Article pubs.acs.org/est
Transcriptional Profiling Suggests that Multiple Metabolic Adaptations are Required for Effective Proliferation of Pseudomonas aeruginosa in Jet Fuel Thusitha S. Gunasekera,† Richard C. Striebich,† Susan S. Mueller,† Ellen M. Strobel,‡ and Oscar N. Ruiz*,‡ †
University of Dayton Research Institute, University of Dayton, Dayton Ohio 45469, United States Aerospace Systems Directorate, Fuels and Energy Branch, Air Force Research Laboratory, 1790 Loop Road, Bldg. 490, Wright-Patterson AFB, Ohio 45433, United States
‡
S Supporting Information *
ABSTRACT: Fuel is a harsh environment for microbial growth. However, some bacteria can grow well due to their adaptive mechanisms. Our goal was to characterize the adaptations required for Pseudomonas aeruginosa proliferation in fuel. We have used DNA-microarrays and RT-PCR to characterize the transcriptional response of P. aeruginosa to fuel. Transcriptomics revealed that genes essential for medium- and long-chain nalkane degradation including alkB1 and alkB2 were transcriptionally induced. Gas chromatography confirmed that P. aeruginosa possesses pathways to degrade different length n-alkanes, favoring the use of n-C11−18. Furthermore, a gamut of synergistic metabolic pathways, including porins, efflux pumps, biofilm formation, and iron transport, were transcriptionally regulated. Bioassays confirmed that efflux pumps and biofilm formation were required for growth in jet fuel. Furthermore, cell homeostasis appeared to be carefully maintained by the regulation of porins and efflux pumps. The Mex RND efflux pumps were required for fuel tolerance; blockage of these pumps precluded growth in fuel. This study provides a global understanding of the multiple metabolic adaptations required by bacteria for survival and proliferation in fuel-containing environments. This information can be applied to improve the fuel bioremediation properties of bacteria.
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INTRODUCTION Pseudomonas aeruginosa can adapt to diverse ecological niches due to its metabolic versatility.1 P. aeruginosa has been shown to grow in jet fuel storage systems and petroleum oil fields and is able to degrade a wide range of hydrocarbons.2,3 Thus, P. aeruginosa has been proposed as an important microorganism for bioremediation of light non-aqueous phase liquids (LNAPLs).4,5 Normal alkanes in fuels are metabolized via oxidation and are used as a sole carbon source for energy and growth. The P. aeruginosa genome encodes two membranebound alkane hydroxylases (AlkB1 and AlkB2) and essential electron transfer proteins, rubredoxins (RubA1, RubA2), and FAD-dependent NAD(P)H2 rubredoxin reductases required for alkane degradation.6,7 In addition to AlkB1 and AlkB2 monooxygenases, bacteria also utilize soluble heme-thiolate prokaryotic-P450 monooxygenases to oxidize n-alkanes. Both membrane-bound AlkB and cytochrome P450 act efficiently on medium chain alkanes ranging from C5−C16.8 However, the physiological signal that regulates the alkane degradation pathway of P. aeruginosa is not well known. On the other hand, it is known that in Pseudomonas putida GpO1 the alk genes are regulated by AlkS, which activates the expression of the alkane degradation pathway in presence of alkanes. The © 2013 American Chemical Society
expression of the alkane degradation pathway is also regulated by a catabolic repression control (CRC) system that represses the alk genes depending on the availability of simple carbon sources in the growth environment.9,10 Another difference between P. aeruginosa PAO1 and P. putida GpO1 is that the alkane degradation genes in GpO1 are grouped in two clusters located on the OCT plasmid,11 whereas in P. aeruginosa PAO1 the alk genes are located in the chromosome. Although P. aeruginosa is equipped with the basic machinery to consume fuel as a carbon source, fuel is considered a harsh environment for bacteria to survive. Therefore, bacteria prefer to proliferate in the water phase or fuel−water interface. To encounter these adverse conditions, bacterial cells have developed multiple adaptations, including the ability to change the cell surface hydrophobicity and form biofilms,12 regulate outer membrane porins and membrane permeability, and extrude toxic compounds. Biofilms are the aggregation of cells enclosed in a matrix of polymeric compounds, primarily Received: Revised: Accepted: Published: 13449
