alcanivorax borkumensis bioremediation

Alcanivorax borkumensis is a cosmopolitan marine bacterium that uses oil hydrocarbons as its exclusive source of carbon and energy. [Fig.1B1B ]), which has been proposed to be essential for catalytic activity (23). Controls for DNA contamination experiments were set up by adding the RNA template after the reverse transcriptase step and before activating the Taq polymerase. Of all the Alcanivorax species and other oil-degrading microbes, Alcanivorax borkumensis is one of the most important worldwide. Specifically, AtfA2 was almost totally devoid of DGAT activity. Substrate specificity of naphthalene dioxygenase: effect of specific amino acids at the active site of the enzyme. Metals content was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). 30, 23422352. (15) van Beilen J, Neunschwander M, Smits T, Roth C, Balada S, Witholt B. 2018 Apr 1;164:27-33. doi: 10.1016/j.colsurfb.2018.01.021. Alkanes were added to the buffer using alkane stock solutions in DMSO. Marchut-Mikolajczyk, O., Kwapisz, E., Wieczorek, D., Antczak, T., 2015. In the double knockout mutant WS activity was completely abolished, whereas a very low but significant basic level of DGAT activity was maintained (Table (Table33). Imaging was performed with an H-500 transmission electron microscope (TEM; Hitachi, Japan) in the bright-field mode at an acceleration voltage of 75 kV at room temperature. Biotechnol. Methylibium petroleiphilum. strain ADP1) (38), the wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT), was discovered. 104, 142148. In turn, the molecules become accessible for degradation to other microbes. This page was last edited on 29 September 2015, at 18:47. Column tests for evaluation of the enzymatic biodegradation capacity of hydrocarbons (C. Bench-scale production of enzymes from the hydrocarbonoclastic bacteria Alcanivorax borkumensis and biodegradation tests. official website and that any information you provide is encrypted The calculated global consumption rate (GCR) was about 566.1442.1 mg kg-1 d-1 for contaminated soil with 6170.7 mg-1 kg-1 of TPH while the maximum consumption was 363.4 mg-1 kg-1. The polymerization of the resin was performed at 70C for 48 h. Sections 70 to 80 nm thick were made with an Ultracut Microtome (LEICA Mikroskopie und Systeme GmbH, Germany) using a diamond knife and were subsequently placed on a 200-mesh copper grid. Overall, degrading capacities of crude enzymes produced were also tested in the presence of contaminated soil which contains a range of contaminants as described in Table 1. Federal government websites often end in .gov or .mil. FOIA Taken together, the selected strain is a potential candidate for the degradation of motor oil (known to be generally more resistant to degradation) in minimal time. Setti, L., Lanzarini, G., Pifferi, P.G., Spagna, G., 1993. This bacterium is significant because it is involved in the bioremediation of oil-contaminated aquatic environments . Similarly, in the type strain Alcanivorax borkumensis SK2 the increased cell damage at 10 MPa was consistent with the intracellular accumulation of the piezolyte ectoine, . Higher enzymatic activities were obtained during the degradation of BTEX with 0.93 U/mL of alkane hydroxylase, 57.8 U/mL of lipase and 60.1 U/mL of esterase when using 10% (v/v) of inoculum concentration after 7 days of degradation (Fig. Supplemental material for this article may be found at http://jb.asm.org/. La pollution de l'eau de source ponctuelle est rglemente par la Clean Water Act. Alcanivorax borkumensis (A. borkumensis) is a Gram-negative, rod-shaped hydrocarbonoclastic ("oil-degrading") bacterium. 30(6):734-742. Fatty acid analysis of whole cells was done by gas liquid chromatography (GC) according to reference (24) after derivatization to fatty acid methyl esters by sulfuric acid-catalyzed methanolysis. The contaminated soil used in this study was provided by TechnoRem Inc from a confidential site in Quebec. 