A novel strategy for partial purification of alkane hydroxylase from P. chrysogenum SNP5 through reconstituting its native membrane into liposome.

Integral proteins or enzymes are still challenging to purify into their native state because of their need for an amphipathic environment and cofactors. Alkane hydroxylase (AlkB) is a membrane-bound enzyme that catalyzes the hydroxylation of a range of alkanes that have a broad spectrum of applications. In the current study, a novel approach has been explored for partial purification of alkane hydroxylase (AlkB) in its native state through restructuring the lipid bilayer of Penicillium chrysogenum SNP5 into a liposome to extend the native and protective environment to AlkB enzyme. Three different methods i.e., reverse-phase evaporation method (RPEM), detergent-based method (DBM), and ethanol injection method (EIM) have been used for reconstituting its native membrane into liposome. On characterizing liposomes through fluorescence imaging, AFM, and particle size analysis, the reverse-phase evaporation method gave the best results based on the size distribution (i.e., 100-300 nm), the morphology of liposomes, and maximum AlkB specific activity (i.e., 140.68 U/mg). The maximum reconstitution efficiency of 29.48% was observed in RPEM followed by 17.3% in DBM and 12.3% in EIM. On the characterization of the purified AlkB, the molecular weight was measured of 44.6 KDa and the thermostability of liposomes synthesized with the RPEM method was obtained maximum at 55 °C. This approach may open a new strategy for the purification of integral enzymes/proteins in their native state in the field of protein purification and its applications in diversified industries.

Biotransformation of grease waste into fatty acid by Penicillium chrysogenum SNP5 through media engineering and artificial neural network.

Degradation of grease waste remains a challenging task. Current work deals with the biotransformation of grease waste into fatty acids under submerged fermentation using Penicillium chrysogenum SNP5 through media formulation and artificial neural network (ANN). Fermentation media was formulated to ameliorate the uptake of hydrocarbon by enhancing alkane hydroxylase (AlkB) activity, extracellular release of fatty acids and inhibiting beta-oxidation of fatty acid by regulating transketolase. Further, the process parameters of fermentation were optimized through Artificial Neural Network (ANN) using three critical variables viz; inoculum size (spores/ml), pH, and incubation time (days) while media engineering was done with the optimal supplementation of various medium components such as glucose, YPD, MnSO4, tetrahydrobiopterin (THB) and phloretin. The maximum conversion of 66.5% of grease waste into fatty acid was achieved at optimum conditions: inoculums size 3.36 × 107 spores/ml, incubation time 11.5 days, pH 7.2 along with formulated media composed of 1% grease in czapek-dox medium supplemented with 55.5 mM glucose, 0.5% YPD, 16.6 mM hexadecane, 1 mM MnSO4, 1 mM THB, and 1 mM phloretin. The presence of long-chain fatty acids in purified extracts such as oleic acid and octadecanoic acid as end products has valued the evolved process as another source of alternative fuel.

M. tuberculosis AlkX Encoded by rv3249c Regulates a Conserved Alkane Hydroxylase System That Is Important for Replication in Macrophages and Biofilm Formation.

Mycobacterium tuberculosis is a highly specialized human pathogen. The success of M. tuberculosis is due to its ability to replicate within host macrophages, resist host immune responses, and ultimately enter a persistent state during a latent tuberculosis infection. Understanding how M. tuberculosis adapts to and replicates in the intracellular environment of the host is crucial for the development of novel, targeted therapeutics. We report the characterization of an M. tuberculosis mutant lacking Rv3249c, a TetR transcriptional regulator. We show that Rv3249c directly represses the adjacent alkB-rubA-rubB operon encoding an alkane hydroxylase/rubredoxin system. For consistency with related systems, we have named the rv3249c gene alkX. The alkX mutant survived better than wild-type M. tuberculosis inside macrophages. This could be phenocopied by overexpression of the alkB-rubA-rubB locus. We hypothesized that the improved intracellular survival phenotype is a result of increased fitness of the mutant; however, we found that the alkX mutant had a defect when grown on some host-associated carbon sources in vitro. We also found that the alkX mutant had a defect in biofilm formation, also linked to the overexpression of the alkB-rubAB genes. Combined, these results define the primary role of AlkX as a transcriptional repressor of the alkB-rubAB operon and suggest the operon contributes to intracellular survival of the pathogen. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is the leading cause of death worldwide due to a single infectious agent. It is important to understand how M. tuberculosis adapts to and replicates in the intracellular environment of the host. In this study, we characterized the TetR transcriptional regulator Rv3249c and show that it regulates a highly conserved alkane hydroxylase/rubredoxin system. Our data demonstrate that the AlkBRubAB system contributes to the success of the bacterium in host macrophages.

