Alkane Hydroxylase Research Articles

N-Alkane Assimilation by Pseudomonas aeruginosa and Its Interactions with Virulence and Antibiotic Resistance

Pseudomonas aeruginosa strains with potential for degrading n-alkanes are frequently cultured from hydrocarbon-contaminated sites. The initial hydroxylation step of long-chain n-alkanes is mediated by the chromosomally encoded AlkB1 and AlkB2 alkane hydroxylases. The acquisition of an additional P. putida GPo1-like alkane hydroxylase gene cluster can extend the substrate range assimilated by P. aeruginosa to <C12 n-alkanes. Efficient niche colonization of hydrocarbon-contaminated sites is facilitated by avid iron-uptake systems, such as pyoverdine, and the production of several compounds with antimicrobial activities. A GPo1-like gene cluster can facilitate detoxification and solvent tolerance in P. aeruginosa. The overproduction of various multidrug efflux pumps, in particular, the MexAB-OprM system, can also contribute to solvent tolerance, which is often associated with reduced susceptibility or full resistance to certain clinically relevant antibiotics. These characteristics, together with the remarkable conservation of P. aeruginosa virulence determinants among human, animal, and environmental isolates, necessitate further studies from a One Health perspective into the acquired antibiotic resistance mechanisms of environmental P. aeruginosa strains and possible ways for their dissemination into the human population.

SlMYB72 interacts with SlTAGL1 to regulate the cuticle formation in tomato fruit.

The cuticle is the first physical barrier covering the surface of tomatoes and plays an important role in multiple stress responses. But the molecular regulatory networks of cuticle formation are not fully understood. In this study, we found that SlMYB72 can interact with SlTAGL1 to regulate the formation of fruit cuticle in tomato. Downregulating the expression of SlMYB72 inhibits the formation of fruit cuticle, resulting in a reduced fruit cuticle thickness, accelerated postharvest water loss, and increased susceptibility to Botrytis cinerea. RNA sequencing analysis showed that downregulation of the SlMYB72 gene decreased the expression levels of genes related to fatty acid and cuticle metabolism. SlMYB72 regulates the cuticle formation by directly binding to the promoter of long-chain acyl-coA synthetases (SlLACS1) and medium-chain alkane hydroxylase (SlMAH1). Moreover, SlMYB72 interacts with SlTAGL1, which can enhance the transcriptional activation of SlMYB72 on the SlMAH1 promoter. Overall, our study expands our understanding of the regulation of cuticle formation by SlMYB72 and provides new insights into fruit shelf life extension via manipulation of cuticle content.

Degradation of diesel fuel by Pseudomonas aeruginosa B031 with expression of the alkB gene in a column bioreactor

Diesel fuel pollutants contain toxic hydrocarbons dominated by aliphatic and aromatic hydrocarbons. Hydrocarbon pollutants can be degraded in an environmentally friendly manner through bioremediation using hydrocarbonoclastic bacteria, i.e., Pseudomonas aeruginosa B031, which harbors the alkB gene that encodes an alkane hydroxylase that degrades alkane chains in hydrocarbons. This study compared the ability and efficiency of P. aeruginosa B031 to degrade diesel pollutants in a batch system and a continuous system using a column bioreactor, as well as the expression of alkB. P. aeruginosa B031 could more efficiently degrade diesel fuel in a continuous system in a column bioreactor than in the batch system. The concentrations of phenol, total organic carbon, chemical oxygen demand, and biological oxygen demand in the column bioreactor underwent a greater decrease than those in the batch system, namely 1.5-fold, 1.7-fold, 1.4-fold, and 1.3-fold, respectively. The decrease in these concentrations was followed by changes in functional groups, as shown via Fourier transform infrared spectroscopy. The number of bacteria and the concentration of exopolysaccharide increased in the column bioreactor by 4-fold and 2.3-fold more than the increase in the batch system. The ability of P. aeruginosa B031 to degrade diesel fuel in the column bioreactor was also demonstrated by the higher expression of alkB than that in the control.

