Enzymic activities and gene expression of enzymes of the acyl-CoA elongase during rapeseed development

Enzymic activities and gene expression of oleoyl-CoA elongase were studied during seed development using two different rapeseed cultivars, high-erucic-acid rapeseed (HEAR) and low-erucic-acid rapeseed (LEAR). The overall elongase activities were maximal in HEAR between the fourth and eighth weeks after pollination (WAP) and absent in LEAR. The 3-ketoacyl-CoA synthase (condensing enzyme, CE) mRNA levels and the developmental profiles in the two cultivars were different since maximal expression levels were detected in HEAR and LEAR at WAP 4 and WAP 6, respectively. Anti-CE antibodies revealed two proteins of 60 and 67 kDa in both cultivars and an additional reacting protein of 57 kDa in HEAR.     

Acyl-CoA elongase expression during seed development in Brassica napus

The Bn-FAE1.1 and Bn-FAE1.2 genes encode the 3-ketoacyl-CoA synthase, a component of the elongation complex responsible for the synthesis of very long chain monounsaturated fatty acids (VLCMFA) in the seeds of Brassica napus. Bn-FAE1 gene expression was studied during seed development using two different cultivars: Gaspard, a high erucic acid rapeseed (HEAR), and ISLR4, a low erucic acid rapeseed (LEAR). The mRNA developmental profiles were similar for the two cultivars, the maximal expression levels being measured at 8 weeks after pollination (WAP) in HEAR and at 9 WAP in LEAR. Differential expression of Bn-FAE1.1 and Bn-FAE1.2 genes was also studied. In each cultivar the same expression profile was observed for both genes, but Bn-FAE1.2 was expressed at a lower level than Bn-FAE1.1. Secondly, VLCMFA synthesis was measured using particulate fractions prepared from maturating seeds harvested weekly after pollination. The oleoyl-CoA and ATP-dependent elongase activities increased from the 4th WAP in HEAR and reached the maximal level at 8 WAP, whereas both activities were absent in LEAR. In contrast, the 3-hydroxy dehydratase, a subunit of the elongase complex, had a similar activity in both cultivars and reached a maximum from 7 to 9 WAP. Finally, antibodies against the 3-ketoacyl-CoA synthase revealed a protein of 57 kDa present only in HEAR. Our results show: (i) that both genes are transcribed in HEAR and LEAR cultivars; (ii) that they are coordinately regulated; (iii) that Bn-FAE1.1 is quantitatively the major isoform expressed in seeds; (iv) that the Bn-FAE1 gene encodes a protein of 57 kDa responsible for the 3-ketoacyl-CoA synthase activity.     

Acyl-CoA elongase expression during seed development in Brassica napus

The Bn-FAE1.1 and Bn-FAE1.2 genes encode the 3-ketoacyl-CoA synthase, a component of the elongation complex responsible for the synthesis of very long chain monounsaturated fatty acids (VLCMFA) in the seeds of Brassica napus. Bn-FAE1 gene expression was studied during seed development using two different cultivars: Gaspard, a high erucic acid rapeseed (HEAR), and ISLR4, a low erucic acid rapeseed (LEAR). The mRNA developmental profiles were similar for the two cultivars, the maximal expression levels being measured at 8 weeks after pollination (WAP) in HEAR and at 9 WAP in LEAR. Differential expression of Bn-FAE1.1 and Bn-FAE1.2 genes was also studied. In each cultivar the same expression profile was observed for both genes, but Bn-FAE1.2 was expressed at a lower level than Bn-FAE1.1. Secondly, VLCMFA synthesis was measured using particulate fractions prepared from maturating seeds harvested weekly after pollination. The oleoyl-CoA and ATP-dependent elongase activities increased from the 4th WAP in HEAR and reached the maximal level at 8 WAP, whereas both activities were absent in LEAR. In contrast, the 3-hydroxy dehydratase, a subunit of the elongase complex, had a similar activity in both cultivars and reached a maximum from 7 to 9 WAP. Finally, antibodies against the 3-ketoacyl-CoA synthase revealed a protein of 57 kDa present only in HEAR. Our results show: (i) that both genes are transcribed in HEAR and LEAR cultivars; (ii) that they are coordinately regulated; (iii) that Bn-FAE1.1 is quantitatively the major isoform expressed in seeds; (iv) that the Bn-FAE1 gene encodes a protein of 57 kDa responsible for the 3-ketoacyl-CoA synthase activity.     