Significance of both alkB and P450 alkane-degrading systems in Tsukamurella tyrosinosolvens: proteomic evidence.

The Tsukamurella tyrosinosolvens PS2 strain was isolated from hydrocarbons-contaminated petrochemical sludge as a long chain alkane-utilizing bacteria. Complete genome analysis showed the presence of two alkane oxidation systems: alkane 1-monooxygenase (alkB) and cytochrome P450 monooxygenase (P450) genes with established high homology to the well-known alkane-degrading actinobacteria. According to the comparative genome analysis, both systems have a wide distribution among environmental and clinical isolates of the genus Tsukamurella and other members of Actinobacteria. We compared the expression of different proteins during the growth of Tsukamurella on sucrose and on hexadecane. Both alkane monooxygenases were upregulated on hexadecane: AlkB-up to 2.5 times, P450-up to 276 times. All proteins of the hexadecane oxidation pathway to acetyl-CoA were also upregulated. Accompanying proteins for alkane degradation involved in biosurfactant synthesis and transport of organic and inorganic molecules were increased. The change in the carbon source affected the pathways for the regulation of translation and transcription. The proteomic profile showed that hexadecane is an adverse factor causing activation of general and universal stress proteins as well as shock and resistance proteins. Differently expressed proteins of Tsukamurella tyrosinosolvens PS2 shed light on the alkane degradation in other members of Actinobacteria class. KEY POINTS: • alkB and P450 systems have a wide distribution among the genus Tsukamurella. • alkB and P450 systems have coexpression with the predominant role of P450 protein. • Hexadecane causes significant changes in bacterial proteome.

Enhanced production of alkane hydroxylase from Penicillium chrysogenum SNP5 (MTCC13144) through feed-forward neural network and genetic algorithm.

Alkane hydroxylase (AlkB), a membrane-bound enzyme has high industrial demand; however, its economical production remains challenging due to its intrinsic nature and co-factor dependency. In the current study, various critical process parameters for optimum production of AlkB have been optimized through feed forward neural network (FFNN) and genetic algorithm (GA) models using Penicillium chrysogenum SNP5 (MTCC13144). AlkB specific activity under preliminary un-optimized conditions i.e., 1% hexadecane, 7.4 pH, 11 days incubation time, 28 °C incubation temperature and 1 ml of inoculum size was 100 U/mg. 'One variable at a time' (OVAT) strategy was used to identify optimum physicochemical parameters and then its output data was fed to develop a model of FFNN with '6-12-1' topology. Outputs of FFNN were further optimized through GA to minimize errors and intensify search level. This has provided superior predictive performances with 0.053 U/mg overall mean absolute percentage error (MAPE), 6.801 U/mg root mean square errors (RMSE), and 0.987 overall correlation coefficient (R). The AlkB specific activity improved by 3.5-fold, i.e., from 100 U/mg under preliminary un-optimized conditions to 351.32 U/mg under optimum physicochemical conditions obtained through FFNN-GA hybrid method, i.e., hexadecane (carbon source): 1.56% v/v, FeSO4: 0.63 mM, incubation temperature: 27.40 °C, pH: 7.38, incubation time: 12.35 days and inoculums size: 1.33 ml. The developed process would be a stepping stone to fulfill the high industrial demands of  Alkane hydroxylase.