Novel Approach of Tackling Wax Deposition Problems in Pipeline Using Enzymatic Degradation Process: Challenges and Potential Solutions

Anthropogenic activities have led to hydrocarbon spills, and while traditional bioremediation methods are costly and time-consuming, recent research has focused on engineered enzymes for managing pollutant. The potential of enzymes for resolving wax flow problems in the petroleum industry remains unexplored. This paper offers a comprehensive review of the current state of research activities related to the bioremediation of petroleum-polluted sites and the biodegradation of specific petroleum hydrocarbons. The assayed enzymes that took part in the degradation were discussed in detail. Lipase, laccase, alkane hydroxylase, alcohol dehydrogenase, esterase, AlkB homologs and cytochrome P450 monooxygenase are among the enzymes responsible for the degradation of more than 50% of the hydrocarbons in contaminated soil and wastewater and found to be active on carbon C8 to C40. The possible biodegradation mechanism of petroleum hydrocarbons was also elucidated. The enzymes’ primary metabolic pathways include terminal, subterminal, and ω-oxidation. Next, given the successful evidence of the hydrocarbon treatment efficiency, the authors analyzed the opportunity for the enzymatic degradation approach if it were to be applied to a different scenario: managing wax deposition in petroleum-production lines. With properties such as high transformation efficiency and high specificity, enzymes can be utilized for the treatment of viscous heavy oil for transportability, evidenced by the 20 to 99% removal of hydrocarbons. The challenges associated with the new approach are also discussed. The production cost of enzymes, the characteristics of hydrocarbons and the operating conditions of the production line may affect the biocatalysis reaction to some extent. However, the challenges can be overcome by the usage of extremophilic enzymes. The combination of technological advancement and deployment strategies such as the immobilization of a consortium of highly thermophilic and halotolerant enzymes is suggested. Recovering and reusing enzymes offers an excellent strategy to improve the economics of the technology. This paper provides insights into the opportunity for the enzymatic degradation approach to be expanded for wax deposition problems in pipelines.

Novel, active, and uncultured hydrocarbon-degrading microbes in the ocean.

Given the vast quantity of oil and gas input to the marine environment annually, hydrocarbon degradation by marine microorganisms is an essential ecosystem service. Linkages between taxonomy and hydrocarbon degradation capabilities are largely based on cultivation studies, leaving a knowledge gap regarding the intrinsic ability of uncultured marine microbes to degrade hydrocarbons. To address this knowledge gap, metagenomic sequence data from the Deepwater Horizon (DWH) oil spill deep-sea plume was assembled to which metagenomic and metatranscriptomic reads were mapped. Assembly and binning produced new DWH metagenome-assembled genomes that were evaluated along with their close relatives, all of which are from the marine environment (38 total). These analyses revealed globally distributed hydrocarbon-degrading microbes with clade-specific substrate degradation potentials that have not been reported previously. For example, methane oxidation capabilities were identified in all Cycloclasticus. Furthermore, all Bermanella encoded and expressed genes for non-gaseous n-alkane degradation; however, DWH Bermanella encoded alkane hydroxylase, not alkane 1-monooxygenase. All but one previously unrecognized DWH plume member in the SAR324 and UBA11654 have the capacity for aromatic hydrocarbon degradation. In contrast, Colwellia were diverse in the hydrocarbon substrates they could degrade. All clades encoded nutrient acquisition strategies and response to cold temperatures, while sensory and acquisition capabilities were clade specific. These novel insights regarding hydrocarbon degradation by uncultured planktonic microbes provides missing data, allowing for better prediction of the fate of oil and gas when hydrocarbons are input to the ocean, leading to a greater understanding of the ecological consequences to the marine environment.IMPORTANCEMicrobial degradation of hydrocarbons is a critically important process promoting ecosystem health, yet much of what is known about this process is based on physiological experiments with a few hydrocarbon substrates and cultured microbes. Thus, the ability to degrade the diversity of hydrocarbons that comprise oil and gas by microbes in the environment, particularly in the ocean, is not well characterized. Therefore, this study aimed to utilize non-cultivation-based 'omics data to explore novel genomes of uncultured marine microbes involved in degradation of oil and gas. Analyses of newly assembled metagenomic data and previously existing genomes from other marine data sets, with metagenomic and metatranscriptomic read recruitment, revealed globally distributed hydrocarbon-degrading marine microbes with clade-specific substrate degradation potentials that have not been previously reported. This new understanding of oil and gas degradation by uncultured marine microbes suggested that the global ocean harbors a diversity of hydrocarbon-degrading bacteria, which can act as primary agents regulating ecosystem health.