August 12, 2013 October 24, 2013 October 28, 2013 October 28, 2013 dx.doi.org/10.1021/es403163k | Environ. Sci. Technol. 2013, 47, 13449−13458
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exopolysaccharides (EPS).13,14 Biofilms are important for bacteria to survive when environmental conditions deteriorate. Bacteria are capable of producing biofilms on a number of different surfaces including fuel tank walls, fuel lines, and the fuel−water interfaces.15,16 P. aeruginosa secretes alginate that produces the specific characteristic observed in the biofilms formed in the lungs of cystic fibrosis patients.14 EPS helps to avoid direct contact of cells with fuel. In this study, we show that genes responsible for alginate biosynthesis and alginate transport across the cell inner membrane have been transcriptionally induced, indicating that the fuel environment can promote biofilm formation. Jet fuel contains aromatic and cyclic hydrocarbons which are toxic to the cell.17−19 Also, fuel can capture heavy metals during transport and storage, which may also affect bacteria. It has been proposed that membrane proton antiporter-pumps of the resistance-nodulation-division (RND) family can function in the extrusion of toxic compounds including antimicrobials, organic solvents, and heavy metals.20 The tripartite efflux pumps MexAB-OprM, MexCD-OprJ, and MexEF-OprN have been shown to provide broad resistance to antibiotics and different aromatic compounds in P. aeruginosa.20,21 Homologous proteins in E. coli encoded by the acrAB/EF-Tol genes have shown similar activity.19,22 Deletion of these efflux pump systems rendered the cell susceptible to toxic molecules.20 Outer membrane porins have been shown to have an essential role in the adaptation of bacteria to different environments. Porins can transport everything from glucose and carbohydrates to phosphate, polyphosphate, and even organic solvents, such as toluene and naphthalene.23−25 Previous studies showed that P. aeruginosa cells lacking the outer membrane proteins OprF were highly resistant to the toxic effects of toluene.23 The protein OprG has also been linked to the transport of solvents, such as naphthalene into the cell.24,25 Although the fundamental aspects of the metabolic machinery and pathways involved in the utilization of hydrocarbons may be known, the transcriptional expression profiles of genes involved in survival, adaptation, and proliferation of bacterial cells in fuel have not been well characterized. Therefore, we initiated a whole genome expression analysis using Affymetrix microarray chips against P. aeruginosa PAO1 in order to study the transcriptional profile of the fuel degrading strain P. aeruginosa ATCC 33988, which was originally isolated from a fuel tank. We utilized the available microarray chip for the PAO1 strain, which also encodes functional genes responsible for fuel degradation.2,26,27 In addition, we have used quantitative reverse transcription polymerase chain reaction (qRT-PCR) to confirm the results obtained through microarray analyses. Chemical analyses using gas chromatography−mass spectrometry (GC-MS) allowed us to describe the degradation profile of jet fuel and the progression and consumption rate of fuel hydrocarbons, including n-alkanes, and cyclic and aromatic hydrocarbons. Finally, we performed functional assays in the presence of fuel to determine the formation of biofilms and the role of efflux pumps in the resistance of toxic compounds and proliferation of bacteria in fuel. Here, we provide a comprehensive description of the transcription profile of P. aeruginosa when exposed to jet fuel. The results demonstrate how multiple metabolic pathways and adaptations take place for effective proliferation of bacteria in fuel. This study provides a new global understanding of the genetic and metabolic plasticity of bacterial cells. This knowledge can be used in combination with
genetic engineering, biotechnological, and environmental engineering approaches to enhance characteristics such as nutrient uptake, biofilm formation, surfactant production, and tolerance to toxic compounds in order to improve the potential of bacteria for detoxification and bioremediation of hydrocarbons and LNAPLs.