2019 Oct 18;10:2387. doi: 10.3389/fmicb.2019.02387. After being washed three times in phosphate-buffered saline (PBS) (pH 7.3), the cells were fixed with 2.5% (wt/vol) glutaraldehyde in 0.1 M PBS (pH 7.3) for 16 h. After being washed three more times in PBS, each time for 20 min, the cells were postfixed with 1% (wt/vol) osmium tetroxide in 0.1 M PBS (pH 7.3) for 90 min and washed once with PBS for 15 min. Technol. The plasmids were then linearized by HindIII digestion; fused to the HindIII restricted mobilizable suicide plasmid pSUP202, yielding plasmid pSUP202::pET23a::atfA1Km or pSUP202::pET23a::atfA2Sm, respectively; and transformed into E. coli S17-1. Alcanivorax spp. Thus, sequence comparison of AtfA1 and AtfA2 from A. borkumensis and WS/DGAT from A. baylyi shows that amino acid residues conserved in AtfA1 and WS/DGAT, but not AtfA2, might specify those domains that are important for DGAT activity, whereas amino acids conserved among all three enzymes indicate regions that are probably necessary for general acyltransferase catalytic activity (Fig. Epub 2018 Jan 23. Cells were harvested, washed once with cLB mating medium (LB medium supplemented with 2% [wt/vol] pyruvate, 0.0445% [wt/vol] Na2HPO4 2H2O, 0.25% [wt/vol] NaNO3, 1.15% [wt/vol] NaCl, 0.375% [wt/vol] KCl, 0.0735% [wt/vol] CaCl2 2H2O), and concentrated 10-fold in cLB mating medium. Careers. The Glycine-Glucolipid of Alcanivorax borkumensis Is Resident to the Bacterial Cell Wall. Opin. 8600 Rockville Pike Ecol. Thus, toluene had been claimed as the most easily biodegradable among the six compounds of BTEX. Expression of atfA1 and atfA2 was analyzed by RT-PCR in samples derived from cells cultivated with 1% (wt/vol) sodium pyruvate as the carbon source. Alcanivorax borkumensis is a hydrocarbon degrading bacterium linked to oil degradation around oil spill sites. 152, 12761284. Federal government websites often end in .gov or .mil. 60, 429439. Cellulosimicrobium cellulans exhibited hydrocarbon degradability of 18.86% after 15 days (Nkem et al., 2016). Accordingly, aerobic enzymatic degradation can be affected by many physical, chemical and biological conditions (e.g. Substrate interactions during anaerobic biodegradation of BTEX by the mixed cultures under nitrate reducing conditions. Wild-type A. borkumensis SK2 cultivated with pyruvate exhibited a moderate level of WS activity (64.0 pmol mg1 min1) and an approximately fourfold-lower level of DGAT activity (Table (Table3).3). An official website of the United States government. Golyshin PN, Martins Dos Santos VA, Kaiser O, Ferrer M, Sabirova YS, Lnsdorf H, Chernikova TN, Golyshina OV, Yakimov MM, Phler A, Timmis KN. We use COREXIT EC9500A, cetylytrimethylamonium bromide, dioctyl sulfosuccinate sodium salt, l--phosphatidylcholine, sodium dodecyl sulfate, and Tween 20 to investigate the impact of dispersants on Alcanivorax borkumensis. The accumulation of TAGs in large quantities has so far been described only for certain gram-positive bacteria of the actinomycetes group, belonging to the genera Mycobacterium, Nocardia, Rhodococcus, and Streptomyces (2). Anal. 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. Alcanivorax borkumensis (A. borkumensis) is a Gram-negative, rod-shaped hydrocarbonoclastic ("oil-degrading") bacterium. Pollut. Disclaimer, National Library of Medicine Lipids were visualized by staining with iodine vapor. Pollut. Thenmozhi, R., Nagasathya, A., Thajuddin, N., 2011. Epub 2010 Mar 31. Free resources to assist you with your university studies! Although so far assumed to be a characteristic restricted to gram-positive actinomycetes, substantial accumulation of TAGs corresponding to a fatty acid content of more than 23% of the cellular dry weight is the first characteristic of large-scale de novo TAG biosynthesis in a gram-negative bacterium. Federation, W.E., Association, A.P.H., others, 2005. The site is secure. In all tested