dicarboxylic acid, 4-phenanthroic acid, 1-hydroxy-2-naphthoic acid, 6,6’-dihydroxy-2,2’-biphenyl dicarboxylic acid, phthalic acid and those of the lower protocatechuic acid pathway. Accumulation of these products may be the reason that the change in pH corresponded to the change in pyrene concentration (Fig. 2). A review of the data presented in this paper shows that both strains CPY1 and LPY1 shared common metabolic properties. The isolation of a pseudomonad is not unexpected since it is a metabolically versatile organism readily encountered in most environmental systems and demonstrated to degrade various organic pollutants. The unusual observation is not only in the fact that an enteric strain CPY1 could grow with pyrene but also in the fact that it appeared to degrade the PAH better than LPY1. This study, therefore, provides new insight into pyrene degradation with the first evidence for the likely role of Proteus vulgaris in pyrene metabolism. It further demonstrates that pyrene metabolic capability may be more widespread than previously believed and that such functionality may not be confined to contaminated systems and soil microorganisms.
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Please cite this article as: Adebusoye, S.A., et al., Isolation and characterization of bacterial strains with pyrene metabolic functions from cow dung and Terminalia catappa phylloplane. Biocatal. Agric. Biotechnol. (2015), http://dx.doi.org/10.1016/j.bcab.2015.08.015i