Xiaoqiang Jia,Yun He,Lei Huang,Dawei Jiang,Wenyu Lu,*

1Department of Biochemical Engineering,School of Chemical Engineering and Technology,Tianjin University,Tianjin 300072,China

2Key Laboratory of Systems Bioengineering(Tianjin University),Ministry of Education,Tianjin 300072,China

3Synthetic Biology Platform,Collaborative Innovation Center of Chemical Science and Engineering(Tianjin),Tianjin 300072,China

Keywords:Biodegradation Metabolite n-Hexadecane Pyrene Rhodococcus sp.T1

A B S T R A C T The high-molecular weight polycyclic aromatic hydrocarbons(PAHs)pyrene and typical long chain alkane nhexadecane are both difficult to degrade.In this study,n-hexadecane and pyrene degrading strain Rhodococcus sp.T1 was isolated from oil contaminated soil.Strain T1 could remove 90.81%n-hexadecane(2 vol%)and 42.79%pyrene(200 mg·L?1)as a single carbon within 5 days,respectively.Comparatively,the degradation of pyrene increased to 60.63%,but the degradation of n-hexadecane decreased to 87.55%when these compounds were mixed.Additionally,identification and analysis of degradation metabolites of Rhodococcus sp.T1 in the above experiments showed that there were significant changes in alanine,methylamine,citric acid and heptadecanoic acid between sole and dual substrate degradation.The optimal conditions for degradation were then determined based on analysis of the pH,salinity,additional nutrient sources and liquid surface activity.Under the optimal conditions of pH 7.0,35 °C,0.5%NaCl,5 mg·L?1of yeast extract and 90 mg·L?1of surfactant,thedegradationincreasedinsingleordualcarbonsources.Toourknowledge,thisisthefirststudytodiscussmetabolite changes in Rhodococcus sp.T1 using sole substrate and dual substrate to enhance the long-chain alkanes and PAHs degradation potential.

1.Introduction

Oil spills occur constantly and could therefore be potential hazards for ecosystem and human health[1].Alkanes and aromatic hydrocarbons,which are the major components of crude oils,are commonly found in oil contaminated environments.These compounds and highly recalcitrant and ubiquitouscompounds are presentin theenvironment.Polycyclic aromatic hydrocarbons are categorized according to their structureandnumber of rings.Commonly foundPAHsincludebenzene,naphthalene,anthracene,phenanthrene,benzopyrene,and pyrene[2],while n-Hexadecane is a typical alkene[3].These compounds are all carcinogenic and difficult to degrade;however,their potential to harm the environment is commonly overlooked[4].

To date,various methods have been developed to control environmental pollution;physical treatments cannot remove the pollutants completely,and the main disadvantage is too costly[5].Whereas,chemical treatments were likely to cause a second pollution to the environment due to the unexpected release of artificial toxic materials into the environment[6].Conversely,bioremediation is efficient,costeffective and versatile for remediation of oil contaminants;therefore,it can be regarded as a valid alternative to classical physicochemical methods[7].Efficient biodegradation techniques have recently been applied to hazardous chemicals from soil[8].Moreover,there have been many reports of the ability of microorganisms to utilize hydrocarbons,especially n-alkanes,as carbon sources[9],and microorganisms play an important role in removal of low or high molecular weight PAHs from oil contaminated sites as the sole source of carbon[10].It is well known that pyrene contains four aromatic rings and is therefore not easily degraded by common bacteria.However,a few species can sufficiently decompose pyrene to be useful in bioremediation applications,includingAlicycliphilussp.,Stenotrophomonassp.,andRhodococcus sp.[11].Walter etal.[12]isolatedRhodococcus sp.UW1,whichwasable to mineralize 72%of pyrene within two weeks.Although there is a wealth of literature describing microorganisms with excellent abilities for degrading hydrocarbons with different chains,most of these can utilize only a narrow range of hydrocarbons[13,14].Typically,PAHs occur together with other long-chain alkanes as complex mixtures in contaminated sites,whichhinders theirremoval[15].As a result,remediation of PAHs and long-chain-alkanes pollution has remained a challenge for researchers.Despite increased attention to methods for biodegradation of PAHs and long-chain-alkanes,little information is available regarding their interaction mechanisms[16-18].