strains, no significant amounts of 3-hydroxy fatty acid monomers were detectable by GC analysis, indicating that PHAs were absent or present only in very small amounts (data not shown). Enhancement of hydrocarbon wastebiodegradation by addition of a biosurfactantfrom Bacillus subtilis O9. Strain MBIC 4326. Once again, n-hexane was added to the pellet (1:1 w/v) and then centrifuged at the same speed and time to recover the pellet. Wild-type cells showed a two-log-fold decrease in viability within the first 10 days of starvation, but after that viability remained relatively constant even over an extended 4-week period of starvation. Bacterial chemotaxis to pollutants and plant-derived aromatic molecules. Ron, E.Z., Rosenberg, E., 2014. Microbiol. Gianfreda, L., Rao, M.A., 2004. The solutions were incubated at 30 C for 7 days with shaking at 100 rpm in an incubator shaker. (1) Yakimov M, Golyshin P, Lang S, Moore E, Abraham W, Lunsdorf H, Timmis K. Alcanivorax borkumensis gen. nov., sp. DOI: 10.1128/JB.184.6.1722-1732.2002, (16) Dutta TK, Harayama S. Biodegredation of n-alkylcycloalkanes and n-alkylbenzenes via New Pathways in Alcanivorax sp. Bioremediation for degradation of hydrocarbons is a widely used alternative for the recovery of contaminated sites. FOIA The donor strain was cultivated at 30C in LB medium for 24 h, and the recipient strain was grown in ONR7a medium containing pyruvate for 48 h at 30C. To export a reference to this article please select a referencing stye below: Energy regards the power derived from a fuel source such as electricity or gas that can do work such as provide light or heat. Alcanivorax borkumensis is a rod-shaped degrading bacterium that is commonly associated with natural bioremediate oil spills. 2022 Aug 23;88(16):e0112622. High levels of WS and lower DGAT activity were detected for AtfA1. Pract. The present study deals with the enzymatic degradation of A. borkumensis which was grown in the presence of xenobiotic petroleum hydrocarbons being reported to be toxic substances, such as hexadecane, motor oil and BTEX. The cell number reached around 108 CFU/mL (Colony Forming Units per mL) in the case of hexadecane, BTEX, motor oil, as well as petroleum hydrocarbons contaminated soil. The alcohol is oxidized by an alcohol dehydrogenase to yield an aldehyde (9). Its widespread and extensive use has led to serious ecological problems mainly due to improper waste management, polluting industries, maritime spills, and leaked oil from transport ships along with leaching landfills. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Microbiol. Storage lipids predominantly consisting of TAGs synthesized during cultivation of A. borkumensis on pyruvate accumulated in the form of electron transparent intracytoplasmic inclusions as shown by TEM (Fig. The present work aims to study the capacity of crude enzymes produced by Alcanivorax borkumensis, which is a hydrocarbonoclastic bacterium, to degrade petroleum hydrocarbons represented by hexadecane with a concentration varying from 5000 mg/L to 7000 mg/L, BTEX with a concentration of the six compounds varying from 30 mg/L to 70 mg/L (1:1:1:1:1:1), motor oil with a concentration varying from 500 mg/L to 1000 mg/L and contaminated soil with a concentration of total petroleum hydrocarbons varying from 2000 mg/L to 6000 mg/L. Before Alcanivorax was one of the types of bacteria that Todd and his . Biosurfactants and Their Applications in the Oil and Gas Industry: Current State of Knowledge and Future Perspectives. strain ZD22. Singh, R., Celin, S.M., 2010. 