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MATERIALS AND METHODS Bacteria Strains and Growth Condition. The P. aeruginosa strain ATCC 33988 originally isolated from a fuel storage tank was used in this study. P. aeruginosa ATCC 33988 was initially grown in LB (Lysogeny broth) medium overnight at 28 °C. Cells were harvested by centrifugation at 10,000 rpm for 5 min. Cells were then washed twice with M9 minimal medium to remove trace amounts of LB before inoculation to M9 minimal medium. Four independent cultures of P. aeruginosa were grown in M9 minimal media with filter sterilized Jet A fuel at 28 °C with aeration. The chemical composition of Jet A was confirmed by GCxGC (Table S6, Supporting Information). As a control, bacteria were grown in M9 minimal media with glycerol as the sole carbon source. Bacteria were grown to mid log phase (0.5−0.6 OD600), and cells were cooled rapidly on ice. Cells were harvested by centrifugation (4 °C) with 10% ethanol/phenol (19:1) solution, and the pellet was frozen immediately on dry ice. RNA Isolation, cDNA Synthesis, and Affymetrix GeneChip Analysis. Total RNA was extracted from cells using the Qiagen Total RNA kit as described by the manufacturer. Any DNA contamination was removed by DNAase treatment. The quality of RNA was initially assessed by electrophoresis through a 1% agarose gel and by the Agilent Bioanalyzer System (Agilent Technologies, Palo Alto, CA). Total RNA, free of genomic DNA, was used to synthesize cDNA. First strand cDNA was synthesized with random primers using SuperScript II Reverse transcriptase. The cDNA was fragmented and labeled with biotin. Subsequently, the labeled-cDNA was hybridized to the Affymterix microarray chips. Arrays were then washed and stained as described in the Affymetrix GeneChip Expression Analysis Technical Manual, using the instructions specifically for P. aeruginosa PAO1. After washing and staining, Microarray chips were scanned using the Affymetrix GeneChip Scanner 3000. The initial data analyses were performed using Affymetrix Microarray Analysis Suite (MAS), version 5.1. Signal values, detection calls (present, absent, marginal), and P values for each detection call were generated using Affymetrix gene chip operating software (GCOS). PCA analyses of microarray chips distinguished four replicates of glycerol chips different to four replicates of jet fuel chips based on their expression profiles. However, to reduce dimensionality, we removed the outlier chip from each condition; three chips from each condition were considered for further analyses. All nine possible comparison analyses were performed using Glycerol CHP files as baseline values to obtain signal log ratio (SLR), change call, and P values associated with the change call. Nonspecific hybridization probes were eliminated by removing signals from a defined cutoff. Data were normalized across the two different conditions. Data were filtered based on the presence call, consensus changed call (CCC), and SLR to obtain significantly up-regulated and down-regulated genes as described previously. 28 Quantitative Reverse Transcription PCR (qRT-PCR). Quantitative RT-PCR was used to validate the microarray data. Gene-specific qRT-PCR primers (Table S1, Supporting 13450
dx.doi.org/10.1021/es403163k | Environ. Sci. Technol. 2013, 47, 13449−13458
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Figure 1. Regulation of P. aeruginosa functional genetic classes when grown in fuel. Functional classifications are according to P. aeruginosa genome project (www.pseudomonas.com). Black and gray bars represent % induced and % repressed genes of total differentially expressed genes, respectively.
μg/mL. Cultures were overlaid with 2 mL of Jet A fuel. Control samples were produced by adding 0.2% glycerol as the carbon source to samples containing efflux pump inhibitor but not fuel. The experiments were performed in triplicate and repeated twice each. Cultures were grown at 28 °C aerobically in a shaking (200 rpm) incubator, and the growth was monitored by measuring OD600.
