Exoproteome analysis of Pseudomonas aeruginosa response to high alkane stress.

Microbial biodegradation serves as an effective approach to treat oil pollution. However, the application of such methods for the degrading long-chain alkanes still encounters significant challenges. Comparative proteomics has extensively studied the intracellular proteins of bacteria that degrade short- and medium-chain alkanes, but the role and mechanism of extracellular proteins in many microorganism remain unclear. To enhance our understanding of the roles of extracellular proteins in the adaptation to long-chain alkanes, a label-free LC-MS/MS strategy was applied for the relative quantification of extracellular proteins of Pseudomonas aeruginosa SJTD-1-M (ProteomeXchange identifier PXD014638). 444 alkane-sentitive proteins were acquired and their cell localization analysis was performed using the Pseudomonas Genome Database. Among them, 111 proteins were found to be located in extracellular or Outer Membrane Vesicles (OMVs). The alkane-induced abundance of 11 extracellular or OMV target proteins was confirmed by parallel reaction monitoring (PRM). Furthermore, we observed that the expression levels of three proteins (Pra, PA2815, and FliC) were associated with the carbon chain length of the added alkane in the culture medium. The roles of these proteins in cell mobility, alkane emulsification, assimilation, and degradation were further discussed. OMVs were found to contain a number of enzymes involved in alkane metabolism, fatty acid beta-oxidation, and the TCA cycle, suggesting their potential as sites for facilitated alkane degradation. In this sense, this exoproteome analysis contributes to a better understanding of the role of extracellular proteins in the hydrocarbon treatment process.

Epicuticular wax chemicals of Lablab purpureus subsp. bengalensis influence short-range attraction and oviposition responses in Aphis craccivora and Aphis gossypii.

Lablab purpureus subsp. bengalensis (Jacq.) Verdc. is an important legume of India and Africa. Both aphids, Aphis craccivora Koch and A. gossypii Glover (Hemiptera: Aphididae), are important herbivorous pests of this legume crop. These viviparous females lay nymphs on the leaf surface of this legume plant. Therefore, it is of considerable interest to study whether leaf surface wax chemicals (long-chain alkanes and free fatty acids) of this legume plant served as short-range attractants and oviposition stimulants in both females to lay nymphs. Twenty-one n-alkanes from n-C12 to n-C35 and 11 free fatty acids from C12:0 to C22:0 were identified in leaf surface waxes. Nonacosane and nonadecanoic acid were the most abundant among n-alkanes and free fatty acids, respectively. Both females were attracted towards one leaf equivalent surface wax against the control solvent (petroleum ether) in short Y-tube olfactometer bioassays. A synthetic blend of tetradecane, pentadecane, tetracosane, tridecanoic acid, tetradecanoic acid, and heneicosanoic acid comparable to one leaf equivalent surface wax served as short-range attractants and oviposition stimulants in A. craccivora; whereas a synthetic blend of tetradecane, hexadecane, docosane, nonadecanoic acid, and arachidic acid comparable to one leaf equivalent surface wax acted as short-range attractants and oviposition stimulants in A. gossypii. These results can provide the basis for efficient pest management strategies of A. craccivora and A. gossypii against L. purpureus subsp. bengalensis using host plant leaf surface wax compounds. Further, SEM studies of antennae and forelegs of both aphids were conducted to observe sensilla structures, which help in chemoreception.

Reactions of C12-C14 n-Alkylcyclohexanes with Cl Atoms: Kinetics and Secondary Organic Aerosol Formation.

Long-chain alkanes are a type of intermediate volatility organic compound (IVOC) in the atmosphere and a potential source of secondary organic aerosols (SOAs). C12-C14 n-alkylcyclohexanes are important compositions of IVOCs, with considerable concentrations and emission rates. The reaction rate constants and SOA formation of the reactions of C12-C14 n-alkylcyclohexanes with Cl atoms were investigated in the present study. The reaction rate constants of the long-chain alkanes obtained via the relative-rate method at 298 ± 0.2 K (in units of ×10-10 cm3 molecule-1 s-1) were as follows: khexylcyclohexane = 5.11 ± 0.28, kheptylcyclohexane = 5.56 ± 0.30, and koctylcyclohexane = 5.74 ± 0.31. The gas-phase products of the reactions were identified as mainly small molecules of aldehydes, ketones, and acids. The particle-phase products were mostly monomers and oligomers, but there were still trimers even under high-NOx conditions. Moreover, under high-NOx conditions (urban atmosphere), the SOA yields of hexylcyclohexane are higher than that under low-NOx conditions (remote atmosphere), indicating that more attention should be given to the SOA formation of Cl-initiated n-alkylcyclohexane oxidations in polluted regions. This research can further clarify the oxidation processes and SOA formation of n-alkylcyclohexanes in the atmosphere.