Breeding high-stearic oilseed rape (Brassica napus) with high- and low-erucic background using optimised promoter-gene constructs

Seed lipids of oilseed rape (Brassica napus) usually contain small proportions (<3%) of stearic acid. The objective of this study was to increase the content of stearic fatty␣acid in rapeseed oil. An antisense down-regulation of the endogenous stearoyl-ACP desaturase (SAD) catalysing the reaction step from stearic to oleic acid in two different genetic backgrounds was studied. The result of down-regulation of the SAD yielded an about 10-fold increase of stearic acid from 3.7% up to 32% in single seeds of transgenic low-erucic acid rapeseed (LEAR), while high-erucic acid rapeseed (HEAR) showed a 4-fold increase of C18:0 from 1% up to 4%. It could be shown in pooled T2 seed material of LEAR rapeseed, that the stearic acid content is highly correlated with the down-regulation of SAD as indicated by the␣stearate desaturation proportion (SDP). The importance of the promoter strength for the alteration of a trait was confirmed in this study as no change in the fatty acid composition of transgenic plants was achieved with gene constructs controlled by the weak FatB4 seed-specific promoter from Cuphea lanceolata.Karim Zarhloul and Christof Stoll have contributed in equal parts to the present work     

Increasing erucic acid content through combination of endogenous low polyunsaturated fatty acids alleles with Ld-LPAAT + Bn-fae1 transgenes in rapeseed (Brassica napus L.)

High erucic acid rapeseed (HEAR) oil is of interest for industrial purposes because erucic acid (22:1) and its derivatives are important renewable raw materials for the oleochemical industry. Currently available cultivars contain only about 50% erucic acid in the seed oil. A substantial increase in erucic acid content would significantly reduce processing costs and could increase market prospects of HEAR oil. It has been proposed that erucic acid content in rapeseed is limited because of insufficient fatty acid elongation, lack of insertion of erucic acid into the central sn-2 position of the triaclyglycerol backbone and due to competitive desaturation of the precursor oleic acid (18:1) to linoleic acid (18:2). The objective of the present study was to increase erucic content of HEAR winter rapeseed through over expression of the rapeseed fatty acid elongase gene (fae1) in combination with expression of the lysophosphatidic acid acyltransferase gene from Limnanthes douglasii (Ld-LPAAT), which enables insertion of erucic acid into the sn-2 glycerol position. Furthermore, mutant alleles for low contents of polyunsaturated fatty acids (18:2 + 18:3) were combined with the transgenic material. Selected transgenic lines showed up to 63% erucic acid in the seed oil in comparison to a mean of 54% erucic acid of segregating non-transgenic HEAR plants. Amongst 220 F₂ plants derived from the cross between a transgenic HEAR line and a non-transgenic HEAR line with a low content of polyunsaturated fatty acids, recombinant F₂ plants were identified with an erucic acid content of up to 72% and a polyunsaturated fatty acid content as low as 6%. Regression analysis revealed that a reduction of 10% in polyunsaturated fatty acids content led to a 6.5% increase in erucic acid content. Results from selected F₂ plants were confirmed in the next generation by analysing F₄ seeds harvested from five F₃ plants per selected F₂ plant. F₃ lines contained up to 72% erucic acid and as little as 4% polyunsaturated fatty acids content in the seed oil. The 72% erucic acid content of rapeseed oil achieved in the present study represents a major breakthrough in breeding high erucic acid rapeseed.     