Information) were designed for several genes representing some of the important biological processes identified by microarray. A SYBR-Green mediated two-step RT-PCR method was used to measure gene expression using the CFX quantitative real-time PCR instrument (BioRAD). PCRamplified target sequences for each gene were amplified from Pseudomonas aeruginosa ATCC 33988 genomic DNA, quantified, and used as external standards for quantification during qPCR. Reverse transcription was carried using 100 ng of total RNA, random primers, and 75 U of iScript MMLV-RT (RNaseH+). The threshold cycle values (Ct) were obtained from amplification curves and the gene expression fold changes was calculated using 2−ΔΔCT method. Fuel Degradation Profiles by GC X GC. Studies were conducted to investigate which specific compounds or compound types in Jet A fuel were preferentially degraded by P. aeruginosa. The bioassays were performed by using 10 μL of Jet A aviation fuel in 1 mL of M9 minimal media containing P. aeruginosa at 0.03 OD in a 10 mL glass vial sealed with a Teflon-lined lid. The samples were maintained in a 28 °C incubator for a period of 13 days, without opening the vial. Multiple sample replicates were incubated at the same time, and then sample vials were removed from the incubator at the time of testing, and finally extracted and analyzed by GC X GC as explain in Table S7 of the Supporting Information. Fluorescent Staining of Cell Aggregates and Biofilm Assay. An Olympus BX50 F4 fluorescence microscope was used to visualize cell aggregations and biofilms. Cells were carefully collected from the fuel−M9 interface and live/dead staining was performed using the BacLight LIVE/DEAD kit (Life Technologies, U.S.A.) to visualize viable cells. A biofilm assay to quantify the formation of bacterial biofilms was conducted as described by Gunasekera et al.29. Efflux Pump Blockage Assay. To demonstrate that the Mex efflux pumps were important in the adaptation and resistance of P. aeruginosa to fuel, we used the known Mexefflux pump inhibitor, c-capped dipeptide Phe-Arg βnaphthylamide dihydrochloride (Sigma-Aldrich P4157− 250MG), to block the efflux pumps and determine the impact on cell growth in the presence of fuel. P. aeruginosa ATCC 33988 was inoculated at 0.03 OD600 into 5 mL M9 medium, and the c-capped dipeptide added at 0, 20, 40, 60, 80, and 100
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RESULTS AND DISCUSSION To date, there has been no comprehensive study describing the diverse synergistic cellular adaptations that have to be triggered in P. aeruginosa to survive and thrive in toxic fuel-containing environments. Using DNA microarrays, we surveyed the global expression profile of P. aeruginosa genes to gain a deeper understanding into the complex machinery required for fuel degradation and adaptation. The P. aeruginosa core genome has been shown to be highly conserved across different strains, irrespective of their origins, regardless of whether a strain is an environmental or clinical strain.1 It has also been shown that environmental strain isolates have physiological properties similar to the clinical strain PAO1.2 Moreover, alkane hydroxylase genes are present in both clinical and environmental isolates.30 The PAO1 strain can utilize Jet A fuel (data not shown), dodecane,24 and hexadecane26 as carbon sources. Whole genome sequencing of Pseudomonas aeruginosa ATCC 33988 revealed that this genome is at least 99% similar to the PAO1 strain for the known genes contained in PAO1 chips. DNA sequence analyses of the genes selected for the qPCR study showed 100% sequence homology to the PAO1 strain. However, P. aeruginosa ATCC 33988 grows faster in jet fuel than the clinical strain PAO1 and appears to be better adapted to the fuel environment. Therefore, we used the P. aeruginosa ATCC 33988 strain for this study. The Affymetrix gene-chip of P. aeruginosa PAO1 (Pae_G1a) genome consists of 5900 probe-sets representing 5549 proteincoding sequences, 18 tRNA genes, a representative of the rRNA cluster, 117 genes from different strains other than PA01, and 199 probe sets of intergenic regions. We used clustering algorithms to analyze expression data. Of 5549 genes, 3778 genes were called present in all 6 conditions and 5461 genes were called present or marginally present in at least 2 chips with 13451
