Brassica napus hairy roots and rhizobacteria for phenolic compounds removal

Phenolic compounds are contaminants frequently found in water and soils. In the last years, some technologies such as phytoremediation have emerged to remediate contaminated sites. Plants alone are unable to completely degrade some pollutants; therefore, their association with rhizospheric bacteria has been proposed to increase phytoremediation potential, an approach called rhizoremediation. In this work, the ability of two rhizobacteria, Burkholderia kururiensis KP 23 and Agrobacterium rhizogenes LBA 9402, to tolerate and degrade phenolic compounds was evaluated. Both microorganisms were capable of tolerating high concentrations of phenol, 2,4-dichlorophenol (2,4-DCP), guaiacol, or pentachlorophenol (PCP), and degrading different concentrations of phenol and 2,4-DCP. Association of these bacterial strains with B. napus hairy roots, as model plant system, showed that the presence of both rhizospheric microorganisms, along with B. napus hairy roots, enhanced phenol degradation compared to B. napus hairy roots alone. These findings are interesting for future applications of these strains in phenol rhizoremediation processes, with whole plants, providing an efficient, economic, and sustainable remediation technology.     

Application of Brassica napus hairy root cultures for phenol removal from aqueous solutions

Phenolic compounds present in the drainage from several industries are harmful pollutants and represent a potential danger to human health. In this work we have studied the removal of phenol from water using Brassica napus hairy roots as a source of enzymes, such as peroxidases, which were able to oxidise phenol. These hairy roots were investigated for their tolerance to highly toxic concentrations of phenol and for the involvement of their peroxidase isoenzymes in the removal of phenol. Roots grew normally in medium containing phenol in concentrations not exceeding 100 mg l(-1), without the addition of H(2)O(2). However, roots were able to remove phenol concentrations up to 500 mg l(-1), in the presence of H(2)O(2), reaching high removal efficiency, within 1h of treatment and over a wide range of pH (4-9). Hairy roots could be re-used, at least, for three to four consecutive cycles. Peroxidase activity gradually decreased to approximately 20% of the control, at the fifth cycle. Basic and near neutral isoenzymes (BNP) decreased along time of recycling while acidic isoenzymes (AP) remained without changes. Although both group of isoenzymes would be involved in phenol removal, AP showed higher affinity and catalytic efficiency for phenol as substrate than BNP. In addition, AP retained more activity than BNP after phenol treatment. Thus, AP appears to be a promising isoenzyme for phenol removal and for application in continuous treatments. Furthermore, enzyme isolation might not be necessary and the entire hairy roots, might constitute less expensive enzymatic systems for decontamination processes.     

Cellular damages in the Allium cepa test system, caused by BTEX mixture prior and after biodegradation process

Petroleum and derivatives have been considered one of the main environmental contaminants. Among petroleum derivatives, the volatile organic compounds benzene, toluene, ethylbenzene and xylene (BTEX) represent a major concern due to their toxicity and easy accumulation in groundwater. Biodegradation methods seem to be suitable tools for the clean-up of BTEX contaminants from groundwater. Genotoxic and mutagenic potential of BTEX prior and after biodegradation process was evaluated through analyses of chromosomal aberrations and MN test in meristematic and F₁ root cells using the Allium cepa test system. Seeds of A. cepa were germinated into five concentrations of BTEX, non-biodegraded and biodegraded, in ultra-pure water (negative control), in MMS 4 × 10⁻⁴ M (positive control) and in culture medium used in the biodegradation (blank biodegradation control). Results showed a significant frequency of both chromosomal and nuclear aberrations. The micronucleus (MN) frequency in meristematic cells was significant for most of tested samples. However, MN was not present in significant levels in the F₁ cells, suggesting that there was no permanent damage for the meristematic cell. The BTEX effects were significantly reduced in the biodegraded samples when compared to the respective non-biodegraded concentrations. Therefore, in this study, the biodegradation process showed to be a reliable and effective alternative to treat BTEX-contaminated waters. Based on our results and available data, the BTEX toxicity could also be related to a synergistic effect of its compounds.     

