Molecular Biomonitoring during Rhizoremediation of Oil-Contaminated Soil

Rhizoremediation is the use of microbial populations present in the rhizosphere of plants for environmental cleanup. The idea of this work was that bacteria living in the rhizosphere of a nitrogen-fixing leguminous plant, goat's rue (Galega orientalis), could take part in the degradation of harmful monoaromatic hydrocarbons, such as benzene, toluene and xylene (BTEX), from oil-contaminated soils. In addition to chemical (e.g. pollutant concentration) and physical (e.g. soil structure) information, the knowledge of biological aspects (e.g. bacteria and their catabolic genes) is essential when developing the rhizoremediation into controlled and effective bioremediation practice. Therefore, the need for reliable biomonitoring methods is obvious. The main aims of this thesis were to evaluate the symbiotic G. orientalis - Rhizobium galegae system for rhizoremediation of oil-contaminated soils, to develop molecular methods for biomonitoring, and to apply these methods for studying the microbiology of rhizoremediation. In vitro, Galega plants and rhizobia remained viable in m-toluate concentrations up to 3000 mg/l. Plant growth and nodulation were inhibited in 500 mg/l m-toluate, but were restored when plants were transferred to clean medium. In the greenhouse, Galega showed good growth, nodulation and nitrogen fixation, and developed a strong rhizosphere in soils contaminated with oil or spiked with 2000 mg/l m-toluate. The high aromatic tolerance of R. galegae and the viability of Galega plants in oil-polluted soils proved this legume system to be a promising method for the rhizoremediation of oil-contaminated soils. Molecular biomonitoring methods were designed and/or developed further for bacteria and their degradation genes. A combination of genomic fingerprinting ((GTG)5-PCR), taxonomic ribotyping of 16S rRNA genes and partial 16S rRNA gene sequencing were chosen for molecular grouping of culturable, heterogeneous rhizosphere bacteria. PCR primers specific for the xylE gene were designed for TOL plasmid detection. Amplified enzyme-coding DNA restriction analysis (AEDRA) with AluI was used to profile both TOL plasmids (xylE primers) and, in general, aromatics-degrading plasmids (C230 primers). The sensitivity of the direct monitoring of TOL plasmids in soil was enhanced by nested C23O-xylE-PCR. Rhizosphere bacteria were isolated from the greenhouse and field lysimeter experiments. High genetic diversity was observed among the 50 isolated, m-toluate tolerating rhizosphere bacteria in the form of five major lineages of the Bacteria domain. Gram-positive Rhodococcus, Bacillus and Arthrobacter and gram-negative Pseudomonas were the most abundant genera. The inoculum Pseudomonas putida PaW85/pWW0 was not found in the rhizosphere samples. Even if there were no ecological niches available for the bioaugmentation bacterium itself, its conjugative catabolic plasmid might have had some additional value for other bacterial species and thus, for rhizoremediation. Only 10 to 20% of the isolated, m-toluate tolerating bacterial strains were also able to degrade m-toluate. TOL plasmids were a major group of catabolic plasmids among these bacteria. The ability to degrade m-toluate by using enzymes encoded by a TOL plasmid was detected only in species of the genus Pseudomonas, and the best m-toluate degraders were these Pseudomonas species. Strain-specific differences in degradation abilities were found for P.oryzihabitans and P. migulae: some of these strains harbored a TOL plasmid - a new finding observed in this work, indicating putative horizontal plasmid transfer in the rhizosphere. One P. oryzihabitans strain harbored the pWW0 plasmid that had probably conjugated from the bioaugmentation Pseudomonas. Some P. migulae and P. oryzihabitans strains seemed to harbor both the pWW0- and the pDK1-type TOL plasmid. Alternatively, they might have harbored a TOL plasmid with both the pWW0- and the pDK1-type xylE gene. The breakdown of m-toluate by gram-negative bacteria was not restricted to the TOL pathway. Also some gram-positive Rhodococcus erythropolis and Arthrobacter aurescens strains were able to degrade m-toluate in the absence of a TOL plasmid. Three aspects of the rhizosphere effect of G. orientalis were manifested in oil-contaminated soil in the field: 1) G. orientalis and Pseudomonas bioaugmentation increased the amount of rhizosphere bacteria. G. orientalis especially together with Pseudomonas bioaugmentation increased the numbers of m-toluate utilizing and catechol positive bacteria indicating an increase in degradation potential. 2) Also the bacterial diversity, when measured as the amount of ribotypes, was increased in the Galega rhizosphere with or without Pseudomonas bioaugmentation. However, the diversity of m-toluate utilizing bacteria did not significantly increase. At the community level, by using the 16S rRNA gene PCR-DGGE method, the highest diversity of species was also observed in vegetated soils compared with non-vegetated soils. Diversified communities may best guarantee the overall success in rhizoremediation by offering various genetic machineries for catabolic processes. 3) At the end of the experiment, no TOL plasmid could be detected by direct DNA analysis in soil treated with both G. orientalis and Pseudomonas. The detection limit for TOL plasmids was encountered indicating decreased amount of degradation plasmids and thus, the success of rhizoremediation. The use of G. orientalis for rhizoremediation is unique. In this thesis new information was obtained about the rhizosphere effect of Galega orientalis in BTEX contaminated soils. The molecular biomonitoring methods can be applied for several purposes within environmental biotechnology, such as for evaluating the intrinsic biodegradation potential, monitoring the enhanced bioremediation, and estimating the success of bioremediation. Environmental protection by using nature's own resources and thus, acting according to the principle of sustainable development, would be both economically and environmentally beneficial for society. Keywords: molecular biomonitoring, genetic fingerprinting, soil bacteria, bacterial diversity, TOL plasmid, catabolic genes, horizontal gene transfer, rhizoremediation, rhizosphere effect, Galega orientalis, aerobic biodegradation, petroleum hydrocarbons, BTEX