Mechanism of Crude Oil Biodegradation in Bioreactors: A Model Approach

Oil-degrading bacteria have the ability to degrade alkanes present in crude oil because of a special enzymatic system, the alkane hydroxylase complex (AlkH). The mechanism for the transport and degradation of alkanes present in crude oil remains unclear, especially related to the first step in hydrocarbons oxidation. In this work, we present a novel model of the crude oil biodegradation mechanism by considering the contact between the oil drop and the cell and calculating the mass transfer coefficients in three oleophilic bacteria (B. licheniformis, P. putida and P. glucanolyticus). The mass transfer coefficients are evaluated under critical time conditions, when the kinetics and mass transport are in balance, and the difference in the values obtained (kL α = 1.60 × 10−3, 5.25 × 10−4 and 6.19 × 10−4 m/d, respectively) shows the higher value of the mass transfer coefficient and higher biodegradation potential for B. licheniformis. Because the morphology of the cells has been analyzed by optical and electron microscopy, in the proposed model, the increase in the size of the cells in P. glucanolyticus compared to P. putida exhibits higher values of the mass transfer coefficients and this is attributed, as a novel statement, to a bigger window for alkanes transport (contact area) when the external area of the cell is bigger.

Metagenomic analysis of a thermophilic bacterial consortium and its use in the bioremediation of a petroleum-contaminated soil

Biodegradation is difficult at high temperatures due to the limited capacity of microorganisms to survive and function outside their optimum temperature range. Here, a thermophilic petroleum-degrading consortium was enriched from compost at a temperature of 55 °C. 16S rDNA and metagenomic techniques were used to analyze the composition of the consortium and the mechanisms of degradation. The consortium degraded 17000 mg total petroleum hydrocarbons (TPHs) L−1 with a degradation efficiency of 81.5% in 14 days. The consortium utilized a range of substrates such as n-hexadecane, n-docosane, naphthalene and pyrene and grew well over a wide range of pH (4–10) and salinity (0–90 g L−1). The hydrocarbon-degrading extremophilic consortium contained, inter alia, (relative abundance >1%) Caldibacillus, Geobacillus, Mycolicibacterium, Bacillus, Chelatococcus, and Aeribacillus spp. Metagenomic analysis was conducted to discover the degradation and environmental tolerance functional genes of the consortium. Two alkane hydroxylase genes, alkB and ladA, were found. A microcosm study shows that the consortium promoted the bioremediation of soil TPHs. The results indicate that the consortium may be a good candidate for the high-temperature bioremediation of petroleum-contaminated soils.

The potential of enhanced phytoremediation to clean up multi-contaminated soil – insights from metatranscriptomics

This study aimed to (i) investigate the potential for enhanced phytoremediation to remove contaminants from soil historically co-contaminated with petroleum hydrocarbons (PHs) and heavy metals (HMs) and (ii) analyze the expression of crucial bacterial genes and whole metatranscriptomics profiles for better understanding of soil processes during applied treatment. Phytoremediation was performed using Zea mays and supported by the Pseudomonas qingdaonensis ZCR6 strain and a natural biofertilizer: meat and bone meal (MBM). In previous investigations, mechanisms supporting plant growth and PH degradation were described in the ZCR6 strain. Here, ZCR6 survived in the soil throughout the experiment, but the efficacy of PH removal from all soils fertilized with MBM reached 32 % regardless of the bacterial inoculation. All experimental groups contained 2 % (w/w) MBM. The toxic effect of this amendment on plants was detected 30 days after germination, irrespective of ZCR6 inoculation. Among the 17 genes tested using the qPCR method, only expression of the acdS gene, encoding 1-aminocyclopropane-1-carboxylic acid deaminase, and the CYP153 gene, encoding cytochrome P450-type alkane hydroxylase, was detected in soils. Metatranscriptomic analysis of soils indicated increased expression of methane particulated ammonia monooxygenase subunit A (pmoA-amoA) by Nitrosomonadales bacteria in all soils enriched with MBM compared to the non-fertilized control. We suggest that the addition of 2 % (w/w) MBM caused the toxic effect on plants via the rapid release of ammonia, and this led to high pmoA-amoA expression. In parallel, due to its wide substrate specificity, enhanced bacterial hydrocarbon removal in MBM-treated soils was observed. The metatranscriptomic results indicate that MBM application should be considered to improve bioremediation of soils polluted with PHs rather than phytoremediation. However, lower concentrations of MBM could be considered for phytoremediation enhancement. From a broader perspective, these results indicated the superior capability of metatranscriptomics to investigate the microbial mechanisms driving various bioremediation techniques.