Pilot-scale production and in-situ application of petroleum-degrading enzyme cocktail from Alcanivorax borkumensis.

Petroleum degrading enzymes can be used as an alternative way to improve petroleum bioremediation approaches. Alcanivorax borkumensis is an alkane-degrading bacteria that can produce petroleum degrading enzymes such as alkane hydroxylase and lipase. In this study, pilot-scale Alcanivorax borkumensis fermentation was developed for producing large volumes of petroleum degrading enzymes cocktail (∼900 L). Different process conditions, such as inoculum age 72 h and size 4% v/v, temperature 30 ± 1 °C, agitation speed at 150 rpm and, fermentation period 3 days were determined as the optimum for producing alkane hydroxylase and lipase activity. The oxygen transfer capacity was studied for obtaining better bacterial growth and higher enzyme activities in bioreactor process optimization as well as scale-up. Results showed that the maximum values of oxygen mass transfer coefficient (kLa), oxygen uptake rate (OUR), oxygen transfer rate (OTR), alkane hydroxylase, lipase, and cell count were 196.95 h-1, 0.92 mmol O2/L/h, 1.8 mmol O2/L/h, 222.49 U/mL, 325 U/mL, and 8.6 × 1010 CFU/mL, respectively. Compared with the bench-scale bioreactors, the 150 L fermenter showed a better oxygen transfer rate which affected the cell growth that doubled the number and enzymes production that increased. Then, the enzyme cocktail was used for a field test in a diesel source zone using a 5-spot well pattern. The results showed a significant reduction in concentrations of C10 - C50 (from 36% to > 99%) after one injection of enzyme cocktail, mainly for the contaminated soils located in the saturated zone of the unconfined aquifer. This study confirmed the scaling-up ofalkane-degrading enzyme production to an industrial-scale and its application for effective bioremediation of petroleum contaminated sites.

An alkane monooxygenase (AlkB) family in which all electron transfer partners are covalently bound to the oxygen-activating hydroxylase.

Alkane monooxygenase (AlkB) is a non-heme diiron enzyme that catalyzes the hydroxylation of alkanes. It is commonly found in alkanotrophic organisms that can live on alkanes as their sole source of carbon and energy. Activation of AlkB occurs via two-electron reduction of its diferric active site, which facilitates the binding, activation, and cleavage of molecular oxygen for insertion into an inert CH bond. Electrons are typically supplied by NADH via a rubredoxin reductase (AlkT) to a rubredoxin (AlkG) to AlkB, although alternative electron transfer partners have been observed. Here we report a family of AlkBs in which both electron transfer partners (a ferredoxin and a ferredoxin reductase) appear as an N-terminal gene fusion to the hydroxylase (ferr_ferrR_AlkB). This enzyme catalyzes the hydroxylation of medium chain alkanes (C6-C14), with a preference for C10-C12. It requires only NADH for activity. It is present in a number of bacteria that are known to be human pathogens. A survey of the genome neighborhoods in which is it found suggest it may be involved in alkane metabolism, perhaps facilitating growth of pathogens in non-host environments.

Aerobic naphthenic acid-degrading bacteria in petroleum-coke improve oil sands process water remediation in biofilters: DNA-stable isotope probing reveals methylotrophy in Schmutzdecke.