Use of hairy roots extracts for 2,4-DCP removal and toxicity evaluation by Lactuca sativa test

2,4-Dichlorophenol (2,4-DCP) is widely distributed in wastewaters discharged from several industries, and it is considered as a priority pollutant due to its high toxicity. In this study, the use of different peroxidase extracts for 2,4-DCP removal from aqueous solutions was investigated. Tobacco hairy roots (HRs), wild-type (WT), and double-transgenic (DT) for tomato basic peroxidases (TPX1 and TPX2) were used to obtain different peroxidase extracts: total peroxidases (TPx), soluble peroxidases (SPx), and peroxidases ionically bound to the cell wall (IBPx). All extracts derived from DT HRs exhibited higher peroxidase activity than those obtained from WT HRs. TPx and IBPx DT extracts showed the highest catalytic efficiency values. The optimal conditions for 2,4-DCP oxidation were pH 6.5, H₂O₂ 0.5 mM, and 200 U mL^?1 of enzyme, for all extracts analyzed. Although both TPx extracts were able to oxidize different 2,4-DCP concentrations, the removal efficiency was higher for TPx DT. Polyethylene glycol addition slightly improved 2,4-DCP removal efficiency, and it showed some protective effect on TPx WT after 2,4-DCP oxidation. In addition, using Lactuca sativa test, a reduction of the toxicity of post removal solutions was observed, for both TPx extracts. The results demonstrate that TPx extracts from both tobacco HRs appear to be promising candidate for future applications in removing 2,4-DCP from wastewaters. This is particularly true considering that these peroxidase sources are associated with low costs and are readily available. However, TPx DT has increased peroxidase activity, catalytic efficiency, and higher removal efficiency than TPx WT, probably due to the expression of TPX1 and TPX2 isoenzymes.     

Comparison of the removal of 2,4-dichlorophenol and phenol from polluted water, by peroxidases from tomato hairy roots, and protective effect of polyethylene glycol

Multiple efforts have been directed towards optimized processes in which enzymes, like peroxidases, are used to remove phenolic compounds from polluted wastewater. Here we describe the use of peroxidase isoenzymes from tomato hairy roots, which were able to oxidise 2,4-dichlorophenol (2,4-DCP) and phenol from aqueous solutions. This could be an interesting alternative for the removal of these compounds from contaminated sites. We used different enzyme fractions: total peroxidases (TP), ionically bound to cell wall peroxidases (IBP), basic (BP) and acidic peroxidases (AP). We analyzed the optimum conditions of removal, the effect of Polyethyleneglycol (PEG-3350) on the process and on the enzyme activities, to obtain the maximum efficiency. The optimal H2O2 concentrations for 2,4-DCP and phenol removal were 1 and 0.1mM, respectively. TP, IBP and BP showed better removal efficiencies than AP, for both contaminants. The addition of different concentrations (10-100mg l(-1)) of PEG-3350 to solutions containing 2,4-DCP showed no effect on the removal efficiencies of the isoenzymes. However, PEG (100mg l(-1)) increased the removal efficiency of phenol by BP and IBP fractions. On the other hand, peroxidase activities from BP and IBP fractions were 3 and 13 times higher, respectively, than those detected for the same fractions in phenol treated solutions without PEG. The protective effect of PEG, which depends on the contaminant as well as of the enzyme fraction used, would be important to improve the removal efficiency of phenol by some peroxidase isoenzymes.     

Cysteine- and glutathione-mediated uptake of lead and cadmium into Zea mays and Brassica napus roots

This study examines a new mechanism for the uptake of Pb and Cd into Brassica napus and Zea mays roots. During hydroponic experiments, the uptake of Pb and Cd was enhanced in the presence of cysteine and glutathione, whereas no or very low uptake was observed in EDTA and penicillamine controls. Uptake rates were also enhanced after pre-exposure to cysteine or glutathione and inhibited in the presence of vanadate, suggesting a biological mechanism of uptake. Increasing concentrations of glutathione in solution resulted in decreasing Pb uptake rates, indicating competition for transport between free-glutathione and Pb–glutathione species. Pb uptake in the presence of increasing cysteine concentrations resulted in decreased uptake initially but linearly increasing uptake at higher concentrations. Experimentation showed concentration dependent Pb uptake rates. We speculate that there are specific transporters for these thiol ligands and describe what barriers remain for application of this novel transport mechanism in chelator-assisted phytoremediation.     