Photograph of oil painting portrait of Samuel Loewen; Berlin Portraits Men

circa 1747 in Potsdam - 30 November 1827 in Berlin; father of Rosa Valentin

Shemen Oil Factory

Digital image

Shemen Oil Factory

Digital image

Analysis and computation of the oil film thickness between the piston ring and cylinder liner of an internal combustion engine

A study of the essential features of piston rings in the cylinder liner of an internal combustion engine reveals that the lubrication problem posed by it is basically that of a slider bearing. According to steady-flow-hydrodynamics, viz. Image the oil film thickness becomes zero at the dead centre positions as the velocity, U = 0. In practice, however, such a phenomenon cannot be supported by consideration of the wear rates of pistion rings and cylinder liners. This can be explained by including the “squeeze” action term in the

Crude oil and agricultural futures: An analysis of correlation dynamics

Correlations between oil and agricultural commodities have varied over previous decades, impacted by renewable fuels policy and turbulent economic conditions. We estimate smooth transition conditional correlation models for 12 agricultural commodities and WTI crude oil. While a structural change in correlations occurred concurrently with the introduction of biofuel policy, oil and food price levels are also key influences. High correlation between biofuel feedstocks and oil is more likely to occur when food and oil price levels are high. Correlation with oil returns is strong for biofuel feedstocks, unlike with other agricultural futures, suggesting limited contagion from energy to food markets.

Engine wear and used oil analysis - comparative study in high speed diesel engines

The operational life and reliability of I.C. engines are limited to a certain extent by the break down of the engine components due to wear. It is advantageous to know the condition of an engine and its components without disassembling for detailed measurements. This paper describes the possibility of employing chemical analysis of the used crank case oil to predict the wear of engine components. It is concluded that the acidity and carbon contents of the crank case oil play a significant role in assessing the wear of copper-lead bearings used for the big end of the connecting rod.