Influence of soil pollution on the composition of a microbial community

The abundance dynamics and composition of indigenous soil microbial communities were studied in soils polluted with naphthalene, dioctyl phthalate, diesel fuel, and crude oil. DGGE analysis of the 16S rRNA genes amplified from the total soil DNA revealed that the bacterial community of uncontaminated soil was more diverse and included no dominant species. In the soil samples polluted with the crude oil, diesel fuel, or dioctyl phthalate, Pseudomonas became the dominant bacteria since the third day of the experiment. In the soil polluted with naphthalene, two genera of bacteria (Pseudomonas and Paenibacillus) were dominant in population on the third day of the experiment, while on the 21th day of the experiment Arthrobacter became dominant. During the experiment, the average number of indigenous bacterial degraders increased approximately by two orders of magnitude. While the key genes of naphthalene catabolism, nahAc and nahH, were not detected in the pristine soil, they were found in a significant amount on the third day after naphthalene addition. Three degrader strains harboring the plasmids of naphthalene biodegradation (IncP-9 group) were isolated on the third day from the soil polluted with naphthalene. Two of these plasmids, although isolated from various degraders, were shown to be identical.     

Polychlorinated biphenyl (PCB)-degrading bacteria associated with trees in a PCB-contaminated site

The abundance, identities, and degradation abilities of indigenous polychlorinated biphenyl (PCB)-degrading bacteria associated with five species of mature trees growing naturally in a contaminated site were investigated to identify plants that enhance the microbial PCB degradation potential in soil. Culturable PCB degraders were associated with every plant species examined in both the rhizosphere and root zone, which was defined as the bulk soil in which the plant was rooted. Significantly higher numbers of PCB degraders (2.7- to 56.7-fold-higher means) were detected in the root zones of Austrian pine (Pinus nigra) and goat willow (Salix caprea) than in the root zones of other plants or non-root-containing soil in certain seasons and at certain soil depths. The majority of culturable PCB degraders throughout the site and the majority of culturable PCB degraders associated with plants were identified as members of the genus Rhodococcus by 16S rRNA gene sequence analysis. Other taxa of PCB-degrading bacteria included members of the genera Luteibacter and Williamsia, which have not previously been shown to include PCB degraders. PCB degradation assays revealed that some isolates from the site have broad congener specificities; these isolates included one Rhodococcus strain that exhibited degradation abilities similar to those of Burkholderia xenovorans LB400. Isolates with broad congener specificity were widespread at the site, including in the biostimulated root zone of willow. The apparent association of certain plant species with increased abundance of indigenous PCB degraders, including organisms with outstanding degradation abilities, throughout the root zone supports the notion that biostimulation through rhizoremediation is a promising strategy for enhancing PCB degradation in situ.     

The catabolic pathways of in situ rhizosphere PAH degraders and the main factors driving PAH rhizoremediation in oil-contaminated soil

Rhizoremediation is a potential technique for polycyclic aromatic hydrocarbon (PAH) remediation; however, the catabolic pathways of in situ rhizosphere PAH degraders and the main factors driving PAH rhizoremediation remain unclear. To address these issues, stable-isotope-probing coupled with metagenomics and molecular ecological network analyses were first used to investigate the phenanthrene rhizoremediation by three different prairie grasses in this study. All rhizospheres exhibited a significant increase in phenanthrene removal and markedly modified the diversity of phenanthrene degraders by increasing their populations and interactions with other microbes. Of all the active phenanthrene degraders, Marinobacter and Enterobacteriaceae dominated in the bare and switchgrass rhizosphere respectively; Achromobacter was markedly enriched in ryegrass and tall fescue rhizospheres. Metagenomes of ¹³C-DNA illustrated several complete pathways of phenanthrene degradation for each rhizosphere, which clearly explained their unique rhizoremediation mechanisms. Additionally, propanoate and inositol phosphate of carbohydrates were identified as the dominant factors that drove PAH rhizoremediation by strengthening the ecological networks of soil microbial communities. This was verified by the results of rhizospheric and non-rhizospheric treatments supplemented with these two substances, further confirming their key roles in PAH removal and in situ PAH rhizoremediation. Our study offers novel insights into the mechanisms of in situ rhizoremediation at PAH-contaminated sites.     