Comparative investigation of toxicity induced by UV-A and UV-C radiation using Allium test

Organisms are increasingly exposed to ultraviolet (UV) rays of sunlight, due to the thinning of the ozone layer and its widespread use in sterilization processes, especially against the SARS-CoV-2 virus. The present study was conducted with the purpose of evaluating the damages of UV-A and UV-C radiations in Allium cepa L. roots. The effects of two different types of UV on some physiological, biochemical, cytogenotoxic, and anatomical parameters were investigated in a multifaceted study. Three groups were formed from Allium bulbs, one of which was the control group. One of the other groups was exposed to 254 nm (UV-C) and the other to 365 nm (UV-A) UV. Growth retardation effect of UV was investigated with respect to germination percentage, total weight gain, and root elongation, while cytogenotoxicity arisen from UV exposure was analyzed using mitotic index (MI) and chromosomal aberration (CA) and micronucleus (MN) frequency. Oxidative stress due to UV application was investigated based on the accumulation of malondialdehyde (MDA) and the total activities of superoxide dismutase (SOD) and catalase (CAT) enzymes. Also, anatomical changes induced by UV-A and UV-C were analyzed in root meristematic cells. UV treatments caused significant reductions in growth-related parameters. Both UV treatments caused a significant increase in MDA levels and induction of SOD and CAT enzymes in root meristematic cells. A decrease in MI and an increase in the frequency of MN and CAs were observed in root tip cells, indicating the cytogenotoxic effect of UV application. Anatomical damages such as epidermis cell damage, cortex cell damage, necrotic zones, giant cell nucleus, and indistinct transmission tissue occurred in cells exposed to UV. All of the physiological, biochemical, cytogenetic, and anatomical damages observed in this study were more severe in cells treated with UV-C compared to UV-A. This study suggested that UV exposure triggered growth inhibition, cytogenotoxicity, oxidative stress, and meristematic cell damages in A. cepa roots depending on the wavelength.

     

Effects of lead on root growth, cell division, and nucleolus of Allium cepa

The effects of different concentrations of lead nitrate on root growth, cell division, chromosome morphology and nucleolus in root tip cells of Allium cepa were studied. The concentrations of lead nitrate (Pb(NO(3))(2)) were 10(-7), 10(-6), 10(-5), 10(-4), 10(-3) and 10(-2) m. It was observed that lead reduces root growth and causes mitotic irregularities, including c-mitosis, anaphase bridges, and chromosome stickiness. Also, interphase cells with micronuclei, irregularly shaped nuclei and nuclei with decomposed nuclear material were noticed. The c-mitotic effect reached its maximum in the meristem at above 10(-4) m Pb, when practically almost all the anomalous dividing cells are of this type. After treatment with Pb, there were many similar silver-stained particulate materials scattered in the nucleus in short, rounded meristem cells and in long, oblong root cap cells.     

Bioefficacy,dissipation kinetics and safety evaluation of selected insecticides in Allium cepa L

This paper reports the bioefficacy of selected insecticides against thrips and their pre-harvest intervals (PHI) in onion pertaining to their recommended application rates and maximum residue limits. Profenophos, methomyl and imidacloprid showed comparatively higher bioefficacy against thrips. GC-MS and LC-MS/MS-based residue analysis methods in onion bulbs and composite matrix of bulbs+leaves were thoroughly validated. The residue data for bulb+leaves was assessed with reference to the EU-MRLs applicable for spring onion. Dimethoate was the most stable chemical with PHI of 52.5 days, followed by monocrotophos (24 days) and carbofuran (20.5 days). The PHIs of profenophos, chlorpyrifos, methomyl and cypermethrin were similar and within the range of 10–13 days. Imidacloprid and λ-cyhalothrin had similar PHI of 4.5 days. Spinosad was the fastest-degrading chemical with PHI of 2 days. The combined bioefficacy and residue dynamics information will support label-claim of these insecticides for the management of thrips in onion, help in scheduling their applications in pest management program as per relative PHIs and minimize the residue accumulations at harvest. The dietary exposure was less than the maximum permissible intake for most of the insecticides on all sampling days except for dimethoate and monocrotophos.     