The underlying metabolic differences that control the responses of bacterial cells to pyrene and n-hexadecane,such as changes of intracellular metabolites,remain largely unexplored[3].Metabolomics,which is the study of the interactions of living organisms with their natural environments at the metabolic level[19],has been successfully applied to investigate changes in intracellular metabolites when bacteria suffer from heat shock,oxidative stress,osmotic stress,metal stress and solvent stress[20].A variety of toxic pollutants together will cause greater intracellular metabolic changes in the level of cells[21].Eshelli et al.[22]investigated the metabolomics of the bio-degradation of aflatoxin B1 by Actinomycetes to determine the optimized biodegradation conditions.To elucidate quorum-sensing-associated gene regulon member functions,Scott et al.[23]characterized their regulation,interactions with each other and contributions to the metabolome,while minimizing endogenously generated cytotoxicity.Studying metabolism is inherently challenging because of metabolites'reactivity,structural diversity,and broad concentration ranges[24].However,few studies have investigated metabolomics associated with microbial environmental management,especially bioremediation of PAHs and long-chain-alkanes by prokaryotic microorganisms.

In this study,we investigated the biodegradation processes of twotypicalhighlytoxic,carcinogenic,mutagenic andteratogenic petroleum hydrocarbons(n-hexadecane,pyrene)by the newly isolated Rhodococcus sp.T1.Additionally,we analyzed the metabolic changes in Rhodococcus sp.T1 in response to unique combinations of substrate(n-hexadecane,pyrene)for the first time.The optimal conditions for degradation of the aforementioned compounds by strain T1 were then identified based on the metabolic analysis.Finally,we report the separate impacts of n-hexadecane and pyrene on bacterial growth and degradation of the two compounds.

2.Materials and Methods

2.1.Chemicals

Pyrene,phenanthrene and n-hexadecane(all purity≥99%)were purchased from Aladdin Industrial Corporation(China).Peptone and yeast extract were acquired from Tianjin Guangfu Institute of Fine Chemicals(China).All other chemicals were purchased from Tianjin Yuanli Chemical Co.,Ltd.(China).

2.2.Culture media

Luria-Bertanimedium(LB)contained(perliterofdistilledH2O)10g peptone,5 g yeast extract and 5 g NaCl.For the preparation of nutrient agar plates,15 g·L?1agar was added.

Mineral salt medium(MSM)contained(per liter of dH2O)5 g NaCl,1 g NH4NO3,1 g K2HPO4,1 g KH2PO4,and 0.5 g MgSO4·7H2O.For preparation of MSM agar plates,15 g·L?1agar was added.

All media were adjusted to pH 7.3 and autoclaved at 121°C for 20 min.

2.3.Isolation and screening of degrading strains

Rhodococcus sp.T1 was isolated from oil contaminated soil at Dagang Oilfield as previously described[10],with some modifications.The oil contaminated soil(2 g)was suspended in 250 ml Erlenmeyerflasks containing 100 ml MSM with crude oil 5 g.The samples were then cultured in a shaker at 30 °C and 200 r·min?1for 7 days.Next,1 ml of culture solution was transferred into another fresh culture solution with 2 g·L?1pyrene and incubated for 7 days,after which 4 g·L?1pyrene was added for 7 days.The final cultures were diluted by a factor of 10 and plated onto agar MSM plates containing 0.5%crude oil,then incubated at 30°C for 36-48 h.