Polychlorinated Biphenyl (PCB)-Degrading Bacteria Associated with Trees in a PCB-Contaminated Site

The abundance, identities, and degradation abilities of indigenous polychlorinated biphenyl (PCB)-degrading bacteria associated with five species of mature trees growing naturally in a contaminated site were investigated to identify plants that enhance the microbial PCB degradation potential in soil. Culturable PCB degraders were associated with every plant species examined in both the rhizosphere and root zone, which was defined as the bulk soil in which the plant was rooted. Significantly higher numbers of PCB degraders (2.7- to 56.7-fold-higher means) were detected in the root zones of Austrian pine (Pinus nigra) and goat willow (Salix caprea) than in the root zones of other plants or non-root-containing soil in certain seasons and at certain soil depths. The majority of culturable PCB degraders throughout the site and the majority of culturable PCB degraders associated with plants were identified as members of the genus Rhodococcus by 16S rRNA gene sequence analysis. Other taxa of PCB-degrading bacteria included members of the genera Luteibacter and Williamsia, which have not previously been shown to include PCB degraders. PCB degradation assays revealed that some isolates from the site have broad congener specificities; these isolates included one Rhodococcus strain that exhibited degradation abilities similar to those of Burkholderia xenovorans LB400. Isolates with broad congener specificity were widespread at the site, including in the biostimulated root zone of willow. The apparent association of certain plant species with increased abundance of indigenous PCB degraders, including organisms with outstanding degradation abilities, throughout the root zone supports the notion that biostimulation through rhizoremediation is a promising strategy for enhancing PCB degradation in situ.     

Toxicity of diesel fuel to germination,growth and colonization of <Emphasis Type="Italic">Glomus intraradices</Emphasis> in soil and <Emphasis Type="Italic">in vitro</Emphasis> transformed carrot root cultures

Phytoremediation is the use of selected plants to decontaminate polluted environments. Arbuscular mycorrhizal fungi (AMF) may potentially be useful for phytoremediation, but it is not known how petroleum hydrocarbons influence AMF spore germination and hyphal growth. To address this question, germination of spores and germ tube growth of Glomus intraradices Schenck and Smith and Glomus aggregatum Schenck and Smith were assessed in soil contaminated with up to 3% (w/v) of F2 diesel oil or HAGO reference oil. Hyphal growth, colonization and progeny spore production were assessed in vitro using transformed root cultures of Daucus carota and G. intraradices spores in a F2 diesel contaminated medium. In addition, extraradical hyphal growth of G. intraradices colonizing Daucus carota in the presence of F2 diesel was studied. Neither F2 diesel nor HAGO reference oil affected spore germination or germ tube growth in soil. However, in the presence of plant roots, germ tube growth of G. intraradices was reduced and delayed in the presence of F2 diesel and root colonization was not detected. Hyphal growth of pre-colonized carrot roots by G. intraradices was reduced and delayed in F2 contaminated medium compared to controls. F2 diesel did not inhibit spore germination of these AMF species but did reduce colonization, germ tube and hyphal growth. These results suggest that AMF inoculum can be established in petroleum-contaminated sites. However, it may prove beneficial to plant pre-colonized plants to increase the probability of sufficient AMF colonization and growth. The likely mechanism(s) of petroleum toxicity in this plant-microbe system was discussed.     

Effect of Photosynthetic Bacteria and Compost on Degradation of Petroleum Products in Soil

Addition of diesel fuel and waste engine oil to soil was found to stimulate hydrocarbon-oxidizing microorganisms. Corynebacteria constitute a large group of hydrocarbon-oxidizing microorganisms. Addition of a liquid culture of photosynthetic bacteria to soil facilitates degradation of petroleum products and also stimulates growth of hydrocarbon-oxidizing microorganisms. Combined addition of photosynthetic bacteria and compost to soil polluted with petroleum products produces a greater increase in the number of hydrocarbon-oxidizing bacteria and substantially augments the rate of pollutant degradation.     