There is an increasing interest in treatment of oil sands process water (OSPW) via biofiltration with petroleum coke (PC) as a substratum. In fixed bed biofilters (FBBs) with PC, the dominance of anaerobic digestion of dissolved organics results in poor removal of naphthenic acids (NAs) along with a high degree of methanogenesis. In this study, the operation of FBBs was modified to improve OSPW remediation by supporting the filtering bed with aerobic naphthenic acid-degrading bacteria treating aerated OSPW (FBBbioaugmentation). The results were compared with a biofilter operated under controlled conditions (FBBcontrol). To this end, a consortium of three aerobic NAs-degrading bacterial strains was immobilized on PC as a top layer (10 cm). These bacteria were pre-screened for growth on 15 different NAs surrogates as a sole carbon source, and for the presence of catabolic genes coding alkane hydroxylase (CYP153) and alkane monooxygenase (alkB) enzymes. The results illustrated that biofiltration in FBBbioaugmentation removed 32% of classical NAs in 15 days; while in the FBBcontrol, degradation was limited to 19%. The degradation of fluorophore (aromatic) compounds was also improved from 16% to 39% for single ring (OI), 22% to 29% for double ring (OII), and 15% to 23% for three rings (OIII) compounds. DNA-Stable Isotope Probing revealed that potential hydrocarbons degraders such as Pseudomonas (inoculated), Pseudoxanthomonas (indigenous) were present up to 9.0% in the 13C-labelled DNA fraction. Furthermore, a high abundance of methylotrophs was observed in the Schmutzdecke, with Methylobacillus comprising more than two-third of the total community. This study shows that bioaugmentation rapidly improved OSPW remediation. Aeration mostly contributed to methane consumption in the top layer, thus minimizing its release into the environment.

Column tests for evaluation of the enzymatic biodegradation capacity of hydrocarbons (C10-C50) contaminated soil.

Though many studies pertaining to soil bioremediation have been performed to study the microbial kinetics in shake flasks, the process efficiency in column tests is seldom. In the present study, soil columns tests were carried out to study the biodegradation of soil contaminated with a high concentration of diesel (≈19.5 g/kg) petroleum hydrocarbons expressed as C10-C50. Experiments were done with crude enzymatic cocktail produced by the hydrocarbonoclastic bacterium, Alcanivorax borkumensis. A. borkumensis was grown on a media with 3% (v/v) motor oil as the sole carbon and energy source. The effects of the enzyme concentration, treatment time and oxidant on the bioremediation efficiency of C10-C50 were investigated. A batch test was also carried out in parallel to investigate the stability of the enzymes and the effect of the biosurfactants on the desorption and the bioconversion of C10-C50. Batch tests indicated that the biosurfactants significantly affected the desorption and alkane hydroxylase and lipase enzymes, maintained their catalytic activity during the 20-day test, with a half-life of 7.44 days and 8.84 days, respectively. The crude enzyme cocktail, with 40 U/mL of lipase and 10 U/mL of alkane hydroxylase, showed the highest conversion of 57.36% after 12 weeks of treatment with a degradation rate of 0.0218 day-1. The results show that the soil column tests can be used to optimize operating conditions for hydrocarbon degradation and to assess the performance of the overall bioremediation process.

Efficiency and kinetics of Assam crude oil degradation by Pseudomonas aeruginosa and Bacillus sp.

We report kinetics of Assam crude oil degradation by Pseudomonas aeruginosa AKS1 and Bacillus sp. AKS2, both isolated from Assam refinery sediments. The isolates exhibited appreciable degrees of hydrophobicity, emulsification index and biosurfactant production. Crude oil degradation efficiency of isolates was assessed in (1) liquid medium amended with 1% v/v crude oil and (2) microcosm sediments (125 mg crude oil/ 10 g sand). In liquid culture, biodegradation rate (k) and half-life (t1/2) values were found to be 0.038 day-1 and 18.09 days for P. aeruginosa AKS1, and 0.020 day-1 and 33.97 days in case of Bacillus sp. AKS2, respectively. In microcosm sediments, the estimated k and t 1/2 values were 0.014 day-1 and 50 days for P. aeruginosa AKS1, and 0.011 day-1 and 61.34 days in case of Bacillus sp. AKS2. The level of nutrient treatment in microcosm sand sediment was 125 µg N and 62.5 µg P/g sediment in case of P. aeruginosa AKS1 and 375 µg N and 37.5 µg P/g sediment in case of Bacillus sp. AKS2. In microcosms without inorganic nutrients, values of k and t1/2 were found to be 0.007 day-1 and 100 days for P. aeruginosa AKS1 and for Bacillus sp. AKS2, the respective values were 0.005 day-1 and 150.68 days. Our data provides important information for predictive hydrocarbon degradation in liquid medium and contaminated sediments.