2.4.Denaturing gradient gel electrophoresis(DGGE)analysis

Denaturing gradient gel electrophoresis was utilized to dynamically monitor changes in the flora structure.Amplification was performed as previously described[25].Genomic DNA was extracted using a PowerSoil DNA isolation kit(MO BIO Laboratories)under the guidance ofthemanufacturer'sinstructions.NestedPCRwasemployedtoamplify theV3regionof16SrRNAwithprimers338F(5′-CGCCCGCCGCGCGCGG CGGGCGGGGCGGGGGCACGGGGGG-ACTCCTACGGGAGGCAGCAG-3′)and 518R(5′-ATTACCGCGGCTGCTGG-3′)under standard reaction conditions with Taq DNA polymerase.Denaturing gradient gel electrophoresis was performed with a DCode?universal mutation detection system(BioRad Laboratories,Hercules,CA)using a denatured gradient ranging from 30%to 60%.

2.5.Identification of degrading strains

The morphological,physiological,and phylogenetic properties of the strains were assayed based on 16S rRNA analysis as previously described[26].The 16S rRNA gene fragments were amplified using the PCR universal primers 27F(5′-AGAGTTTGATCCTGGCTCAG-3′)and 1492R(5′-GGTTACCTTGTTACGACTT-3′)understandardreactionconditions with Taq DNA polymerase(TransGen Biotech).Theproducts were then sequenced by GENEWIZ(Suzhou)Biological Technology Co.,Ltd.,and imported into the CLUSTALX program for assembly and alignment.

2.6.Degradation of n-Hexadecane and pyrene

Changes of strain T1 during growth under different substrates were identified by cell density,which wasmonitored spectrophotometrically by measuring the optical density at 600 nm(OD600).

Afterculturein100 mlofLBmediumfor24hat35°C and shakingat 200 r·min?1,cells were harvested by centrifugation at 1000 ×g for 5 min.Next,2 ml aliquots of cell suspensions(OD600=2.0)were used to inoculate 100 ml of MSM amended with 2%n-hexadecane(v/v)and/or 200 mg·L?1pyrene in shaking flasks.Samples were then incubated at 35 °C and shaken at 200 r·min?1for 5 days.Each carbon source was tested with one blank control trial and three parallel experiments.Residual carbon was extracted using dichloromethane and analyzed by gas chromatography-mass spectrometry(GC/MS).

The GC-MS system consisted of a gas chromatograph(Agilent 6890 N)equipped with a 30 m HP-5MS(0.25 mm × 0.25 μm film),an Agilent 5975C MSD and an Agilent 7683B(Agilent Technologies,Palo Alto,USA)operating at 70 eV.Helium was employed as the carrier gas and the average flow rate was 35.0 ml·min?1.Separation on the column was achieved by using an initial temperature of 40°C for 3 min,followed by an increase to 300 °C at 6 °C·min?1.The sample size was 1 μl and the diversion rate was 1:1.

2.7.Determination of intracellular metabolites

Sample quenching and extraction of intracellular metabolites were conducted as described by Wentzel et al.[27],with some modifications.Briefly,cells were sampled after the degradation experiment was completed.Specifically,at day 5,10 ml culture samples weretaken from the fermentation vessel and immediately filtered.The cells on the filters were subsequently washed with 10 ml 0.9%NaCl solution,then transferred to a 50 ml tube containing 25 ml 60%methanol solution that had been pre-chilled in an ethanol bath at?40 °C.The mixture was subsequently centrifuged at 3000×g for 3 min,after which the supernatant was removed.Finally,the samples were frozen at?80°C until further processing.

Cell drying and metabolite derivatization were then performed as described by Xia et al.[28].Briefly,cell pellets that had been stored at?80 °C were suspended with 2.5 ml of cold methanol-water solution(50 vol%,?30 °C)and thawed in an ice bath for 4 min.Next,the mixture was vigorously mixed with a vortex mixer(HYQ-3110,Crystal Technology,Jiangsu,China)for1 min,thenfrozenat?80°C for 30 min.This freeze-thaw cycle was repeated three times,after which the samples werecentrifugedat10000×g for15minat?20°C.Thesupernatant was then collected and amended with 2.5 ml cold methanolwater solution(50 vol%,?40 °C)to extract the cell pellet again.The two extracts were combined,after which 10 ml of 0.1 mg·ml?1succinic d4(St.Louis,MO)andD-sorbitol-13C6(St.Louis,MO)were added to 100 ml of extract.