Short-range attraction and oviposition stimulant of a biocontrol agent, Galerucella placida Baly (Coleoptera: Chrysomelidae) toward weed leaf surface waxes.

Two Polygonaceae weeds, Rumex dentatus L. and Polygonum glabrum Willd. are abundant in wheat- and rice-fields, respectively, in India. Galerucella placida Baly (Coleoptera: Chrysomelidae) is a biocontrol agent of these two weeds. The importance of long-chain alkanes and free fatty acids present in leaf surface waxes of these weeds was assessed as short-range attractant and ovipositional stimulant in G. placida females. Extraction, TLC, GC-MS and GC-FID analyses demonstrated 19 n-alkanes from n-C14 to n-C35 and 14 free fatty acids from C12:0 to C22:0 in leaf surface waxes. Hentriacontane was predominant among alkanes in both weeds, while oleic acid and docosanoic acid were predominant among free fatty acids in R. dentatus and P. glabrum, respectively. Females of G. placida were attracted toward one leaf equivalent surface wax of both weeds against the control solvent (petroleum ether) in a short Y-tube olfactometer bioassay. But, the insect could not differentiate between one leaf equivalent surface wax of R. dentatus and P. glabrum, indicating that both weed leaves were equally attractive in females. A synthetic blend of either 2.44, 35.57 and 23.58 µg ml-1 of octadecane, heptacosane and nonacosane, respectively, resembling the amounts present in one leaf equivalent surface wax of R. dentatus or 4.08, 19.54 and 23.58 µg ml-1 of octadecane, palmitoleic acid and docosanoic acid, respectively, resembling the amounts present in one leaf equivalent surface wax of P. glabrum acted as short-range attractant and ovipositional stimulant in G. placida. These results could be a basis for host plant specificity of the biocontrol agent.

Long-chain alkanes in the atmosphere: A review.

As a representative species of intermediate volatile organic compounds (IVOCs), long-chain alkanes are considered to be important precursors of secondary organic aerosols (SOA) in the atmosphere. This work reviews the previous studies on long-chain alkanes in the atmosphere: (1) the detection methods and filed observations of long-chain alkanes in both gas and particle phases are summarized briefly; (2) the laboratory studies of long chain alkanes are reviewed, the kinetic data, reaction mechanism, SOA yields, and physicochemical properties of SOA are included in detail; (3) the research progress related to model simulations of long-chain alkanes are also discussed. In addition, based on available research results, several perspective contents are proposed that can be used as a guideline for future research plans.

Leaf Surface Wax Chemicals in Trichosanthes anguina (Cucurbitaceae) Cultivars Mediating Short-Range Attraction and Oviposition in Diaphania indica.

Larval Diaphania indica (Saunders) (Lepidoptera: Crambidae) cause complete defoliation of Trichosanthes anguina L. and reduce crop yield in India. Females lay eggs on the leaf surface, and therefore leaf surface waxes are potentially involved in host selection. Alkanes and free fatty acids are the major constituents of leaf surface waxes, so a study was conducted to determine whether these wax constituents from three T. anguina cultivars (MNSR-1, Baruipur Long, and Polo No.1) could act as short-range attractants and oviposition stimulants in D. indica females. Twenty n-alkanes from n-C14 to n-C36 and 13 free fatty acids from C12:0 to C21:0 were detected in the leaf surface waxes of these cultivars. Heptadecane and stearic acid were predominant among n-alkanes and free fatty acids, respectively, in these cultivars. Females showed attraction towards one leaf equivalent surface wax of each of these cultivars against solvent controls (petroleum ether) in Y-tube olfactometer bioassays. A synthetic blend of heptadecane, eicosane, hexacosane, and stearic acid, a synthetic blend of hexacosane and stearic acid, and a synthetic blend of pentadecane and stearic acid comparable to amounts present in one leaf equivalent surface wax of MNSR-1, Baruipur Long, and Polo No.1, respectively, were short-range attractants and oviposition stimulants in D. indica. Female egg laying responses were similar to each of these blends, providing information that could be used to developing baited traps in integrated pest management (IPM) programs.