2010. If you are the original writer of this dissertation and no longer wish to have your work published on the UKDiss.com website then please: Our academic writing and marking services can help you! Int. Bioresour. A 2-l portion of the organic phase was analyzed after split injection (1:5); hydrogen (constant flow, 0.6 ml min1) was used as carrier gas. Biochim. Kem MP, Zane HK, Springer SD, Gauglitz JM, Butler A. Metallomics. The use of fertilizer to increase the activity of A. borkumensis has become an accepted technique for crude oil spill clean ups (17). Se cree actualmente para ser ms importantes organismos que degradan petrleo del Crude oil is challenging to clean up as it has over 17000 distinct compounds (3). The current study aimed to use Alcanivorax borkumensis crude enzymes preparation as an agent for enhanced microbial hydrocarbons biodegradation in contaminated water and soil. Int J Biol Macromol. Horowitz, A., Atlas, R.M., 1977. Opin. We therefore tested the presence of potential storage lipid species in alkane-grown and pyruvate-grown cells of A. borkumensis by means of GC and TLC analyses. A. borkumensis can only degrade select alkanes in crude oil (3). The resulting PCR products were cloned as HindIII-XhoI fragments into the expression vector pET23a colinear to the T7 promoter, yielding pET23a::atfA1 or pET23a::atfA2, respectively, and were then transformed into E. coli BL21(DE3). (Fig.2B),2B), accounting for a fatty acid content of ca. . FEMS Microbiol. 129, 153158. Toxic Radioact. These so-called hydrocarbonoclastic bacteria so far comprise the genera Alcanivorax (42), Cycloclasticus (10), Marinobacter (15), Neptunomonas (21), Oleiphilus (16), Oleispira (41), and Thalassolituus (40). eCollection 2019. An official website of the United States government. Disclaimer, National Library of Medicine Biodegradation of benzene and its derivatives by a psychrotolerant and moderately haloalkaliphilic Planococcus sp. In fact, biodegradation progression is affected by the bacterial strain used for bioremediation and the capacity of each bacterial isolate to use efficiently the available nutrients and produce enzymes. The family includes the type genus Alcanivorax and the genus Kangiella. Fig. Cells of Escherichia coli were grown in LB medium at 37C (30). 4(3): 141-147. The nature of the alternative TAG biosynthesis route in A. borkumensis is not known so far. Of particular importance is Alcanivorax, a marine bacterium that can assimilate various linear or branched alkanes (Liu et al., 2011; Wu et al., 2008; Yakimov et al., 2007). Genome sequence completed of Alcanivorax borkumensis, a hydrocarbon-degrading bacterium that plays a global role in oil removal from marine systems. Limited emulsification has been implicated in causing the transition from the exponential to a linear growth phase (19). How does bioremediation of spilled oil work? 94, 355362. Alcanivorax borkumensis is an alkane-degrading bacteria that can produce petroleum degrading enzymes such as alkane hydroxylase and lipase. Hexadecanol, derived from oxidation of the growth substrate hexadecane, was the only fatty alcohol component of WEs isolated from alkane-grown cells detectable by GC analysis (data not shown). 106(2-3): 215-220. Microbiol. Das, N., Chandran, P., 2010. Enhanced bioremediation of oil spills in the sea. In clear contrast to this, dramatic differences between AtfA1 and AtfA2, regarding their specificity for different acylglycerol substrates, were observed (Table (Table5).5). During growth on hexadecane, intracellular storage lipids consisted of TAGs and WEs (Fig. (Fig.2C2C). (Fig.2B).2B). Neither atfA1 nor atfA2 was clustered with genes with any known relevance for storage lipid biosynthesis (Fig. These results suggested that the enzymatic activities implicated in the biodegradation processes can be preserved for 7 days which offer a wide range of application. The higher activities ranged from 40 to 71 U/mL of lipase for the different studied substrates. official website and that any information you provide is encrypted Such lipophilic dyes are not specific and cannot differentiate between PHAs, TAGs, WEs, and other neutral lipids, such as hydrocarbon inclusions. Characterization of two alkane hydroxylase genes from the marine hydrocarbonoclastic bacterium Alcanivorax borkumensis. J. It thrives