Following lyophilization(ALPHA 1-2LD PLUS,Christ,Germany),the samples were subjected to two-stage chemical derivatization[29].First,samples were freeze-dried by dissolution in 50 ml methoxamine hydrochloride(20 mg·ml?1in pyridine)followed by incubation at 40°C for 80 min.Next,80 ml N-methyl-N-(trimethylsilyl)trifluoroacetamidewasaddedtothesamples,whichweresubsequently incubated at 40°C for 80 min.Finally,derivatized metabolite samples were analyzed by GC-MS using the method described in Section 2.6.

2.8.Preliminary optimization of biodegradation by strain T1

Optimization was conducted to maximize the degradation rates of n-hexadecane and pyrene.To accomplish this,the biodegradation experiments were performed in flasks at 35 °C and 200 r·min?1under different pH,salinity,nutrient concentrations and liquid surface activity,during which time samples were collected once every five days.Specifically,pH values of 6 to 8 were evaluated by adjusting the sample solutions with 1 mol·L?1NaOH or 1 M HCl.Additionally,NaCl concentrations of 0.5%to 2%,were investigated.The effects of nutrient sources were measured by amending samples with yeast extracts at 5 mg·L?1to20mg·L?1.Finally,the liquidsurfaceactivity wasadjusted by adding rhamnolipid at 60 mg·L?1to 120 mg·L?1.

2.9.Statistical analysis

2.9.1.Analysis of degradation rate

To evaluate the n-hexadecane and pyrene biodegradation rate,we computed the integral for the peak area as the total substrates content.Ineachsample,includingthecontrolandexperimentalgroups,thepeak area of the internal standard phenanthrene and the hydrocarbons residues were isometric.The n-hexadecane and pyrene degradation rates were computed using the integral for the peak area of phenanthrene and the hydrocarbons residues.After computing the integral for the peak area of the hydrocarbons residues,the corrective area of the different samples was calculated using phenanthrene as the internal standard.

Then-hexadecaneandpyrenebiodegradationrateformula isshown asthe following:AHrefers to thepeakarea of thealkaneresidues,while ATrefers to the peak area of phenanthrene.

2.9.2.Analysis of metabolites

ThesimplemetabolomicsdatasetwasprocessedinMZmineforpeak recognition,identification,and deconvolution.Metabolite identification was accomplished by comparing the mass spectra with a National Institute of Standards and Technology(NIST)mass spectral library[20].After centering and scaling,the datasets derived from metabolite profiling were analyzed by principal component analysis(PCA)using Matlab.Four biological replicates were used to perform a multivariate analysis for each sample.

The significance of metabolites in abundance in different MSM was identified by a two-tailed Student's t-test performed with Microsoft Excel.The level of metabolites in different medium was considered significant at p＜0.05.

3.Results and Discussion

3.1.Isolation and description of Rhodococcus sp.T1

The domestication period shown in Fig.1 was divided into three parts.The first period was the time at which 2%(w/v)crude oil was added(day 7),the second was the time at which 2 g·L?1pyrene was added(day 14)and the final period was when 4 g·L?1pyrene was added(day 21)and domestication was ended.Theoretically,bands in the same place in the DGGE profile are the same organism[30].As shown in Fig.1,the composition of the microbial consortium changed significantly in 21 days.In the original sample,many bacteria were distinguished,but the bands were dim.In the final profile(when domestication was over),four bands could be seen;however,the bands had become much darker,suggesting that there were four functionalbacteriainthissystem[30].Fourfunctionalbacteriaweredetected after domestication via PCR-DGGE analysis.From the first to the fourth band,they showed the highest identity with Cellulosimicrobium sp.,Pseudomonas sp.,Rhodococcus sp.and Bacillus sp.(Fig.1).Band 3,which was closest to Rhodococcus sp.,became increasingly darker after pyrene was added,and when domestication was over,this bacterium was dominant in the system.Strain Rhodococcus sp.T1 exhibited the highest degradation capacity of pyrene in the four isolates(Fig.S1)and was therefore selected for subsequent analyses.