Nutrients, surfactants, and aeration in constructed wetlands affect bacterial persistence and metabolic activity during the remediation of crude oil-contaminated water

The use of constructed wetlands (CWs) is one of the best options to treat wastewater. In CWs, microorganisms play a major role in the degradation of organic pollutants but the concentration of nutrients, surfactant, and aeration (NSA) in oil-contaminated water is one of the factors that affect the persistence and metabolic functioning of hydrocarbon-degrading microorganisms. In the present investigation, the influence of the addition of NSA on the persistence of the augmented bacteria, copy of (alkane hydroxylase gene) alkB gene, and its expression level in the water, soil, and plants of CWs were evaluated. The CWs mesocosms were developed by the vegetation of Typha latifolia and Cyperus laevigatus and inoculated with the bacterial consortium (Pseudomonas putida TYRI39, Acinetobacter junii TYRH47, Acinetobacter sp. CYRH17, Pseudomonas sp. CYSI27, and Pseudomonas sp. TYRH42). The mesocosms were provided with nutrients (20 mg l− 1 N, 2.6 mg l− 1 P, and 16.4 mg l− 1 K) in liquid form, surfactant Tween-20 (0.2%, v/v) in liquid form, and aeration (≥ 7.0 ± 1 mg l− 1 DO) using aeration pump. The addition of NSA in CWs enhanced the persistence and metabolic functioning of the inoculated bacteria in the water, rhizospheric soil, and plants. The maximum hydrocarbon removal (97%) was observed in the water treated by CWs having C. laevigatus, bacteria, and NSA, and it is correlated with the copy numbers of alkB and its expression level. The application of NSA in CWs not only improved bacterial persistence and catabolic gene expression but also increased plant development and hydrocarbon removal.

Autochthonous bioaugmentation strategies for the successful bioremediation of high-salt petrochemical wastewater using a biosurfactant-producing Enterobacter cloacae Z11

Petrochemical wastewater contains a high concentration of refractory aliphatic and aromatic hydrocarbons. Bioremediation of these hazardous materials using autochthonous bioaugmentation (ABA) strategy is attracting increasing interest. Herein, an indigenous biosurfactant-producing bacteria Z11 was isolated and identified as Enterobacter sp. The reintroduction of Z11 into petrochemical wastewater led to a substantial reduction (P < 0.01) in TPHs, decreasing from 8099 to 1723 mg/L, as well as a significant decrease in COD, decreasing from 8064.2 to 1689.2 mg/L. At a critical micelle concentration of 200 mg/L, the biosurfactant synthesized by Z11 exhibited an extraordinary decrease in surface tension from 67.5 to 29 mN/m. The biosurfactant demonstrated favorable surface activity over a wide pH, temperature, and salinity range. Based on a series of chemical analyses, the biosurfactant was classified as phospholipid. The autochthonous bioaugmentation technique developed by Z11 indicated a significant increase (P < 0.05) in the degradation of n-alkanes of varying chain lengths and polycyclic aromatic hydrocarbons. The analysis of the microbial composition demonstrated that the addition of Z11 caused a rise in bacterial populations capable of hydrocarbon decomposition, such as Pseudomonas sp. and Stenotrophomonas sp. The activities of two enzymes involved in degradation, alkane hydroxylase and alcohol dehydrogenase, increased dramatically and showed a significant (P < 0.05) correlation with Enterobacter sp. Z11. The proposed mechanism could be elucidated that functional genes regulate the secretion of biodegradation enzymes and biosurfactants to promote the deep degradation of petroleum hydrocarbons. This study provides technical support for the reintroduction of autochthonous microorganisms capable of producing biosurfactants in implementing in situ ABA strategies.

Ecological indicators and biological resources for hydrocarbon rhizoremediation in a protected area.