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the rhamnosyltransferase involved in rhamnolipid biosurfactant production was up-regulated in our experiment over 5-fold, suggesting that rhamnolipid plays a role in fuel uptake. Degradation of alkanes involves hydroxylation of the terminal methyl group to render a primary alcohol, followed by further oxidation to an aldehyde, and then conversion to fatty acids. Fatty acids are conjugated to co-enzyme A (CoA) and further processed by β-oxidation to generate acetyl CoA. Activation of medium chain (C5−C11) or long chain alkane molecules (C12 and greater) require the expression of the alkane hydroxylase genes, which encode membrane bound monooxygenases. The alkB1 and alkB2 genes are known to be present in P. aeruginosa, and our results showed that both genes were up-regulated by 6.75- and 2.33-fold (Table S2, Supporting Information), respectively, when grown in jet fuel. In agreement with a previous study,35 the alkB1 and alkB2 genes were differentially expressed during the exponential phase of growth. However, the genes encoding two soluble electron transfer proteins rubredoxins (PA5350, PA5351) and rubredoxin reductase (PA5349) were not transcriptionally induced by fuel. These results are in agreement with Marin et al.,35 in which they showed that rubredoxins and rubredoxin reductase encoding genes are constitutively expressed even in the presence of alkanes. Rubredoxin transfers electrons from NADH-dependent flavoprotein to rubredoxin reductase and to AlkB and the cytochromes. AlkB1 and AlkB2 have overlapping substrates ranging from medium chain length alkanes to long chain alkanes. The AlkB1 and AlkB2 were shown to be particularly active on C10−C22/C24 alkanes but were differentially express at different stages of the growth phase.32 In addition to AlkB1 and AlkB2, cytochrome P450 alkane hydroxylases (PA2475, PA 3331), which are ubiquitous among all kingdoms of life,7 were also induced in P. aeruginosa when grown in fuel. Several ferrodoxins and ferrodoxin reductase (PA4331) were up-regulated when Pseudomonas was grown in fuel, indicating the possible involvement of these enzymes in jet fuel degradation (Table S2, Supporting Information). Both AlkB and Cytochrome P450 monooxygenase oxidize medium-chain (C5−C11) and long-chain alkanes (>C11). The up-regulation of alkB1 (6.75-fold), alkB2 (2.33-fold), and P450 (4.89- and 3.85fold) indicates that these enzymes were activated by the presence of fuel and likely involved alkane degradation. In addition to alkB and P450 genes, a number of enzymes involved in terminal oxidation pathways were induced including, alcohol dehydrogenase, aldehyde dehydrogenase, and acyl-CoA-synthetase (Table S2, Supporting Information). The fatty acid degradation pathway plays a critical role in consumption of jet fuel by P. aeruginosa. The initial oxidation step of beta-oxidation is catalyzed by acyl-CoA dehydrogenase. We observed that multiple acyl-CoA dehydrogenases were induced in the presence of fuel. A number of enzymes in the enoyl-CoA hydratase family that catalyze the second step of βoxidation pathway were also induced (Table S2, Supporting Information). Selective Consumption of n-Alkanes in Jet-A Fuel by P. aeruginosa. Jet fuel composition analyses performed by two-dimensional gas chromatography (GC × GC) provided new insights into the type of hydrocarbons degraded by P. aeruginosa ATCC 33988. These results further confirmed activity of n-alkane degradation pathways detected by microarray. The results showed that n-C9, C10, and C11 presented a slower rate of decomposition over the 13 days, while a much greater decline in concentrations occurred for n-C12 through n-
an alpha-1 value of 0.05. Of 5570 annotated Open Reading Frames (ORFs) in the P. aeruginosa PAO1 genome (www. pseudomonas.com31), here we found that under fuel growth, 2963 genes were induced and 617 genes were repressed over 2fold. Additionally, differentially expressed genes were assigned to different functional classes (Figure 1). Analyses showed that 47% of the induced and 22.5% of the repressed genes were hypothetical with unknown functions in P. aeruginosa. Of the induced known genes, 12.4% were membrane proteins, 14.6% were putative enzymes, and 14.4% were transcriptional regulators (Figure 1). Up-regulation of large number transcriptional regulators during fuel growth allowed cells to adapt to the fuel environment rapidly. Among the down-regulated genes, the functional class of translation and post-translational modification were most notable (Figure 1). Almost every gene involved in the formation of ribosomal small (S) and ribosomal Large (L) proteins were significantly down-regulated probably due to slower growth rates in fuel compared to glycerol. In addition, a large number of genes related to translational and post-translational modification, amino acid biosynthesis and metabolism, energy metabolism, cell division, and cell wall were down-regulated. The microarray results were further validated using quantitative reverse transcription−PCR (qRT-PCR) for several candidate genes that represented the different pathways studied through microarray (Figure 2). The results showed that the mRNA expression levels detected by qRT-PCR were consistent with the expression levels obtained by microarray.
Figure 2. Validation of microarray gene expression data using qRTPCR. A few candidate genes that represented some of the important biological processes identified by microarray including alkane degradation (alkB1, alkB2), EPS biosynthesis mechanism (pelA, pelD, pelE, algD), efflux pumps, and porin regulation (oprF, oprG, oprN, oprJ), iron transport, and metabolism (Fur, pchF, pfeR) were selected to be tested by qRT PCR. Microarray fold-change data were compared against the qRT-PCR data.