Fig.1.DGGE profiles of bacterial communities during domestication(A:The DGGEprofile when 2%(w/v)crude oil was added(day 7);B:The DGGE profile when 2 g·L?1pyrene was added(day 14);C:The DGGE profile when 4 g·L?1pyrene was added(day 21)).

Alignment of this sequence through matching with reported 16S rRNA gene sequences in GenBank revealed high similarity(99%)to Rhodococcus 16S rRNA genes(Fig.2).Strain T1 was aerobic,gram positive and globular upon microscopic-examination.Colonies cultivated for 3 days were orange,round,smooth,and semi-humid with smooth edges,indicating they may have been Rhodococcus sp.Recent research has documented that a large number of Rhodococcus sp.are able to utilize hydrocarbons as their only carbon source.For example,Li et al.[2]isolated Rhodococcus sp.JZX-01,which could degrade long-chain hydrocarbons(C31-C38)and branched alkanes.Rhodococcus ruber were isolated from the original sediment sample and found to exert high activity in the degradation of phenanthrene and fluorene during liquid cultivation[31].Strain T1 should have an outstanding ability to degradelong-chainalkanesandPAHsinenvironmentallypollutedareas.

3.2.Degradation of n-hexadecane and pyrene by Rhodococcus sp.T1

The characteristics of degradation of n-hexadecane and pyrene under 35°C,pH 7 and 0.5%of NaCl are shown in Figs.3 and 4.As shown in Fig.3,the OD600values of the strain indicated that its growth characteristics were influenced by different substrates.The population of bacteria derived from the solutions of n-hexadecane and a mixture of pyrene and n-hexadecane continually increased through the 120-hour period,whereas cells cultured in medium containing pyrene alone showed no obvious changes.These findings indicated that pyrene prolonged the lag phase of strain T1.Although strain T1 showedahightolerancetopyrene,itsuppressedtheirgrowthwhenex-posed to a pyrene environment for long periods.Therefore,cells grew better in mixed substrate than in substrate containing pyrene alone,but worse than in substrate containing n-hexadecane alone(Fig.3).

Fig.2.Phylogenetic tree based on 16S rRNA gene sequence of strain T1.

Fig.3.Changes in growth of strain T1 under different substrate.

Fig.4.Degradation rate of pyreneand n-hexadecaneinfivedaysunder different substrate(A:the degradation rate of pyrene in 100 ml MSM amended with 200 mg·L?1pyrene;B:the degradation rate of pyrene in 100 ml MSM amended with 200 mg·L?1pyrene and 2 vol%n-hexadecane;C:the degradation rate of n-hexadecane in 100 ml MSM amended with 2 vol%n-hexadecane;D:the degradation rate of n-hexahecane in 100 ml MSM amended with 200 mg·L?1pyrene and 2 vol%n-hexadecane.

Fig.4 shows the differences in degradation rates of different substrates by the isolated strain.Strain T1 led to good removal of n-hexadecane alone,with a degradation rate of 90.81%in five days,while the degradation rate of pyrene was 42.79%.Apparently,combination of the two substrates led to more pyrene removal and less n-hexadecane degradation,as indicated by removal rates of 60.63%and 87.55%,respectively.It should be noted that the removal rate of nhexadecane was reduced by 3.26%in mixed carbon source system of pyreneand n-hexadecane,but that the degradation of pyreneincreased by 17.84%.These findings indicate that the presence of n-hexadecane promotes pyrene degradation,whereas pyrene has hard effect on the removal of n-hexadecane by microorganisms.