The role of Lathyrus sativus flower surface wax in short-range attraction and stimulant for nymph laying by an adult viviparous aphid.

Aphis craccivora Koch (Hemiptera: Aphididae) is an important pest of Lathyrus sativus L. plants, and causes retarded plant growth and loss of seed production. The insect sucks cell sap from flowers and lays nymphs on flowers. Hence, an attempt has been made to observe whether flower surface wax compounds (alkanes and free fatty acids) from two cultivars (BIO L 212 Ratan and Nirmal B-1) of L. sativus could act as short-range attractant and stimulant for nymph laying by adult viviparous females. The n-hexane extracts of flower surface waxes were analyzed by TLC, GC-MS and GC-FID analyses. Twenty one and 22 n-alkanes between n-C12 and n-C36 were detected in BIO L 212 Ratan and Nirmal B-1, respectively; whereas 12 free fatty acids between C12:0 and C22:0 were identified in both cultivars. Pentadecane and tridecanoic acid were predominant n-alkane and free fatty acid, respectively. One flower equivalent surface wax of both cultivars served as short-range attractant and stimulant for nymph laying through Y-tube choice experiments and I-tube viviparity assays, respectively, by adult viviparous females. A synthetic blend of nonacosane, tridecanoic acid and linoleic acid, and a synthetic blend of tetradecane, pentadecane, nonacosane, tridecanoic acid and linoleic acid resembling in amounts as present in one flower equivalent surface wax of BIO L 212 Ratan and Nirmal B-1, respectively, served as short-range attractant and stimulant for nymph laying by adult viviparous females. This study suggests that both these blends could be used in lures in the development of baited traps in pest management programmes.

Characterization and identification of long-chain hydrocarbon-degrading bacterial communities in long-term chronically polluted soil in Ogoniland: an integrated approach using culture-dependent and independent methods.

Escalating oil consumption has resulted in an increase in accidental spills of petroleum hydrocarbons, causing severe environmental degradation, notably in vulnerable regions like the Niger Delta. Complex mixture of these hydrocarbons particularly long-chain alkanes presents unique challenges in restoration of polluted environment due to their chemical properties. This study aimed to investigate the long-chain hydrocarbon-degrading bacterial communities within long-term chronically polluted soil in Ogoniland, by utilizing both traditional cultivation methods and modern culture-independent techniques. Results revealed that surface-polluted soil (SPS) and subsurface soil (SPSS) exhibit significantly higher total organic carbon (TOC) ranging from 5.64 to 5.06% and total petroleum hydrocarbons (TPH) levels ranging from 36,775 ppm to 14,087 ppm, compared to unpolluted soil (UPS) with 1.97% TOC and 479 ppm TPH, respectively. Analysis of carbon chain lengths reveals the prevalence of longer-chain alkanes (C20-28) in the surface soil. Culture-dependent methods, utilizing crude oil enrichment (COE) and paraffin wax enrichment (PWE), yield 47 bacterial isolates subjected to a long-chain alkane degradation assay. Twelve bacterial strains demonstrate significant degradation abilities across all enriched media. Three bacterial members, namely Pseudomonas sp. (almA), Marinomonas sp. (almA), and Alteromonas (ladA), exhibit genes responsible for long-chain alkane degradation, demonstrating efficiency between 50 and 80%. Culture-independent analysis reveals that surface SPS samples exhibit greater species richness and diversity compared to subsurface SPSS samples. Proteobacteria dominates as the phylum in both soil sample types, ranging from 22.23 to 82.61%, with Firmicutes (0.2-2.22%), Actinobacteria (0.4-3.02%), and Acidobacteria (0.1-3.53%) also prevalent. Bacterial profiles at genus level revealed that distinct variations among bacterial populations between SPS and SPSS samples comprising number of hydrocarbon degraders and the functional predictions also highlight the presence of potential catabolic genes (nahAa, adh2, and cpnA) in the polluted soil. However, culture-dependent analysis only captured a few of the dominant members found in culture-independent analysis, implying that more specialized media or environments are needed to isolate more bacterial members. The findings from this study contribute valuable information to ecological and biotechnological aspects, aiding in the development of more effective bioremediation applications for restoring oil-contaminated environments.