Spillage from oil refineries, pipelines, and service stations consistently leads to soil, food and groundwater contamination. Bacterial-assisted phytoremediation is a non-invasive and sustainable solution to eliminate or decrease the concentration of xenobiotic contaminants in the environment. In the present study, a protected area interested by a fuel discharge was considered to assess a bioremediation intervention. From the spill point, a plume of contamination flowed South-West into the aquifer, eventually reaching a wetland area. Soils, groundwaters and plants belonging to the species Scirpus sylvaticus (L.) were sampled. In the majority of the soil samples, concentrations of total petroleum hydrocarbons, both C ≤ 12 and C > 12, exceeded legal limits set forth in Directive 2000/60/EC. The analysis of diatom populations, used as ecological indicators, evidenced morphology alterations and the presence of Ulnaria ulna and Ulnaria biceps species, previously detected in hydrocarbon-polluted waters. Tests for phytotoxicity and phytodegradation, carried out in soil mesocosms, planted with Zea mays and Helianthus annuus, demonstrated that both species significantly contributed to the removal of total petroleum hydrocarbons. Removal of C ≤ 12 and C > 12 petroleum hydrocarbons was in the range of 80%-82% for Z. mays and 71%-72% for H. annuus. Microbial communities inhabiting high organic carbon and vegetated soils were more active in hydrocarbon degradation than those inhabiting subsoils, as evidenced by soil slurry experiments. The abundance of functional genes encoding toluene-benzene monooxygenase (tbmD) and alkane hydroxylase (alkB), quantified in environmental samples, confirmed that the plant rhizosphere recruited a microbial community with higher biodegradation capacity. Bacterial strains isolated from the sampling site were able to grow on model hydrocarbons (hexane, hexadecane and o-, m-, p-xylene) as sole carbon and energy sources, indicating that a natural bio-attenuation process was on-going at the site. The bacterial strains isolated from rhizosphere soil, rhizoplane and endosphere showed plant growth promoting traits according to in vitro and in vivo tests on Z. mays and Oryza sativa, allowing to forecast a possible application of bacterial assisted rhizoremediation to recover the protected area.

Degradation and enhanced oil recovery potential of Alcanivorax borkumensis through production of bio-enzyme and bio-surfactant

Microbial enhanced oil recovery (EOR) has become the focus of oilfield research due to its low cost, environmental friendliness and sustainability. The degradation and EOR capacity of A. borkumensis through the production of bio-enzyme and bio-surfactant were first investigated in this study. The total protein concentration, acetylcholinesterase, esterase, lipase, alkane hydroxylase activity, surface tension, and emulsification index (EI) were determined at different culture times. The bio-surfactant was identified as glycolipid compound, and the yield was 2.6 ± 0.2 g/L. The nC12 and nC13 of crude oil were completely degraded, and more than 40.0 % of nC14–nC24 was degraded by by A. borkumensis. The results of the microscopic etching model displacement and core flooding experiments showed that emulsification was the main mechanism of EOR. A. borkumensis enhanced the recovery rate by 20.2 %. This study offers novel insights for the development of environmentally friendly and efficient oil fields.

Exploring untapped bacterial communities and potential polypropylene-degrading enzymes from mangrove sediment through metagenomics analysis.

The versatility of plastic has resulted in huge amounts being consumed annually. Mismanagement of post-consumption plastic material has led to plastic waste pollution. Biodegradation of plastic by microorganisms has emerged as a potential solution to this problem. Therefore, this study aimed to investigate the microbial communities involved in the biodegradation of polypropylene (PP). Mangrove soil was enriched with virgin PP sheets or chemically pretreated PP comparing between 2 and 4 months enrichment to promote the growth of bacteria involved in PP biodegradation. The diversity of the resulting microbial communities was accessed through 16S metagenomic sequencing. The results indicated that Xanthomonadaceae, unclassified Gaiellales, and Nocardioidaceae were promoted during the enrichment. Additionally, shotgun metagenomics was used to investigate enzymes involved in plastic biodegradation. The results revealed the presence of various putative plastic-degrading enzymes in the mangrove soil, including alcohol dehydrogenase, aldehyde dehydrogenase, and alkane hydroxylase. The degradation of PP plastic was determined using Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), Scanning Electron Microscopy (SEM), and Water Contact Angle measurements. The FTIR spectra showed a reduced peak intensity of enriched and pretreated PP compared to the control. SEM images revealed the presence of bacterial biofilms as well as cracks on the PP surface. Corresponding to the FTIR and SEM analysis, the water contact angle measurement indicated a decrease in the hydrophobicity of PP and pretreated PP surface during the enrichment.