Characterization and Transcriptome Analysis of a Long-Chain n-Alkane-Degrading Strain Acinetobacter pittii SW-1.

Strain sw-1, isolated from 7619-m seawater of the Mariana Trench, was identified as Acinetobacter pittii by 16S rRNA gene and whole-genome sequencing. A. pittii sw-1 was able to efficiently utilize long-chain n-alkanes (C18-C36), but not short- and medium-chain n-alkanes (C8-C16). The degradation rate of C20 was 91.25%, followed by C18, C22, C24, C32, and C36 with the degradation rates of 89.30%, 84.03%, 80.29%, 30.29%, and 13.37%, respectively. To investigate the degradation mechanisms of n-alkanes for this strain, the genome and the transcriptome analyses were performed. Four key alkane hydroxylase genes (alkB, almA, ladA1, and ladA2) were identified in the genome. Transcriptomes of strain sw-1 grown in C20 or CH3COONa (NaAc) as the sole carbon source were compared. The transcriptional levels of alkB and almA, respectively, increased 78.28- and 3.51-fold in C20 compared with NaAc, while ladA1 and ladA2 did not show obvious change. The expression levels of other genes involved in the synthesis of unsaturated fatty acids, permeases, membrane proteins, and sulfur metabolism were also upregulated, and they might be involved in n-alkane uptake. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) confirmed that alkB expression was significantly induced by C20, C24, and C32, and almA induction extent by C24 and C32 was higher than that with C20. Furthermore, ladA2 expression was only induced by C32, and ladA1 expression was not induced by any of n-alkanes. In addition, A. pittii sw-1 could grow with 0%-3% NaCl or 8 out of 10 kinds of the tested heavy metals and degrade n-alkanes at 15 °C. Taken together, these results provide comprehensive insights into the degradation of long-chain n-alkanes by Acinetobacter isolated from the deep ocean environment.

Enhanced degradation of different crude oils by defined engineered consortia of Acinetobacter venetianus RAG-1 mutants based on their alkane metabolism.

Microbial consortia offer an attractive biodegradation strategy for removing hydrocarbons from oil-contaminated sites. In this study, we explored the degradation properties of Acinetobacter venetianus strain RAG-1 (RAG-1). RAG-1 effectively degrades three crude oils with excellent emulsification activity and cell surface hydrophobicity, while exhibiting broad environmental tolerance. RAG-1 accepts a range of alkane substrates (C10-C38) using three alkane hydroxylases (AlkMa, AlkMb, and AlmA). Bacterial mutant with alkMa or alkMb deletion enhanced degradation of C10-C20 or C22-C32 n-alkanes, respectively. Based on the substrate metabolism of the mutants, adjustable and targeted consortia consisting of ΔalkMa/almA and ΔalkMb were constructed, achieving enhanced degradation (10 days) of light crude oil (73.42% to 88.65%), viscous crude oil (68.40% to 90.05%), and high waxy crude oil (47.46% to 60.52%) compared with the single wild-type strain. The degradation properties of RAG-1 and the engineered consortia strategy may have potential use in microbial biodegradation applications.

Potential for and Distribution of Enzymatic Biodegradation of Polystyrene by Environmental Microorganisms.