Bioefficacy, dissipation kinetics and safety evaluation of selected insecticides in Allium cepa L

This paper reports the bioefficacy of selected insecticides against thrips and their pre-harvest intervals (PHI) in onion pertaining to their recommended application rates and maximum residue limits. Profenophos, methomyl and imidacloprid showed comparatively higher bioefficacy against thrips. GC-MS and LC-MS/MS-based residue analysis methods in onion bulbs and composite matrix of bulbs+leaves were thoroughly validated. The residue data for bulb+leaves was assessed with reference to the EU-MRLs applicable for spring onion. Dimethoate was the most stable chemical with PHI of 52.5 days, followed by monocrotophos (24 days) and carbofuran (20.5 days). The PHIs of profenophos, chlorpyrifos, methomyl and cypermethrin were similar and within the range of 10-13 days. Imidacloprid and λ-cyhalothrin had similar PHI of 4.5 days. Spinosad was the fastest-degrading chemical with PHI of 2 days. The combined bioefficacy and residue dynamics information will support label-claim of these insecticides for the management of thrips in onion, help in scheduling their applications in pest management program as per relative PHIs and minimize the residue accumulations at harvest. The dietary exposure was less than the maximum permissible intake for most of the insecticides on all sampling days except for dimethoate and monocrotophos.     

Induction of chromosome aberrations in the Allium cepa test system caused by the exposure of seeds to industrial effluents contaminated with azo dyes

Numerous potentially mutagenic chemicals have been studied mainly because they can cause damaging and inheritable changes in the genetic material. Several tests are commonly used for biomonitoring pollution levels and to evaluate the effects of toxic and mutagenic agents present in the natural environment. This study aimed at assessing the potential of a textile effluent contaminated with azo dyes to induce chromosomal and nuclear aberrations in Allium cepa test systems. A continuous exposure of seeds in samples of the textile effluent in different concentrations was carried out (0.3%, 3%, 10%, and 100%). Cells in interphase and undergoing division were examined to assess the presence of chromosome aberrations, nuclear changes, and micronuclei. Our results revealed a mutagenic effect of the effluent at concentrations of 10% and 100%. At lower concentrations, the effluent (3% and 0.3%) did not induce mutagenic alterations in the test organism A. cepa. These findings are of concern, since cell damage may be transmitted to subsequent generations, possibly affecting the organism as a whole, as well as the local biota exposed to the effluent discharge. If the damage results in cell death, the development of the organism may be affected, which could also lead to its death.     

Turmeric (Curcuma longa L.) tends to reduce the toxic effects of nickel (II) chloride in Allium cepa L. roots

Environmental Science and Pollution Research - The immense protection potential of plant-derived products against heavy metal toxicity has become a considerable field of research. The goal of the...     

Cytotoxic and genotoxic evaluation of bisphenol S on onion root tips by Allium cepa and comet tests

Environmental Science and Pollution Research - Bisphenol S (BPS) is an analog of bisphenol A, which is used as substitute of BPA in many products like airport luggage tags, baby bottles, plastics,...     

Tolerance and metabolism of phenol and chloroderivatives by hairy root cultures of Daucus carota L

Hairy root cultures are shown to be suitable experimental systems to screen higher plants for tolerance to various inorganic and organic pollutants, and for determining the role of the root matrix in the uptake and further metabolism of contaminants. A number of clones were obtained by infection of carrot tissues with Agrobacterium rhizogenes and two (the fastest and the slowest growing root clones) were chosen for further experimentation. Both clones showed a similar degree of tolerance towards phenol and its chlorinated derivatives, i.e. the growth of root biomass was maintained in concentrations of phenol equivalent to 1000 micromol/l, whilst the chlorophenols were tolerated only at concentrations 20 times lower (50 micromol/l). Transformed carrot roots were able to remove more than 90% of the exogenous phenolic compounds from the culture medium within 120 h after treatment. Metabolism of these compounds occurred in the root tissue and was accompanied by an increase in peroxidase activity.     