Wang et al.[16]reported that Bacillus strain NG80-2 from a deep subterranean oil-reservoir could degrade long-chain n-alkanes.The ability to degrade n-alkanes ranging from C15 to C36,but not those less than C14 indicates that NG80-2 has only evolved a mechanism for degradation of long-chain n-alkanes,but not short chains.Wang et al.[32]reported a pyrene-degrading consortium that could grow with nine of 11 kinds of PAHs,and could remove 60%of pyrene(1 g·L?1)within 2 weeks.However,most bacteria can only use a narrow range of hydrocarbons or have a long biodegrading period.Strain T1 achieved a relatively higher biodegradation rate in the mixture of pyrene and n-Hexadecane.

3.3.Intracellular metabolic changes

The metabolites of Rhodococcus sp.T1 from nine samples of three different media(three replicated from each medium)were analyzed by GC-MS.A total of 110 intracellular metabolites were detected,80 of which were identified,including amino acids,organic acids,amines,and fatty acids.Some major metabolites are presented in Table S1.Andphthalic acid was detected in the pyrene metabolismof bothsingle andmixture substrate;therefore,pyreneis presumablymetabolized via the phthalic acid pathway by Rhodococcus sp.T1[14].The main metabolic pathway for the degradation of alkanes by microorganisms is the oxidation of alkanes to alcohols,the continuous dehydrogenation of alcohols to fatty acids,and then to the tricarboxylic acid cycle[33].

The PCA score plot was used to determine the similarity and differences between the samples[20].Using PCA,we found that there were considerable differences in the intracellular metabolism of cells under different substrates,especially pyrene.In the score plot of PCA,samples from differentsubstrateswere clearly divided intothree groups(Fig.5),indicatingthat themetabolismof Rhodococcus sp.T1exhibited different characteristics when distinctive substrates were used.

Fig.5.PCA score plot of intracellular metabolites.

As shown in the PCA loading plot,alanine,methylamine,citric acid and heptadecanoic acid contributed significantly to the cluster formation(Fig.6).Because these four metabolites are far from the center,they are the key metabolites that affect the three subgroups on the scorechartandthereforecanbeutilizedasbiomarkers[34].Therelative abundance of these biomarkers of different carbon sources is diverse in cell metabolites.As shown in Fig.7,the accumulation of these metabolites differed markedly under different carbon source selection pressures(p＜0.05).

Fig.6.PCA loading plot of intracellular metabolites.

Amino acids are essential metabolites that are primarily produced by cell metabolism synthesis and protein decomposition[35].In the present study,the relative abundance of alanine increased gradually as the proportion of pyrene in the substrate decreased.Methylamine is a decarboxylation product of glycine,and it has been reported that amines can accumulate rapidly under environmental stress and enhancetheantioxidantcapacitytoprotectcells[36].Theaccumulation of methylamine was highest when pyrene was the sole carbon source,and the accumulation of methylamine was roughly the same content in the other two groups.Citric acid is mainly involved in the tricarboxylic acid cycle.The comparative index of citric acid decreases as the proportion of pyrene in the substrate decreases,which is similar to alanine.The relative abundance of heptadecanoic acid was similar to that of methylamine among the tested substrates.Previous studies have shownthatincreasesinfattyacidscouldenhanceresistancetotheenvironmental changes[37].

Fig.7.The relative abundance of four biomarkers.

Pyrene had a more significant effect on cell metabolism than nhexadecane in that it inhibited amino acid synthesis,reduced energy metabolism and accumulated fatty acids in the cell.The biomarkers involved in the main metabolic pathways,citric acid and alanine,decreased with increasing proportions of pyrene,which may have been because of the poor solubility in water or the toxicity of pyrene[38,39].The abundance of methylamine and heptadecanoic acid both increased with increasing proportions of pyrene,which may indicate that these two biomarkers can reduce the damage caused by extreme environments[36,37].