Using Big Data to manage safety-related risk in the upstream oil and gas industry: A research agenda

Despite considerable effort and a broad range of new approaches to safety management over the years, the upstream oil & gas industry has been frustrated by the sector’s stubbornly high rate of injuries and fatalities. This short communication points out, however, that the industry may be in a position to make considerable progress by applying “Big Data” analytical tools to the large volumes of safety-related data that have been collected by these organizations. Toward making this case, we examine existing safety-related information management practices in the upstream oil & gas industry, and specifically note that data in this sector often tends to be highly customized, difficult to analyze using conventional quantitative tools, and frequently ignored. We then contend that the application of new Big Data kinds of analytical techniques could potentially reveal patterns and trends that have been hidden or unknown thus far, and argue that these tools could help the upstream oil & gas sector to improve its injury and fatality statistics. Finally, we offer a research agenda toward accelerating the rate at which Big Data and new analytical capabilities could play a material role in helping the industry to improve its health and safety performance.

Evaluation of chlorothalonil and paraffinic oil alone and in combinations with registered fungicides for the control of yellow Sigatoka (Mycosphaerella musicola) of bananas in Northern Queensland, Australia

The efficacy of chlorothalonil and paraffinic oil alone and in combinations with the registered fungicides propiconazole, tebuconazole, difenoconazole, epoxiconazole and pyrimethanil was evaluated in a field experiment over two cropping cycles in 2013 and 2014 in Northern Queensland, Australia, for control of yellow Sigatoka (caused by Mycosphaerella musicola) of banana. The predominantly applied by the banana industry treatment mancozeb with paraffinic oil was included for comparison. The results from the two cropping cycles suggested that all chemicals used with paraffinic oil were as effective or more effective than when applied with chlorothalonil, and chlorothalonil alone. Difenoconazole and epoxiconazole with paraffinic oil followed by propiconazole with paraffinic oil were the most effective treatments. Pyrimethanil and tebuconazole plus chlorothalonil were the least effective treatments. None of the chemical treatments was phytotoxic or reduced yield.

Engine wear and used oil analysis-A comparative study in high speed diesel engines

The operational life and reliability of I.C. engines are limited to a certain extent by the break down of the engine components due to wear. It is advantageous to know the condition of an engine and its components without disassembling for detailed measurements. This paper describes the possibility of employing chemical analysis of the used crank case oil to predict the wear of engine components. It is concluded that the acidity and carbon contents of the crank case oil play a significant role in assessing the wear of copper-lead bearings used for the big end of the connecting rod.

Superhydrophobic fabrics for oil-water separation based on the metal organic charge transfer complex CuTCNAQ

This work reports on the fabrication of a superhydrophobic nylon textile based on the organic charge transfer complex CuTCNAQ (TCNAQ = 11,11,12,12-tetracyanoanthraquinodimethane). The nylon fabric that is metallized with copper undergoes a spontaneous chemical reaction with TCNAQ dissolved in acetonitrile to form nanorods of CuTCNAQ that are intertwined over the entire surface of the fabric. This creates the necessary micro and nanoscale roughness that is required for the Cassie-Baxter state thereby achieving a superhydrophobic/superoleophilic surface without the need for a fluorinated surface. The material is characterised with SEM, FT-IR and XPS spectroscopy and investigated for its ability to separate oil and water in two modes, namely under gravity and as an absorbent. It is found that the fabric can separate dichloromethane, olive oil and crude oil from water and in fact reduce the water content of the oil during the separation process. The fabric is reusable and tolerant to conditions such as seawater, hydrochloric acid and extensive time periods on the shelf. Given that CuTCNAQ is a copper based semiconductor may also open up the possibility of other applications in areas such as photocatalysis and antibacterial applications.

Effect of depletion of vitamin A, followed by supplementation with retinyl acetate or retinoic acid, on regeneration of rat liver

After partial hepatectomy the net increase in tissue weight and in RNA, DNA and proteins in the regenerating liver was markedly less in vitamin A-deleted or retinoic acid-supplemented male rats, compared with the corresponding normal control or retinyl acetate-supplemented ones.