Polystyrene (PS) is one of the main polymer types of plastic wastes and is known to be resistant to biodegradation, resulting in PS waste persistence in the environment. Although previous studies have reported that some microorganisms can degrade PS, enzymes and mechanisms of microorganism PS biodegradation are still unknown. In this study, we summarized microbial species that have been identified to degrade PS. By screening the available genome information of microorganisms that have been reported to degrade PS for enzymes with functional potential to depolymerize PS, we predicted target PS-degrading enzymes. We found that cytochrome P4500s, alkane hydroxylases and monooxygenases ranked as the top potential enzyme classes that can degrade PS since they can break C-C bonds. Ring-hydroxylating dioxygenases may be able to break the side-chain of PS and oxidize the aromatic ring compounds generated from the decomposition of PS. These target enzymes were distributed in Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes, suggesting a broad potential for PS biodegradation in various earth environments and microbiomes. Our results provide insight into the enzymatic degradation of PS and suggestions for realizing the biodegradation of this recalcitrant plastic.

Kurstakin molecules facilitate diesel oil assimilation by Acinetobacter haemolyticus strain 2SA through overexpression of alkane hydroxylase genes.

Biodegradation is a cost-effective process commonly used to eliminate many xenobiotic hydrocarbons such as diesel oils. However, their hydrophobic character reduces the biodegradation efficiency. In order to overcome this hurdle, kurstakins isolated from Bacillus thuringiensis strain 7SA were used as emulsifying agents. The influence of kurstakin molecules on diesel oil degradation by Acinetobacter haemolyticus strain 2SA was evaluated in the presence and absence of the aforementioned lipopeptide. The degradation rates and gene expressions of alkane hydroxylases were evaluated at days 4, 10, 14 and 21. Results showed that kurstakin molecules increased the hydrophobicity of 2SA. Moreover, diesel oil degradation activities were higher in the presence of kurstakin with 29%, 35%, 29% and 23% improvement at 4th, 10th, 14th and 21st day respectively. Statistical analysis indicated that the difference between the degradation rates in the presence and absence of kurstakin was significant with p = 0.03. The detection of three different hydroxylase genes namely alkB, almA and cyp153 in 2SA genome, might have allowed more efficient degradability of alkanes. According to the real-time PCR results, cyp153 was the most induced gene during diesel oil degradation in the presence and absence of kurstakin. Yet, the three genes demonstrated higher levels of expression in the presence of kurstakin when compared to its absence. This study showed that kurstakins enhance the diesel oil biodegradation rate by increasing the hydrophobicity of 2SA. In addition to their anti-fungal activities, kurstakins can be used as biosurfactant to increase biodegradation of diesel oil.

Integrated Comparative Genomic Analysis and Phenotypic Profiling of Pseudomonas aeruginosa Isolates From Crude Oil.

Pseudomonas aeruginosa is an environmental microorganism that can thrive in diverse ecological niches including plants, animals, water, soil, and crude oil. It also one of the microorganism widely used in tertiary recovery of crude oil and bioremediation. However, the genomic information regarding the mechanisms of survival and adapation of this bacterium in crude oil is still limited. In this study, three Pseudomonads strains (named as IMP66, IMP67, and IMP68) isolated from crude oil were taken for whole-genome sequencing by using a hybridized PacBio and Illumina approach. The phylogeny analysis showed that the three strains were all P. aeruginosa species and clustered in clade 1, the group with PAO1 as a representitive. Subsequent comparative genomic analysis revealed a high degree of individual genomic plasticity, with a probable alkane degradation genomic island, one type I-F CRISPR-Cas system and several prophages integrated into their genomes. Nine genes encoding alkane hydroxylases (AHs) homologs were found in each strain, which might enable these strains to degrade alkane in crude oil. P. aeruginosa can produce rhamnolipids (RLs) biosurfactant to emulsify oil, which enables their survival in crude oil enviroments. Our previous report showed that IMP67 and IMP68 were high RLs producers, while IMP66 produced little RLs. Genomic analysis suggested that their RLs yield was not likely due to differences at genetic level. We then further analyzed the quorum sensing (QS) signal molecules that regulate RLs synthesis. IMP67 and IMP68 produced more N-acyl-homoserine lactones (AHLs) signal molecules than that of PAO1 and IMP66, which could explain their high RLs yield. This study provides evidence for adaptation of P. aeruginosa in crude oil and proposes the potential application of IMP67 and IMP68 in microbial-enhanced oil recovery and bioremediation.