Additional insights from very-high-resolution 13C NMR spectra of long-chain n-alkanes.

New information has been obtained from very-high-resolution (13) C NMR studies of a series of long-chain n-alkanes. These compounds are fundamentally important in the petroleum industry and are essential to the life of some plants, flowers, and insects. At least partial resolution of the ten different (13) C NMR signals of n-C20 H42 is observed at 11.7 T for solutions in C6 D6 or C6 D5 CD3 . A (13) C T1 inversion-recovery experiment provides much more detailed information than in previous studies of long-chain n-alkanes, demonstrates a steady increase in the relaxation times of the ten different carbons proceeding from the middle to the end of the chain because of segmental motion, and thus confirms the assignments for the interior carbons. In contrast, there is significant overlap for the signals for C-7 and the more interior carbons in a solution of n-C16 or longer chain alkanes in CDCl3 . Not only are the chemical shifts sensitive to the solvent used, but also the relative chemical shifts change. Signals for the interior carbons of the odd-number alkanes in CDCl3 are better resolved than in the spectra of their even-number counterparts. Some mixed aromatic solvent systems give increased dispersion of the cluster of C-6 through C-10 signals of n-C20 H42 , n-C21 H44 , and n-C22 H46 . However, none of the solvents used could even partially resolve the C-10 and C-11 signals of n-C21 H44 or n-C22 H46 at 11.7 T, which may result from a different distribution of conformers for n-C21 H44 or n-C22 H46 than for n-C20 H42 and shorter n-alkanes.

Directly Evolved AlkS-Based Biosensor Platform for Monitoring and High-Throughput Screening of Alkane Production.

Biosynthetic alkane using acyl-ACP aldehyde reductase (AAR) and aldehyde-deformylating oxygenase (ADO) from cyanobacteria is considered a promising alternative for the production of biofuels and chemical feedstocks. However, the lack of suitable screening methods to improve the catalytic efficiency of AAR and ADO has hindered further improvements in alkane production. Herein, a novel alkane biosensor was developed based on transcriptional factor AlkS by directed evolution, which shows sensitive dynamic response curves for exogenous long-chain alkanes as well as in situ monitoring of endogenously produced alkanes. The evolved biosensor enables high-throughput screening of alkane-producing strains from the AAR and ADO mutant library, which led to a 13-fold increase in the production of long-chain alkanes, including a 22-fold increase of C15. This study is the first to improve the alkane production through biosensors, which provides a good reference for the establishment of microbial cell factories for alkane production.

Molecular docking analysis of long-chain alkanes with the ß-lactamase BEL-1 from P. aeruginosa.

Pseudomonas aeruginosa is a gram-negative opportunistic bacterium that is a concern worldwide due to its innate antibiotic resistance properties. This warrants the development of non-antibiotic compounds that can potentially address the growing concern. Therefore, it is of interest to document the molecular docking analysis data of three long-chain alkanes, namely, eicosane, triacontane, and nonadecane with ß-lactamase BEL-1. Data shows that nonadecane have good binding features and drug-likeness when compared to triacontane and nonadecane. It is well known that nonadecane is a compound that is abundantly available from natural resources for further consideration.

Oily sludge treatment in subcritical and supercritical water: A review.

Oily sludge, an inherent byproduct of the petroleum industry, presents dual characteristics of petroleum resources and hazardous waste. Owing to the unique physicochemical properties of sub-/supercritical water, hydrothermal technologies have been increasingly used for oily sludge treatment. This review is the first to focus on oily sludge treatment using sub-/supercritical water. Eight hydrothermal technologies used for different purposes are summarized herein: pressurized hot water extraction (PHWE) for hydrocarbon separation, thermal hydrolysis (TH) for dewaterability improvement, hydrothermal carbonization (HTC) for hydrochar production, wet air oxidation (WAO) for biodegradability improvement, hydrothermal liquefaction (HTL) for bio-oil production, supercritical water upgrading (SCWU) for light oil production, supercritical water oxidation (SCWO) for complete degradation, and supercritical water gasification (SCWG) for H2-rich syngas production. Moreover, a general reaction pathway for sub-/supercritical water treatment of oily sludge is presented, with a particular focus on the chemical mechanism at temperatures above 350 °C. Lastly, two reaction maps are included to illustrate the reaction pathways of two groups of identifiable model compounds in oily sludge: aliphatic and aromatic hydrocarbons. This review provides detailed information that can promote a better understanding of various hydrothermal technologies, a guideline for selecting the suitable hydrothermal process for a particular oily sludge, and recommendations for further researches.