in halophilic, aerobic environments and is found in the upper layers of freshwater or marine environments such as the Mediterranean Sea, Pacific Ocean and Arctic Sea . Marinobacter hydrocarbonoclasticus (72 h at 30C), Alcanivorax jadensis T9 (72 h at 30C), and Thalassolituus oleivorans (96 h at room temperature) were grown aerobically in ONR7a medium containing 1% (wt/vol) sodium pyruvate. Exploring Prospectivity on the Flanks of the Troll Field. Microbiol. . Chapter 1 Introduction Environ. Toxicol. P.N.G. Some environmental aspects of marine hydrocarbon bacteriology. Aquatic Microbiology. St, PstI-digested DNA. In contrast, inactivation of atfA2 caused a relatively low reduction of both acyltransferase activities. Biodegradacin de hidrocarburos por un consorcio microbia-no de la rizsfera de una planta nativa de pantano. A putative active site motif is boxed. Acinetobacter baumannii isolated from crude oil exhibited 62.8% of TPH biodegradation after 7 days (Mishra et al., 2004). The identification of two gene products homologous to the WS/DGAT from A. baylyi strain ADP1 indicated the potential capability of A. borkumensis to synthesize and accumulate TAGs and/or WEs. In gram-negative bacteria, TAGs have so far been reported only as a minor constituent in Acinetobacter species (23, 28). More than 89% of enzymatic removal was obtained in the presence of different compounds namely BTEX, motor oil, hexadecane and contaminated soil. Functional expression of atfA2 was also documented by the fact that somehow reduced WS activity levels were observed in vitro in atfA2 knockout mutants (Table (Table33). As a result, A. borkumensis cannot utilize aromatic alkanes as a primary fuel source (9). 106, 215220. Not very much is known about the exact method used by Alcanivorax to biodegrade oil, but some The structures of the inclusions were generally highly variable, many of them being quite long and sprawling, occasionally reaching a length of 500 nm. Enzyme Microb. 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. Careers. The performance of the extracellular and intracellular crude enzymes from A. borkumensis was evaluated in batch tests in Milli-Q water. The aldehyde is subsequently oxidized to yield a fatty acid via an aldehyde dehydrogenase (9). 2002. Abstract: Three bacterial isolates identified as Alcanivorax borkumensis SK2, Rhodococcus erythropolis HS4 and Pseudomonas stutzeri SDM, based on 16S rRNA gene sequences, were isolated from crude oil enrichments of natural seawater. These results are advantageous compared with Pseudomonas aeruginosa isolated from hydrocarbon-polluted environment which was able to utilize 81% of used engine oil within 4 weeks compared to 7 days in the current study (Thenmozhi et al., 2011). A. borkumensis is the type species, an almost exclusively hydrocarbonoclastic bacteria, which dominates marine environments suffering from oil contamination through its ability of nutrient scavenging, oligotrophic growth, and biofilm formation. One unit of enzyme activity was defined as the amount of enzyme which liberated 1 mol of fatty acids per minute. oil), polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), heterocyclic compounds (such as pyridine or quinoline . Biodegradation of BTEX (benzene, toluene, ethyl benzene and xylene) compounds by bacterial strain under aerobic conditions. This permits A. borkumensis to break down cycloalkanes only when there are aliphatic alkanes present, since the expression of the necessary genes is induced by the presence of aliphatic alkanes. Both enzymes could comparably utilize a broad range of short-, medium-, and long-chain linear alcohols with the highest specificity for medium-chain alcohols (1-decanol) (Table (Table4).4). Cells were harvested, washed three times in ONR7a medium without any carbon source, resuspended in the same medium, and incubated at 30C. The strain was stored at 4 C on agar plates coated with a film of hexadecane. 