Effect of photosynthetic bacteria and compost on degradation of petroleum products in soil

Addition of diesel fuel and waste engine oil to soil was found to cause biostimulation of hydrocarbon-oxidizing microorganisms. Corynebacteria constitute a large group of hydrocarbon-oxidizing microorganisms. Addition of a liquid culture of photosynthetic bacteria to soil not only facilitates degradation of petroleum products, but also stimulates growth of hydrocarbon-oxidizing microorganisms. Combined addition of photosynthetic bacteria and compost to soil polluted with petroleum products causes even a more significant increase in the count of hydrocarbon-oxidizing bacteria and substantially increases the rate of pollutant degradation.     

Rhizosphere Microbial Characterization in Petroleum-Contaminated Soil

Contamination of soil with petroleum compounds is of concern worldwide. Although there are a variety of physical and chemical technologies available to remediate petroleum waste sites, biological methods are often used due to lower cost and public acceptance. Growth and enhanced activity of microbial communities in contaminated soil is a key factor for the success of bioremediation. Establishing vegetation in petroleum-contaminated soil may enhance microbial activity and remediation success even further by providing root exudates to the rhizosphere microorganisms. In this study, microorganisms were characterized in petroleum-contaminated soils and sediments quantitatively and qualitatively based on enumeration and metabolic diversity assessments. Contaminated soils and sediments were obtained from a phytoremediation field demonstration project in California. Microbial numbers in the unvegetated soil, based on plate counts and most probable number of hydrocarbon degraders, were significantly lower than the vegetated soils. Metabolic microbial characterization using BIOLOG was also conducted and based on principle component analysis (PCA), there was a distinct difference between the metabolic diversity of microbial communities in vegetated and unvegetated soils. Results from this research indicate that the presence and type of plants, and level of contamination may greatly influence microbial communities in polluted soils.     

Bioremediation of soil polluted with fuels by sequential multiple injection of native microorganisms: Field-scale processes in Poland

Research was conducted to estimate impact of the multiple bioaugmentation on the treatment of soil contaminated by fuels - diesel oil and aircraft fuel. The bacteria used to inoculate the remediation plots were isolated from the polluted soil and proliferated in field conditions. The amount of biomass applied to the polluted soil was set to ensure the total number of bacteria in soil 10⁷-10⁸ cfu/g d.w. The multiple inoculation of soil with indigenous bacteria active in diesel oil and engine oil (plot A) degradation increased bioremediation effectiveness by 50% in comparison to the non-inoculated control soil and by 30% in comparison to the soil that was inoculated only once. The multiple inoculation of soil with indigenous microorganisms was then applied in bioremediation of the soil polluted with double high concentration of diesel oil (soil B) and in bioremediation of the soil polluted with aircraft fuel (soil C). The process efficiency was 80% and 98% removal of TPH for soil B and C, respectively.     

Screening of Australian Native Grasses for Rhizoremediation of Aliphatic Hydrocarbon-Contaminated Soil

Rhizoremediation involves the breakdown of contaminants in soil resulting from microbial activity that is enhanced in the plant root zone. The objective of this study was to identify Australian native grass species as suitable candidates for rhizoremediation application. Seeds of nine perennial Australian native grasses were sown in soil from a mine site and artificially contaminated with a 60:40 diesel/oil mixture at concentrations of 1% (w/w), 0.5% (w/w), and 0% (control). Seedling emergence was not adversely affected by the presence of hydrocarbon contamination for all but one grass species. Three promising species (Brachiaria decumbens, Cymbopogon ambiguus, and Microlaena stipoides var. Griffin) were assessed for growth characterization in contaminated and uncontaminated soils. The evaluated species survived for 120 days in the contaminated soil and, in some instances, produced considerably more root biomass in the presence of contamination. C. ambiguus showed growth stimulation in the presence of contamination (1% and 0.5% w/w) with significantly increased root biomass production compared with the control (p = 0.0001). B. decumbens and M. stipoides showed tolerance, without adverse growth effects in the presence of diesel/oil at the exposed concentrations. Stimulation of the rhizosphere microbial population that is capable of degrading diesel/oil was found for all of the species tested, using a most probable number method for enumeration. This investigation has identified suitable candidates for further investigation of their rhizoremediation potential.     