Insights into the degradation of diesel-contaminated seawater through natural microbiota and culturable microorganisms: The overlook role of extracellular polymeric substances (EPS)

The role of EPS produced during biodegradation diesel-contaminated seawater, as well as the effect of EPS on natural microbiota and culturable microorganisms, remains unknown. Here, natural microbiota (pre and post enrichment with diesel) from 5 islands’ seawater in Jiaozhou Bay were explored through metagenomic and culture-dependent approaches. EPS response degradation was mainly characterized by the following aspects: 1) The post-enrichment samples contained significantly fewer dominant bacterial genera than the pre-enrichment samples, and EPS or PS (polysaccharides) promoted degradation of C16-C24 hydrocarbon fractions; 2) EPS caused higher increases in aromatic compound degradation functional genes copies; 3) EPS enhanced the enzymatic activity of alkane hydroxylase and ethanol dehydrogenase; 4) Diesel degradation by mono and mixed cultures of culturable bacteria (B1 and B2) with extracted EPS showed a higher biomass, biosurfactant production, expression of PAH-RHDα GN and cytochrome P450 genes, degradability and emulsification capacity. The above innovations provided new perspectives for oil-spill bioremediation.

Valorisation of low-density polythene (LDPE) waste into fuels and chemicals through alkane hydroxylase and microbial treatment

Polythene exhibits low reactivity and slow biodegradability due to its highly reduced hydrocarbon structure. In this study, a process has been developed for the transformation of polythene waste into fuel and its precursor molecule by microbial and enzymatic treatments. In first step polythene was pretreated with Paenibacillus alvei. Through fermentation process for 15 days and products released after fermentation were subsequently bio-transformed into alcohols and acids corresponding to its hydrocarbons, employing Alkane hydroxylase (AlkB). On fermentation using Paenibacillus alvei, 16.8% weight loss was observed after 15 days and surface modification was analyzed through AFM and SEM. The major products released during microbial pretreatment analyzed through Chromatography–High-Resolution Mass Spectrometry (GC-HRMS) were pentane, hexane, heptadecane, dodecane, tetracontane, bromotetradecane, methyl esters, hexadecane, etc. which were converted in second step into hexadecanol, octadecanol, octadecanoic acid, and heptadecanoic acid, etc. with the help of Alkane hydroxylase on enzymatic transformation. These findings suggested that the microbial pretreatment results in a release of alkanes, some fatty acids, and esters using Paenibacillus alvei Among these, AlkB facilitated the transformation of C16 and C15 hydrocarbons into hexadecanol, and pentadecanol respectively. This process holds the potential as an alternative to traditional fuels, and an optimization process can be pursued to enhance the overall yield.

Multi-omics association study of hexadecane degradation in haloarchaeal strain Halogranum rubrum RO2-11

Haloarchaea with the capacity to degrade alkanes is promising to deal with petroleum pollution in hypersaline environments. However, only a limited number of haloarchaeal species are investigated, and their pathway and mechanism for alkane degradation remain unclear. In this study, Halogranum rubrum RO2-11, a haloarchaeal strain, verified the ability to degrade kerosene and hexadecane in 184 g/L NaCl, with 53% and 52% degradation rates after 9 and 4 days, respectively. Genome sequencing and gene annotation indicated that strain RO2-11 possesses a complete potential alkane-degrading pathway, of which alkane hydroxylases may include CYP450, AlmA, and LadA. Transcriptome and metabolome analyses revealed that the upregulation of related genes in TCA cycle, lysine biosynthesis, and acetylation may help improve hexadecane degradation. Additionally, an alternative degrading pathway of hexadecane based on dual-terminal β-oxidation may occur in strain RO2-11. It is likely to be the first report of alkane degradation by the genus Halogranum, which may be helpful for applications of oil-pollution bioremediation under high-salt conditions.

Bioremedial potential of bacteria isolated from soil in automobile garages and fuel stations in Mbarara City, Southwestern Uganda