3.4.Optimizing the degradation of n-hexadecane and pyrene

As described in Section 3.3,some factors may affect biodegradation,suchaslackofcarbonsource,poorsubstratewatersolubilityorextreme environments.Basedontheresultsofmetabolomicsanalysis,theexperiments were designed to optimize the degradation conditions by adjustingpHandsaltconcentrations,addingadditionalnutrientsources and increasing the liquid surface activity.

The optimization experiments revealed that(1)substrate degradation could be promoted by appropriate pH and salinity concentrations,and that the optimal pH and salinity were 7 and 0.5%NaCl,respectively(Fig.S2(a)andFig.S2(b)).AppropriatepHandsalinitycanimprovethe environmentsoasto promote biodegradation[40].(2)Thedegradation of pyrene and n-hexadecane by T1 is promoted by a certain amount of yeast powder(5 mg·L?1)whether these compounds are alone or mixed,but the degradation rate was reduced by the addition of an additional carbon source during the experimental period(Fig.S2(c)).Yeast powder can promote growth of the strain as a nutrient source to improve the degradation rate;however,too much yeast will inhibit degradation because the strain will utilize the yeast as a carbon source instead of the target compound[41].(3)Surfactants promote the biodegradation of petroleum contaminants by increasing bioavailability[42].Rhamnolipids were applied in the present study because they are less toxic to bacteria[43].The addition of 90 mg·L?1rhamnolipid in the experiment resulted in the highest degradation rates of pyrene and n-hexadecane,but more surfactant inhibited degradation(Fig.S2(d)).Rhamnolipids can also be considered additional nutrients;therefore,their presence will reduce the degradation rate of hydrocarbons if excessive[44].

Based on these results,the optimum biodegradation conditions for strain T1 degradation of n-hexadecane and pyrene were pH 7,0.5%NaCl,5 mg·L?1yeast extract,and 90 mg·L?1rhamnolipid,which resulted in degradation rates of 48.77%and 94.6%for pyrene and n-Hexadecane,respectively,under sole substrate conditions,or 68.29%and 88.94%,respectively,under mixed substrate conditions.In general,the degradation of pyrene and n-hexadecane could be promoted by suitable environmental conditions generated by appropriate amounts of nutrients and surfactants.However,utilization of pyrene and n-hexadecane by the strain will decrease in the presence of excessive amounts of additional carbons because the additives are preferred by the bacteria.

4.Conclusions

Strain T1,which was identified as Rhodococcus sp.,could effectively degrade n-hexadecane,pyrene and their mixtures.In this study,substrate interactions were investigated during biodegradation of mixturesofn-hexadecaneandpyrene.n-hexadecaneenhancedthebiodegradation of pyrene,while pyrenehad hard effecton thebiodegradation of n-hexadecane in their mixtures.Additionally,there were significantdifferencesinmetabolitesbetweensubstrates.Furtherinvestigation revealed that the optimum conditions for biodegradation of nhexadecane and pyrene by strain T1 were:pH 7,0.5%NaCl,5 mg·L?1yeast extract,and 90 mg·L?1rhamnolipid,which led to degradation rates of 48.77%and 94.96%for pyrene and n-hexadecane,respectively,under sole substrate conditions and 68.29%and 88.94%under mixed substrate.

It is important to understand the differences in substrate interactions identified in the present study to enable better understanding and utilization of this organism for remediation of complicated compounds and scale-up[38].It should be noted that the unique ability of Rhodococcus sp.T1 to degrade these highly recalcitrant compounds indicates that this strain has great potential for application in practical bioremediation of complicated PAHsand alkanes.An artificial microbial consortium is currently being constructed by our laboratory to overcome the low efficiency of natural microorganisms,and a higher degradation of PAHs is expected in the future.

Supplementary Material

Supplementary data tothis article canbefoundonlineathttps://doi.org/10.1016/j.cjche.2018.03.034.