Bioremediation of Soils Contaminated with Nigerian Petroleum Products Using Composted Municipal Wastes

The efficiency of ready-to-use, source-separated, composted municipal organic wastes of Nigerian origin on degradation of soil total petroleum hydrocarbons (TPHs) in soils polluted with petroleum products (crude oil, diesel, and spent engine oil) was assessed in screen house experiments. The effect of compost:soil ratios and combined effect of compost-phytoremediation technique were also studied. TPH was determined spectrophotometrically, after extraction with 1:1 acetone-dichloromethane mixture at 425 nm. Soil pH, electrical conductivity, and phytotoxicity to seed germination and growth of maize (Zea mays L.) served as risk assessments on soil quality and evidence of recovery for the oil-impacted soil. Results showed that the treatments increased soil pH and electrical conductivity but reduced TPH. Reductions in TPH by compost technology ranged from 40% to 75.87%. Toxicity to seed germination reduced from 100% to 16.12%. Positive correlations were obtained for plant agronomical parameters and growth period, for all treatments, with coefficients in the range of .905 to .996, p < .05. This study revealed that ready-to-use composted waste has the potential for bioremediation of soils polluted with petroleum and petroleum products. This study is a contribution to the data bank of relatively simple bioremediation methods, suitable for workers in the developing countries, where there is no easy access to high-technology facilities. However, further development of this technique to achieve zero residual TPH is recommended.     

Effect of the tropical grass Brachiaria brizantha (Hochst. ex A. Rich.) Stapf on microbial population and activity in petroleum-contaminated soil

The effect of the tropical pasture grass Brachiaria brizantha on numbers of bacteria, fungi and degraders of alkanes, aromatics, cycloalkanes and crude oil in petroleum hydrocarbon contaminated and uncontaminated savannah soil was evaluated. Substrate induced soil respiration and soil pH were compared between planted and unplanted soil. B. brizantha had a mostly increasing effect on microbial numbers. As an exception, growth of bacteria was not or negatively affected. Microbial respiration and pH were always lower in planted than in unplanted soil. Low pH may result from enhanced oil degradation in planted soil leading to an accumulation of organic acids. A comparable stimulation of crude oil degraders and fungi in planted soil points to the importance of fungi. Since they tolerate lower pH values than bacteria, they are considered to play a central role in oil degradation. Given that the enhancement of crude oil degradation under the influence of B. brizantha could not clearly be correlated to microbial numbers and activity, other factors like oxygen availability, plant enzymes and synergistic degradation by microbial consortia have to be considered.     

Strong impact on the polycyclic aromatic hydrocarbon (PAH)-degrading community of a PAH-polluted soil but marginal effect on PAH degradation when priming with bioremediated soil dominated by mycobacteria

Bioaugmentation of soil polluted with polycyclic aromatic hydrocarbons (PAHs) is often disappointing because of the low survival rate and low activity of the introduced degrader bacteria. We therefore investigated the possibility of priming PAH degradation in soil by adding 2% of bioremediated soil with a high capacity for PAH degradation. The culturable PAH-degrading community of the bioremediated primer soil was dominated by Mycobacterium spp. A microcosm containing pristine soil artificially polluted with PAHs and primed with bioremediated soil showed a fast, 100- to 1,000-fold increase in numbers of culturable phenanthrene-, pyrene-, and fluoranthene degraders and a 160-fold increase in copy numbers of the mycobacterial PAH dioxygenase gene pdo1. A nonpolluted microcosm primed with bioremediated soil showed a high rate of survival of the introduced degrader community during the 112 days of incubation. A nonprimed control microcosm containing pristine soil artificially polluted with PAHs showed only small increases in the numbers of culturable PAH degraders and no pdo1 genes. Initial PAH degradation rates were highest in the primed microcosm, but later, the degradation rates were comparable in primed and nonprimed soil. Thus, the proliferation and persistence of the introduced, soil-adapted degraders had only a marginal effect on PAH degradation. Given the small effect of priming with bioremediated soil and the likely presence of PAH degraders in almost all PAH-contaminated soils, it seems questionable to prime PAH-contaminated soil with bioremediated soil as a means of large-scale soil bioremediation.