Efficient cyclic oxidation of macro long-chain alkanes in soil using Fenton oxidation with recyclable Fe.

Recyclable Fe in soil was prepared by using fermented food waste supernatant. The efficient cyclic oxidation of long-chain alkanes in oil-contaminated soils could be achieved by Fenton oxidation with the recyclable Fe. The oxidation efficiency of macro long-chain alkanes (C27-C30) from the first cycle (63.4%) to the last cycle (60.1%) showed no significant decrease during three-cycle Fenton oxidation with the recyclable Fe. However, for the oil-absorbing Fe prepared by HA and Fe-SOM prepared by Cs, the oxidation efficiency of C27-C30 could not be efficiently cyclic oxidized during three-cycle Fenton oxidation. Further analysis showed that the proportion of Fe(III) in the recyclable Fe was higher than that in the oil-absorbing Fe or the Fe-SOM, where the iron content was similar. Moreover, more fulvic-like acid and humic-like acid were found in the recyclable Fe, and thus many Fe(III) ions simultaneously combined with the fulvic-like acid and humic-like acid through -C-O-C and CË­O bonds in the recyclable Fe. It was the recyclable Fe with such a stable structure that could still maintain high catalytic activity and efficiently cyclic oxidize macro long-chain alkanes during three-cycle Fenton oxidation, which is valuable for its repeated use.

Comprehensive genomics and expression analysis of eceriferum (CER) genes in sunflower (Helianthus annuus).

Sunflower occupies the fourth position among oilseed crops the around the world. Eceriferum (CER) is an important gene family that plays critical role in very-long-chain fatty acids elongation and biosynthesis of epicuticular waxes under both biotic and abiotic stress conditions. The aim of present study was to investigate the effect of sunflower CER genes during drought stress condition. Thus, comparative analysis was undertaken for sunflower CER genes with Arabidopsis genome to determine phylogenetic relationship, chromosomal mapping, gene structures, gene ontology and conserved motifs. Furthermore, we subjected the sunflower cultivars under drought stress and used qRT-PCR analysis to explore the expression pattern of CER genes during drought conditions. We identified thirty-seven unevenly distributed CER genes in the sunflower genome. The phylogenetic analysis revealed that CER genes were grouped into seven clades in Arabidopsis, Helianthus annuus, and Gossypium hirsutum. Expression analysis showed that genes CER10 and CER60 were upregulated in sunflower during drought conditions, indicating that these genes are activated during drought stress. The results obtained will serve to characterize the CER gene family in sunflower and exploit the role of these genes in wax biosynthesis under limited water conditions. KEY MESSAGE: Cuticular waxes protect the plants from drought stress, so we observed the expression of wax bio synthesis genes in recently sequences genome of Helianthus annuus. We observed that expression of wax biosynthesis genes CER10 and CER60 was upregulated when the plants were subjected to drought stress.

Rapid degradation of long-chain crude oil in soil by indigenous bacteria using fermented food waste supernatant.