Cytochrome P450 family member CYP96B5 hydroxylates alkanes to primary alcohols and is involved in rice leaf cuticular wax synthesis.

Odd-numbered primary alcohols are components of plant cuticular wax, but their biosynthesis remains unknown. We isolated a rice wax crystal-sparse leaf 5 (WSL5) gene using a map-based cloning strategy. The function of WSL5 was illustrated by overexpression and knockout in rice, heterologous expression in Arabidopsis and transient expression in tobacco leaves. WSL5 is predicted to encode a cytochrome P450 family member CYP96B5. The wsl5 mutant lacked crystalloid platelets on the surface of cuticle membrane, and its cuticle membrane was thicker than that of the wild-type. The wsl5 mutant is more tolerant to drought stress. The load of C23 -C33 alkanes increased, whereas the C29 primary alcohol reduced significantly in wsl5 mutant and WSL5 knockout transgenic plants. Overexpression of WSL5 increased the C29 primary alcohol and decreased alkanes in rice leaves. Heterologous expression of WSL5 increased the C29 primary alcohol and decreased alkanes, secondary alcohol, and ketone in Arabidopsis stem wax. Transient expression of WSL5 in tobacco leaves also increased the production C29 primary alcohol. WSL5 catalyzes the terminal hydroxylation of alkanes, yielding odd-numbered primary alcohols, and is involved in the formation of epidermal wax crystals on rice leaf, affecting drought sensitivity.

Elucidation of multiple alkane hydroxylase systems in biodegradation of crude oil n-alkane pollution by Pseudomonas aeruginosa DN1.

AIMS: The purpose of this study was to elucidate the characteristics of multiple alkane hydroxylase systems in Pseudomonas aeruginosa DN1, including two homologues of AlkB (AlkB1 and AlkB2 ), a CYP153 homologue (P450), and two homologues of Alm-like (AlmA1 and AlmA2 ). METHODS AND RESULTS: DN1 was capable of utilizing diverse n-alkanes with chain lengths from 8 to 40 C atoms as the sole carbon source, and displayed high degradation efficiency (＞85%) of crude oil and a majority of n-alkanes using gas chromatography method. RT-qPCR analysis showed that the five enzyme genes could be induced by n-alkanes ranging from medium-chain length to long-chain length which indicated the dissimilarity of expression between those genes when grown on different n-alkanes. Notably, the expression of alkB2 gene was upregulated in the presence of all of the tested n-alkanes, particularly responded to long-chain n-alkanes like C20 and C32 . Meanwhile, long-chain n-alkanes (C20 -C36 ) significantly elevated cyp153 expression level, and the expression of two almA genes was only upregulated in the presence of n-alkanes with chain lengths of 20C's and longer. Furthermore, the disruption of those genes demonstrated that AlkB2 appeared to play a key role in the biodegradation of substrates of a broad-chain length ranges, besides other alkane hydroxylase systems ensured the utilization of n-alkanes with chain lengths of from 20 to 40 C atoms. CONCLUSION: The five functional alkane hydroxylase genes make DN1 an attractive option for its versatile alkane degradation, which is primarily dependent on the expression of alkB2 . SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings suggest that P. aeruginosa DN1 is a predominately potential long-chain n-alkane-degrading bacterium with multiple alkane hydroxylase systems in crude oil-contaminated environment.

Characterization and Transcriptional Regulation of n-Alkane Hydroxylase Gene Cluster of Rhodococcus jostii RHA1.