3.2 Biodegradation kinetics of contaminated soil. Ultrathin sections of cells were analyzed by transmission electron microscopy. The inoculum and hydrocarbons concentration have a remarkable effect on the biodegradation with the crude enzymes. Colloids Surf B Biointerfaces. doi:10.1016/j.petrol.2015.02.038. DOI: 10.1099/00207713-48-2-339, (2) Hara A, Syutsubo K, Harayama S. Alcanivorax which prevails in oil-contaminated seawater exhibits broad substrate specificity for alkane degradation. Environmental Microbiology. The method used to determine the PAHs is MA. The results of this study will be important in the decision of dispersant use in the future. Fedorak, P.M., Westlake, D.W.S., 1981. Gertler C, Gerdts G, Timmis KN, Yakimov MM, Golyshin PN. For these reasons, hexadecane is used in this study as a model molecule to study aliphatic hydrocarbon biodegradation since it has been always considered as a model (Schoefs et al., 2004). The removal percentage of C16H34 decreased from 73.75% to 59.74% after 7 days of enzymatic degradation, while increasing the concentration of hexadecane from 5000 to 7000 mg/L when using 10% (v/v) of inoculum concentration (Fig. Alcanivorax borkumensis is a marine bacteria. Bacto agar (Difco, Fisher Scientific, Mississauga, Ontario) (15 g/L) was added to the first solution for the preparation of solid media (Yakimov et al., 1998). The genome comprises numerous putative acyltransferase genes of yet unknown function (31; see Table S1 in the supplemental material), but none of these exhibits reasonable homology to any of the above-mentioned eukaryotic TAG-synthesizing enzymes. The https:// ensures that you are connecting to the 7H2O. 87, 8186. Dans le cas de dversements de ptrole dans l'ocan, une bioremdiation naturelle continue a tendance se produire, dans la mesure o des bactries . A research team shows the. This is corroborated by the fact that no obvious intracellular lipophilic inclusions remain in mutant cells impaired in TAG and WE biosynthesis. Environ. (A) Genotypic characterization of A. borkumensis mutants. HZ01 from the crude oil-contaminated seawater at the Daya Bay, southern China. Although many marine bacteria are capable of degrading petroleum hydrocarbons, only a few of them seem to be important for petroleum biodegradation in natural marine environments. Safety. Alcanivorax borkumensis is a hydrocarbon degrading bacterium linked to oil degradation around oil spill sites. In this study, pilot-scale Alcanivorax borkumensis Petroleum degrading enzymes can be used as an alternative way to improve petroleum bioremediation approaches. Air. . Interestingly, atfA1 is situated in close proximity to the origin of replication of the chromosome, probably resulting in a high gene dosage. Parales, R.E., Harwood, C.S., 2002. doi:10.1111/j.1462-2920.2009.01948.x. Biotechnol. In general, there are four methods of cleaning up an oil spill. J. Liu, C., Wang, W., Wu, Y., Zhou, Z., Lai, Q., Shao, Z., 2011. Split/Splitless injector and detector (FID) temperatures were 260 C and 260 C, respectively, and 1 mL of the sample was injected. Microbiol. Please enable it to take advantage of the complete set of features! The present study describes the analysis of biosynthesis and accumulation of storage lipids in A. borkumensis and the use of biochemical characterization and directed mutagenesis to examine the role of the two WS/DGAT-homologous proteins in this pathway. Bradford, M.M., 1976. +, RT-PCR assay; , control for DNA contamination experiments. Remarkably, this novel enzyme exhibits no homology to known acyltransferases involved in TAG or WE biosynthesis in eukaryotes, and it is widely distributed among TAG-accumulating actinomycetes (23). Bull. Technol. Degradation of aromatics and saturates in crude oil by soil enrichments. DOI: 10.1111/j.1462-2920.2004.00567.x, (11) Teimoori A, Ahmadian S, Mdadkar-Sobhani A, Bambai B.

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