The objective of this study is to explore how to stimulate soil indigenous bacteria for the degradation of long-chain crude oil by adding fermented food waste supernatant (FS). Four concentrations of FS (0 mL, 0.1 mL, 1 mL, and 3 mL) were added to two oil-contaminated soils S1 and S2 for 30 days of bioremediation experiments. The results showed that the biodegradation of long-chain alkanes (C29 - C24) could reach up to 1756 mg/kg (49.3%, S1) and 3937 mg/kg (43.9%, S2), which were 3.1 and 3.2 times that of the non-nutrient system. In addition, the logarithmic growth rate of the indigenous hydrocarbon degraders (IHD) reached 41.5%. The long-chain crude oil can be rapidly degraded by indigenous bacteria with FS added in a short time. The glucose and acetic acid accelerated the consumption of ammonia nitrogen (NH4+-N) in the prophase of bioremediation and the molar ratio of consumed carbon (contained in glucose and acetic acid) to consumed NH4+-N (C/N) was high by adding FS. Thus, the IHD can multiply rapidly. The analysis of microbial diversity revealed that the IHD (genera Acinetobacter and Aquabacterium) became the dominant bacteria. Long-chain alkanes became the main carbon sources for IHD after 14 days in soil S1 and 16 days in soil S2. Thus, the rapid biodegradation of long-chain crude oil was achieved. The genus Aquabacterium which was uncultivable on crude oil medium became the dominant bacteria. This study provides an environment-friendly and sustainable remediation technology for bioremediation of oil-contaminated soils.

Differential Protein Expression During Growth on Medium Versus Long-Chain Alkanes in the Obligate Marine Hydrocarbon-Degrading Bacterium Thalassolituus oleivorans MIL-1.

The marine obligate hydrocarbonoclastic bacterium Thalassolituus oleivorans MIL-1 metabolizes a broad range of aliphatic hydrocarbons almost exclusively as carbon and energy sources. We used LC-MS/MS shotgun proteomics to identify proteins involved in aerobic alkane degradation during growth on medium- (n-C14) or long-chain (n-C28) alkanes. During growth on n-C14, T. oleivorans expresses an alkane monooxygenase system involved in terminal oxidation including two alkane 1-monooxygenases, a ferredoxin, a ferredoxin reductase and an aldehyde dehydrogenase. In contrast, during growth on long-chain alkanes (n-C28), T. oleivorans may switch to a subterminal alkane oxidation pathway evidenced by significant upregulation of Baeyer-Villiger monooxygenase and an esterase, proteins catalyzing ketone and ester metabolism, respectively. The metabolite (primary alcohol) generated from terminal oxidation of an alkane was detected during growth on n-C14 but not on n-C28 also suggesting alternative metabolic pathways. Expression of both active and passive transport systems involved in uptake of long-chain alkanes was higher when compared to the non-hydrocarbon control, including a TonB-dependent receptor, a FadL homolog and a specialized porin. Also, an inner membrane transport protein involved in the export of an outer membrane protein was expressed. This study has demonstrated the substrate range of T. oleivorans is larger than previously reported with growth from n-C10 up to n-C32. It has also greatly enhanced our understanding of the fundamental physiology of T. oleivorans, a key bacterium that plays a significant role in natural attenuation of marine oil pollution, by identifying key enzymes expressed during the catabolism of n-alkanes.

High Frequency of Thermodesulfovibrio spp. and Anaerolineaceae in Association with Methanoculleus spp. in a Long-Term Incubation of n-Alkanes-Degrading Methanogenic Enrichment Culture.

In the present study, the microbial community and functional gene composition of a long-term active alkane-degrading methanogenic culture was established after two successive enrichment culture transfers and incubated for a total period of 1750 days. Molecular analysis was conducted after the second transfer (incubated for 750 days) for both the active alkanes-degrading methanogenic enrichment cultures (T2-AE) and the background control (T2-BC). A net increase of methane as the end product was detected in the headspace of the enrichment cultures amended with long-chain n-alkanes and intermediate metabolites, including octadecanoate, hexadecanoate, isocaprylate, butyrate, isobutyrate, propionate, acetate, and formate were measured in the liquid cultures. The composition of microbial community shifted through the successive transfers over time of incubation. Sequences of bacterial and archaeal 16S rRNA gene (16S rDNA) and mcrA functional gene indicated that bacterial sequences affiliated to Thermodesulfovibrio spp. and Anaerolineaceae and archaeal sequences falling within the genus Methanoculleus were the most frequently encountered and thus represented the dominant members performing the anaerobic degradation of long-chain n-alkanes and methanogenesis. In addition, the presence of assA functional genes encoding the alkylsuccinate synthase α subunit indicated that fumarate addition mechanism could be considered as a possible initial activation step of n-alkanes in the present study. The succession pattern of microbial communities indicates that Thermodesulfovibrio spp. could be a generalist participating in the metabolism of intermediates, while Anaerolineaceae plays a key role in the initial activation of long-chain n-alkane biodegradation.