Gram-positive actinomycete Rhodococcus jostii RHA1 is able to grow on C10 to C19 n-alkanes as a sole source of carbon and energy. To clarify, the n-alkane utilization pathway-a cluster of 5 genes (alkBrubA1A2BalkU) which appeared to be involved in n-alkane degradation-was identified and the transcriptional regulation of these genes was characterized. Reverse transcription-PCR analyses revealed that these genes constituted an operon and were transcribed in the presence of n-alkane. Inactivation of alkB led to the absence of the ability to utilize n-undecane. The alkB mutation resulted in reduction of growth rates on C10 and C12 n-alkanes; however, growths on C13 to C19 n-alkanes were not affected by this mutation. These results suggested that alkB was essential for the utilization of C10 to C12 n-alkanes. Inactivation of alkU showed the constitutive expression of alkB. Purified AlkU is able to bind to the putative promoter region of alkB, suggesting that AlkU played a role in repression of the transcription of alk operon. The results of this study indicated that alkB was involved in the medium-chain n-alkanes degradation of strain RHA1 and the transcription of alk operon was negatively regulated by alkU-encoded regulator. This report is important to understand the n-alkane degradation pathway of R. jostii, including the transcriptional regulation of alk gene cluster.

Bench-scale production of enzymes from the hydrocarbonoclastic bacteria Alcanivorax borkumensis and biodegradation tests.

This study investigates motor oil (3, 5, 7.5 and 10% (v v-1)) as a sole carbon source for the production of Alcanivorax borkumensis in shake flasks and a 5 L bench-scale fermenter in comparison to the standard media. Shake flask studies showed a significant and higher cell growth (p=0.000038), lipase (p = 0.006900) and alkane hydroxylase production (p = 0.000921) by Alcanivorax borkumensis when motor oil was used as the substrate. Based on Tukey post-hoc tests, 5% motor oil concentration was selected as the optimal substrate concentration. The 5 L fermenter experiments conducted using motor oil at 5% (v v-1) concentration, under controlled conditions exhibited significant and higher alkane hydroxylase and lipase activities (55.6 U mL-1 (p = 0.018418) and 208.30 U mL-1 (p = 0.020087), respectively) as compared with those of motor oil at 3% (v v-1) and n-hexadecane at 3% (v v-1) concentration which was used as control. Cell growth was significantly higher when motor oil (3 or 5%) was used as a substrate (p = 0.024705). Enzymatic degradation tested on two different polycyclic aromatic hydrocarbons (PAHs) contaminated groundwaters showed 37.4% removal after 5 days with a degradation rate of 196.6 ppb day-1 and 82.8% removal after 10 days with a degradation rate of 217.54 ppb day-1 for the 1st site and an almost complete biodegradation with 95% removal and 499.02 ppb day-1 removal rate after only 5 days for the 2nd site.

Production and characterization of novel hydrocarbon degrading enzymes from Alcanivorax borkumensis.

This study investigates the production of alkane hydroxylase, lipase and esterase by the marine hydrocarbon degrading bacteria Alcanivorax borkumensis. The focus of this study is the remediation of petroleum hydrocarbons, hexane, hexadecane and motor oil as model substrates. A. borkumensis showed an incremental growth on these substrates with a high cell count. Growth on motor oil showed highest alkane hydroxylase and lipase production of 2.62 U/ml and 71 U/ml, respectively, while growth on hexadecane showed the highest esterase production of 57.5 U/ml. The percentage of hexane, hexadecane, and motor oil degradation during A. borkumensis growth after 72 h, was around 80%, 81.5% and 75%, respectively. Zymogram showed two different bands with a molecular weight of approx. 52 and 40 kDa, respectively with lipase and esterase activity. Alkane hydroxylase reached optimum activity at pH 8.0 and 70 ±â€¯1 °C for hexane and hexadecane and 75 ±â€¯1 °C for motor oil. Lipase and esterase showed optimum activity at 35 ±â€¯1 °C and 40 ±â€¯1 °C, respectively and pH 7.0. The crude enzymes showed higher stability in a wide range of pH, but they were not thermostable at higher temperatures.