Uptake and transformation of phenol and chlorophenols by hairy root cultures of Daucus carota, Ipomoea batatas and Solanum aviculare

Hairy root cultures of Daucus carota L., Ipomoea batatas L. and Solanum aviculare Forst were investigated for their susceptibility to the highly toxic pollutants phenol and chlorophenols and for the involvement of inherent peroxidases in the removal of phenols from liquid media. Roots of D. carota grew normally in medium containing 1000 micromol l(-1) of phenol, whilst normal growth of roots of I. batatas and S. aviculare was only possible at levels up to 500 micromol l(-1). In the presence of chlorophenols, normal root growth was possible only in concentrations not exceeding 50 micromol l(-1), except for I. batatas which was severely affected at all concentrations. Despite the reduction in biomass, the growth of S. aviculare cultures was sustained in medium containing up to 2000 micromol l(-1) of phenol or 2-chlorophenol, and up to 500 micromol l(-1) of 2,6-dichlorophenol. The amounts of phenol removed by the roots within 72 h of treatment were 72.7%, 90.7% and 98.6% of the initial concentration for D. carota, I. batatas and S. aviculare, respectively. For the removal of 2,6-dichlorophenol the values were, respectively, 83.0%, 57.7% and 73.1%. Phenols labelled with 14C were absorbed by the root tissues and condensed with highly polar cellular substances as well as being incorporated into the cell walls or membranes. The results suggest that S. aviculare, an ornamental plant, would be best suited for remediation trials under field conditions.     

Uptake and degradation of DDT by hairy root cultures of Cichorium intybus and Brassica juncea

Hairy root cultures of Cichorium intybus and Brassica juncea were used for their ability to uptake and degrade DDT (1,1,1-trichloro-2,2-bis-(4'-chlorophenyl)ethane). After 24 h of 14C DDT treatment, only 12-13% of the total applied radioactivity was detected in the culture media, indicating the efficient uptake of DDT by the hairy roots. The majority of the applied radioactivity was associated with the roots. DDT was degraded to various other products such as DDD, DDE and DDMU, along with some unknown compounds by hairy root cultures, which were detected by thin layer chromatography (TLC) and autoradiography. The rate of in situ degradation was found to be higher during the initial stages of culture and the residual 14C DDT in the roots was found to decrease from 77% to 61% over a period of 10-days. There was no spontaneous degradation of 14C DDT in media lacking hairy root cultures or in media with autoclaved hairy roots. This suggests that endogenous root enzymes play a role in the breakdown of 14C DDT. These results suggest the potential applicability and advantage of using these plant species for phytoremediation of persistent xenobiotics such as DDT in an eco-friendly and efficient manner for environmental clean up.     

Phenol tolerance, changes of antioxidative enzymes and cellular damage in transgenic tobacco hairy roots colonized by arbuscular mycorrhizal fungi

Phytoremediation has been recognized as a cheap and eco-friendly technology which could be used for the remediation of organic pollutants, such as phenolic compounds. Besides, the extent to which plants react to environmental pollution might depend on rhizosphere processes such as mycorrhizal symbiosis. In the present work, phenol tolerance of transgenic tobacco hairy roots (HR), namely TPX1, colonized with an arbuscular mycorrhizal fungus (AMF) was studied. However, the question is whether AMF symbiosis can moderate adverse effects of phenol to the plant tissues. Thus, the antioxidative response as well as parameters of oxidative damage, like malondialdehyde (MDA) content, were determined. Antioxidative enzymes such as peroxidase, superoxide dismutase, ascorbate peroxidase were higher in TPX1 HR colonized with AMF, compared to wild type HR colonized by AMF, in the presence of increasing concentrations of the pollutant. Besides, MDA levels remained unaltered in TPX1 HR associated with AMF treated with the xenobiotic. These results, suggested that this culture could tolerate phenol and moreover, it has an efficient protective mechanism against phenol-induced oxidative damage, which is of great importance in the selection of species with remediation capacities. Thus, transgenic HR colonized with AMF could be considered as an interesting model system to study different processes which play a key role in the phytoremediation of organic pollutants.     