The use of bacteria to clean up oil-contaminated soils is widely considered the most desirable bioremediation strategy. The majority of research has focused on contaminated soils in large oil-producing countries. However, accidental spills or poor handling and disposal of petroleum hydrocarbons (PHs) products, which happen at automobile maintenance garages and leakages at fuel stations contaminate soil. There is limited information on PH-degrading bacteria in soil patches around automobile garages and fuel stations for their possible bioremediation potential. We characterized bacteria with the alkane hydroxylase (alkB) gene isolated from soil around automobile repair garages in Mbarara City, Uganda, and assessed their efficiency and effectiveness to degrade PHs. The isolation and identification of cultured bacteria were performed using standard microbiology morphological and biochemical tests. Bacterial efficiency was determined by measuring their growth on used engine oil after 14 days using a turbidometer. The effectiveness of the bacteria was measured after 14 days by dichlorophenol-indophenol redox indicator method. Twelve different genera were identified in 45 bacterial isolates. Enterobacter spp. and Aeromonas spp. were the most effective and efficient bacteria in degrading the used diesel oil, respectively. The alkB-3 gene was identified in 15 bacteria isolates. Two isolates of Yersinia spp and Aeromonas spp showed two genes of 157 bp and 250 bp. These findings are suggestive of the catabolic potential of the isolates to degrade alkanes and require further studies to establish the physicochemical requirements and conditions necessary to ensure the maintenance of an even microbial community composition and diversity needed for successful bioremediation approaches.

Enhancing systematic tolerance in Bermuda grass (Cynodon dactylon L.) through amplified alkB gene expression and bacterial-driven hydrocarbon degradation.

This study aimed to access the impact of soil polluted with petroleum (5, 10g petroleum kg-1 soil) on Bermuda grass (Cynodon dactylon L.) with and without applied bacterial inoculants (Arthrobacter oxydans ITRH49 and Pseudomonas sp. MixRI75). Both soil and seed were given bacterial inoculation. The evaluated morphological parameters of Bermuda grass were fresh and dry weight. The results demonstrated that applied bacterial inoculants enhanced 5.4%, 20%, 28% and 6.4%, 21%, and 29% shoot and root fresh/dry weights in Bermuda grass under controlled environment. The biochemical analysis of shoot and root was affected deleteriously by the 10g petroleum kg-1 soil pollution. Microbial inoculants enhanced the activities of enzymatic (catalase, peroxidase, glutathione reductase, ascorbate peroxidase, superoxide dismutase) and non-enzymatic (ɑ-tocopherols, proline, reduced glutathione, ascorbic acid) antioxidant to mitigate the toxic effects of ROS (H2O2) under hydrocarbon stressed condition. The maximum hydrocarbon degradation (75%) was recorded by Bermuda grass at 5g petroleum kg-1 soil contamination. Moreover, bacterial persistence and alkane hydroxylase gene (alkB) abundance and expression were observed more in the root interior than in the rhizosphere and shoot interior of Bermuda grass. Subsequently, the microbe used a biological tool to propose that the application of plant growth-promoting bacteria would be the most favorable choice in petroleum hydrocarbon polluted soil to conquer the abiotic stress in plants and the effective removal of polyaromatic hydrocarbons in polluted soil.

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 Penicilliumchrysogenum 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.

Polyethylene Biodegradation by an Artificial Bacterial Consortium: Rhodococcus as a Competitive Plastisphere Species.

Polyethylene (PE), a widely used recalcitrant synthetic polymer, is a major global pollutant. PE has very low biodegradability due to its rigid C-C backbone and high hydrophobicity. Although microorganisms have been suggested to possess PE-degrading enzymes, our understanding of the PE biodegradation process and its overall applicability is still lacking. In the present study, we used an artificial bacterial consortium for PE biodegradation to compensate for the enzyme availability and metabolic capabilities of individual bacterial strains. Consortium members were selected based on available literature and preliminary screening for PE-degrading enzymes, including laccases, lipases, esterases, and alkane hydroxylases. PE pellets were incubated with the consortium for 200 days. A next-generation sequencing ana-lysis of the consortium community of the culture broth and on the PE pellet identified Rhodococcus as the dominant bacteria. Among the Rhodococcus strains in the consortium, Rhodococcus erythropolis was predominant. Scanning electron microscopy (SEM) revealed multilayered biofilms with bacteria embedded on the PE surface. SEM micrographs of PE pellets after biofilm removal showed bacterial pitting and surface deterioration. Multicellular biofilm structures and surface biodeterioration were observed in an incubation of PE pellets with R. erythropolis alone. The present study demonstrated that PE may be biodegraded by an artificially constructed bacterial consortium, in which R. erythropolis has emerged as an important player. The results showing the robust colonization of hydrophobic PE by R. erythropolis and that it naturally possesses and extracellularly expresses several target enzymes suggest its potential as a host for further improved PE biodeterioration by genetic engineering technology using a well-studied host-vector system.