Evaluation of oil production from oil palm empty fruit bunch by oleaginous micro-organisms

Oil palm empty fruit bunch (EFB) is a readily available, lignocellulosic biomass that has potential to be utilized as a carbon substrate for microbial oil production. In order to evaluate the production of microbial oil from EFB, a technical study was performed through the cultivation of oleaginous micro-organisms (Rhodotorula mucilaginosa, Aspergillus oryzae, and Mucor plumbeus) on EFB hydrolyzates. EFB hydrolyzates were prepared through dilute acid pre-treatment of the biomass, where the liquid fraction of pre-treatment was detoxified and used as an EFB liquid hydrolyzate (EFBLH). The solid residue was enzymatically hydrolyzed prior to be used as an EFB enzymatic hydrolyzate (EFBEH). The highest oil concentrations were obtained from M. plumbeus (1.9 g/L of oil on EFBLH and 4.7 g/L of oil on EFBEH). In order to evaluate the feasibility of large-scale microbial oil production, a techno-economic study was performed based on the oil yields of M. plumbeus per hectare of plantation, followed by the estimation of the feedstock cost for oil production. Other oil palm biomasses (frond and trunk) were also included in this study, as it could potentially improve the economics of large-scale microbial oil production. Microbial oil from oil palm biomasses was estimated to potentially increase oil production in the palm oil industry up to 25%, at a cheaper feedstock cost. The outcome of this study demonstrates the potential integration of microbial oil production from oil palm biomasses with existing palm oil industry (biodiesel, food and oleochemicals production), that could potentially enhance sustainability and profitability of microbial oil production.

Microbial oil production from sugarcane bagasse hydrolysates by oleaginous yeast and filamentous fungi

This study investigated the potential use of sugarcane bagasse as a feedstock for oil production through microbial cultivation. Bagasse was subjected to dilute acid pretreatment with 0.4 wt% H2SO4 (in liquid) at a solid/liquid ratio of 1:6 (wt/wt) at 170 °C for 15 min, followed by enzymatic hydrolysis of solid residue. The liquid fractions of the pretreatment process and the enzymatic hydrolysis process were detoxified and used as liquid hydrolysate (SCBLH) and enzymatic hydrolysate (SCBEH) for the microbial oil production by oleaginous yeast (Rhodotorula mucilaginosa) and filamentous fungi (Aspergillus oryzae and Mucor plumbeus). The results showed that all strains were able to grow and produce oil from bagasse hydrolysates. The highest oil concentrations produced from bagasse hydrolysates were by M. plumbeus at 1.59 g/L (SCBLH) and 4.74 g/L (SCBEH). The microbial oils obtained have similar fatty acid compositions to vegetable oils, indicating that the oil can be used for the production of second generation biodiesel. On the basis of oil yields obtained by M. plumbeus, from 10 million t (wet weight) of bagasse generated annually from sugar mills in Australia, it is estimated that the total biodiesel that could be produced would be equivalent to about 9% of Queensland’s diesel consumption.

Division of the Salmonella-Containing Vacuole and Depletion of Acidic Lysosomes in Salmonella-Infected Host Cells Are Novel Strategies of Salmonella enterica To Avoid Lysosomes

Salmonella has evolved several strategies to counteract intracellular microbicidal agents like reactive oxygen and nitrogen species. However, it is not yet clear how Salmonella escapes lysosomal degradation. Some studies have demonstrated that Salmonella can inhibit phagolysosomal fusion, whereas other reports have shown that the Salmonella-containing vacuole (SCV) fuses/interacts with lysosomes. Here, we have addressed this issue from a different perspective by investigating if the infected host cell has a sufficient quantity of lysosomes to target Salmonella. Our results suggest that SCVs divide along with Salmonella, resulting in a single bacterium per SCV. As a consequence, the SCV load per cell increases with the division of Salmonella inside the host cell. This demands more investment from the host cell to counteract Salmonella. Interestingly, we observed that Salmonella infection decreases the number of acidic lysosomes inside the host cell both in vitro and in vivo. These events potentially result in a condition in which an infected cell is left with insufficient acidic lysosomes to target the increasing number of SCVs, which favors the survival and proliferation of Salmonella inside the host cell.