Improvement of rape (Brassica napus) plant growth and cadmium uptake by cadmium-resistant bacteria

This study focuses on the screening of cadmium-resistance bacterial strains from heavy metal-polluted soils to examine their plant growth promotion and cadmium uptake in rape (Brassica napus). A large number of bacteria were isolated from heavy metal-polluted soil in Nanjing, China. Thirty isolates showing cadmium-resistance on Cd-amended medium were selected and evaluated for their potential to solubilize cadmium carbonate in solution culture. Atomic absorption spectrometer analysis showed variable amounts of water-soluble Cd (ranging from 24 to 117 mg l(-1)) released by the cadmium-resistant bacterial strains from cadmium carbonate. Qualitative analysis confirmed the presence of indole acetic acid as the auxin in the culture of these cadmium-resistant bacterial strains. Root elongation assay conducted on rape under gnotobiotic conditions demonstrated increases (up to 31%) in root elongation of inoculated rape seedlings compared to the control plants. Based upon cadmium-resistance, bio-activation of CdCO3 and growth-promoting activity, three isolates were selected for promoting plant growth and uptake of cadmium from cadmium-amended soil in pot experiments. Inoculation with the isolates was found to increase root dry weight (ranging from 8% to 20%) and shoot dry weight (ranging from 6% to 25%) of rape. An increase in cadmium content varying from 16 to 74%, compared to the non-inoculated control, was observed in rape plants cultivated in soil treated with 100 mgCd kg(-1) (as CdCl2) and inoculated with the isolates. The bacterial isolates were also able to colonize and develop in the rhizosphere soil of rape after root inoculation.     

Characterization of heavy metal-resistant endophytic bacteria from rape (Brassica napus) roots and their potential in promoting the growth and lead accumulation of rape

Two lead (Pb)-resistant endophytic bacteria were isolated from rape roots grown in heavy metal-contaminated soils and characterized. A pot experiment was conducted for investigating the capability of the two isolates to promote the growth and Pb uptake of rape from Pb-amended soil. The two isolates were identified as Pseudomonas fluorescens G10 and Microbacterium sp. G16 based on the 16S rDNA gene sequence analysis. Strains G10 and G16 exhibited different multiple heavy metal and antibiotic resistance characteristics and increased water-soluble Pb in solution and in Pb-added soil. Root elongation assays demonstrated increases in root elongation of inoculated rape seedlings compared to the control plants. Strain G16 produced indole acetic acid, siderophores and 1-aminocyclopropane-1-carboxylate deaminase. Increases in biomass production and total Pb uptake in the bacteria-inoculated plants were obtained compared to the control. The two strains could colonize the root interior and rhizosphere soil of rape after root inoculation.     

Microtox toxicity test: detoxification of TNT and RDX contaminated solutions by poplar tissue cultures

Poplar (Populus deltoidesxnigra DN34) tissue cultures removed 2,4,6-trinitrotoluene (TNT) from an aqueous solution in five days, reducing the toxicity of the solution from highly toxic Microtox EC value to that of the control. 1,3,5-Trinitro-1,3,5-triazacyclohexane (RDX) was taken up by the plant tissue cultures more slowly, but toxicity reduction of the solution was evident. The measurement of toxicity reduction of aqueous solutions containing TNT and RDX was performed using a novel methodology developed for use with the Microtox testing system. Radiolabeled TNT and RDX were used to confirm removal of explosives from hydroponic solutions containing plant tissue cultures and to verify that toxicity did not change in solutions where no plant cultures were present (positive controls). High Performance Liquid Chromatography (HPLC) and Liquid Scintillation Counter (LSC) measurements confirmed removal of TNT and RDX from solutions containing poplar plant tissue cultures and constancy of the plant-free controls. In addition, metabolites were identified in remediated solutions by HPLC, confirming the mechanism by which plants can remediate groundwater, surface water, and soil solutions.