Alkane Degradation Pathways. Alkanes are highly reduced saturated hydrocarbons with high energy content. Therefore, alkanes can serve as a rich carbon and energy source for bacteria to grow. Alkane degradation is a complex process that involves the uptake of alkanes from a hydrophobic environment to the inside of the cell, followed by oxidation using substrate specific enzymes. P. aeruginosa is known to secrete surfactant rhamnolipids that emulsify hydrocarbons, thereby improving the uptake process.32−34 In addition, rhamnolipids increase the hydrophobicity of the cell surface improving adaptability of P. aeruginosa to fuel-containing environment. A complex gene regulatory network is involved in rhamonlipid production. The rhlAB gene operon encoding 13452
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(Figure 3b). Naphthalene and tetralin were entirely undegraded. However, the concentration of decalin was reduced from its original concentration by 25% as compared to an approximate 85% loss of the n-alkane C13. Clearly, cells preferentially consumed the n-alkane in favor of the aromatics (Figure 3b). The results demonstrated that saturated cyclic compounds that have no aromaticity such as decalin are also degraded but to a lesser degree than the n-alkanes. These observations are in general agreement with the studies conducted on oil spills where bacteria consume hydrocarbons in the order of n-alkanes > branched alkanes > low molecular weight aromatics > cyclic alkanes > higher molecular weight aromatics.3 Biosynthesis of Extracellular Polysaccharides and Biofilm Formation. The formation of biofilms in the fuel environment has been indicated previously.15,16 P. aeruginosa successfully colonizes fuel tanks by secreting extracellular polysaccharides and forming biofilms. We observed that P. aeruginosa produced a significant amount of biofilm at the fuel and M9 media interface (Figure 4a). Microscopic analyses showed that most of the cell aggregations were alive (Figure 4b). Under static condition, P. aeruginosa formed a significant (P < 0.01) amount of biofilm when compared to P. aeruginosa grown in glycerol in as short as 3 days (Figure 4c). Using expression microarray, we identified genes likely responsible for extracellular polysaccharide (EPS) and biofilm formation. We noticed that in the presence of fuel, alginate, and Pel protein biosynthesis and export mechanisms were induced (Table S3, Supporting Information). Alginate, a linear polymer of β-1,4linked D-mannuronic acid, is known to play a role in the development of biofilms of P. aeruginosa in the upper and lower airways of cystic fibrosis patients.9,32 With the exception of algC, which does not reside in the algD operon, all the other genes involved in biosynthesis and secretion of alginate were induced in the presence of fuel. The algC gene, which has additional functions in lipopolysaccharide (LPS) biosynthesis,36 was down-regulated in presence of fuel (Table S3, Supporting Information). The algA and algD genes which encode the enzymes required for synthesis of the alginate precursor guanosine diphosphate (GDP) mannuronic acid were upregulated over 4-fold in fuel (Table S3, Supporting Information). Once the alginate precursor is synthesized in the cytoplasm, it is polymerized and exported across the outer membrane. The genes that encode the alginate precursor transport proteins, alg8 and alg44, were induced over 7-fold. The genes encoding three periplasmic proteins (AlgG, AlgK, AlgX) involved in forming a scaffold to protect alginate degradation from alginate lyase were also induced. The gene products of algI, algJ, algF, which serve as the reaction center for O-acetylation in the membrane were also induced in fuel. Additionally, the algE gene that encodes the outer membrane protein that exports alginate from periplasm to the environment was up-regulated. Overall, our study provides novel insight into the production of alginate by P. aeruginosa during fuel growth. Exopolysaccharides make cells more mucoid, enabling cells to attach to fuel tank surfaces and produce biofilms, which prevent direct contact of cells with fuel. The regulation of alginate biosynthesis is a complex process controlled at the transcriptional level, which involves a number of proteins including AlgT, AlgR, MucA, and MucB.9 However, the expression of these transcriptional regulator genes was not changed under fuel condition suggesting that transcription of the alginate biosynthetic genes might be activated by an
C18 alkanes (Figure 3a). These results agreed with the degradability profiles shown for the PAO1 strain by Smits et
Figure 3. Selective consumption of Jet A fuel hydrocarbons by P. aeruginosa. (a) Degradation rate of normal alkanes based on molecular weight. (b) Comparison of the consumption of four different jet fuel hydrocarbons representing four important compound classes in fuel: n-C13 (C13H28), indicative of paraffins and isoparaffins; decalin (C10H20), indicative of cycloparaffins; naphthalene (C10H8), indicative of diaromatics; and tetralin (C10H14), indicative of monoaromatics. The samples were analyzed by GC × GC with dual flame ionization detection (FID) and mass spectrometry detection.
al.,30 which indicated higher degradability of n-C12 to n-C16 compared to shorter chain alkanes
