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.     

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.     

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.     

Structural responses of Daucus carota root-organ cultures and the arbuscular mycorrhizal fungus, Glomus intraradices, to 12 pharmaceuticals

Pharmaceuticals and personal care products may enter the terrestrial environment through the amendment of agricultural soils with manure or biosolids with potential impacts on beneficial soil microbe populations. The beneficial symbiotic relationship between most plant species and arbuscular mycorrhizal fungi is a primary determinant of plant health and soil fertility. As such, there is increasing recognition of the need to study the impacts of anthropogenic stressors on plant-microbe interactions in soil ecotoxicology studies and risk assessment. A case study exploring the use of root-organ cultures to evaluate the effects of 12 common veterinary and human-use pharmaceuticals on the arbuscular mycorrhizal fungus, Glomus intraradices grown on Daucus carota root-organ cultures is presented. The bioassays were conducted over a 28-day exposure period at concentrations up to 1000mugl(-1). Root length and the fungal endpoints of hyphal growth and spore production were evaluated weekly during the study. Sulfamethoxazole and atorvastatin were the most phytotoxic compounds with EC(50) values of 45mugl(-1) and 65mugl(-1), respectively. Three compounds exhibited selective mycotoxicity, whereby the fungal symbiont was adversely affected at concentrations significantly less than that calculated for root length. The EC(50) for G. intraradices hyphal length was 45mugl(-1) for doxycycline, while carbamazepine and 17-alpha-ethynyl estradiol targeted spore production with EC(50) values of 113 and 116mugl(-1), respectively. The assay results indicate that the root lengths responded quickly to the presence of phytotoxic pharmaceuticals in the culture medium. Hyphal length is a sensitive endpoint after 21 days exposure, while spore production requires 28 days exposure before significant differences could be detected. Root-organ cultures provide an effective means to evaluate chemical stressors on arbuscular mycorrhizal fungi and can be used to screen for root-based phytotoxicity.     

Evaluation of phenol detoxification by Brassica napus hairy roots, using Allium cepa test

Introduction  

Meristematic mitotic cells of Allium cepa constitute an adequate material for cytotoxicity and genotoxicity evaluation of environmental pollutants, such as phenol, which is a contaminant frequently found in several industrial effluents.     

Toxicity, uptake and metabolism of 4-n-nonylphenol in root cultures and intact plants under septic and aseptic conditions

4-Nonylphenol, a compound with estrogenic activity, has been shown to occur in sewage sludges and effluents of sludge treatment. This, as well as its use in the formulation of pesticides, may result in the contamination of crop plants and may therefore have an impact on the quality of food or feedstuff. The toxicity, uptake and metabolism of 4-n-nonylphenol (4-n-NP) were investigated as¹⁴C-labeled 4-n-NP in root cultures under septical and aseptical conditions and with intact plants grown in containers with soil and aseptically grown in nutrient media. 4-n-NP was toxic to all plant systems tested. The presence of microorganisms and the developmental state of the plant material appeared to have an influence on the EC₅₀ values. 4-n-NP was taken up by the roots and a metabolism to polar compounds was observed in the cases where sufficiently high uptake rates. With intact plants a transport from roots to the shoots was evident. Metabolism in roots changed quantitatively in the presence of microorganisms. The mineralization of 4-n-NP to₁₄CO₂ only occurred with microorganisms.     

Biodegradation of phenol in refinery wastewater by pure cultures of Pseudomonas aeruginosa NCIB 950 and Pseudomonas fluorescence NCIB 3756

The potential of microorganisms to catabolise and metabolise xenobiotic compounds has been recognised as a potentially effective means of toxic and hazardous wastes disposal. Phenol and its derivatives have long been recognised as some of the most persistent chemicals in petroleum refinery wastewaters, with high toxicity even at low concentrations. Biodegradation of these compounds has been recognised as a potential solution for their disposal owing to its cost effectiveness and simplicity. Two species of pseudomonas, P. aeruginosa and P. fluorescence, were studied for their biodegradation potential on phenol present in a refinery wastewater under a batch fermentation process. Phenol was successfully degraded by both species, and there was high positive correlation between phenol biodegradation and microbial growth. The maximum specific growth rate were obtained for both species from the Haldane model. The study revealed the high potential of these local strains, with P. aeruginosa being more effective, and the possibility of using them in bioremediation of petroleum refinery wastewaters.     

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.     

Tolerance, uptake and removal of nitrobenzene by a newly-found remediation species Mirabilis jalapa L

The growth, photosynthesis rate, and ultrastructure of Mirabilis jalapa L. as a newly-found remediation species under stress of nitrobenzene (NB) and its uptake and removal of NB by the plants were investigated. The results showed that M. jalapa plants could endure contaminated soils by lower than 10.0 mg NB kg⁻¹ because there was no decrease in the total length of the plant roots, the maximum length of the hypocotyle, the length of the first seminal root, the height of the shoots and the dry biomass of the seedlings as well as the photosynthesis rate of the plants compared with those in the control. In particular, the growth of the plants could be significantly (P < 0.01) enhanced by 0.1 mg NB kg⁻¹ under unautoclaved and autoclaved soils. Ultrastructural observations on leaf cells of the plants found that these cells had smooth, clean and continuous cell membranes and cell walls, indicating that there was no obvious damage by NB in comparison with those in the control. Although the absorption of NB in shoots and roots of M. jalapa was weak, plant-promoted biodegradation of NB was considerable and the dominant contribution in the removal of NB from contaminated soils, suggesting the feasibility of M. jalapa applied to phytoremediation of NB contaminated soils.     

Adsorption of phenol by bentonite

The potential of bentonite for phenol adsorption from aqueous solutions was studied. Batch kinetics and isotherm studies were carried out to evaluate the effect of contact time, initial concentration, pH, presence of solvent, and the desorption characteristics of bentonite. The adsorption of phenol increases with increasing initial phenol concentration and decreases with increasing the solution pH value. The adsorption process was significantly influenced by the solvent type in which phenol was dissolved. The affinity of phenol to bentonite in the presence of cyclohexane was greater than that in water and was lowest in the presence of methanol. Methanol was used to extract phenol from bentonite. The degree of extraction was dependent on the amount of phenol adsorbed by bentonite. X-ray diffraction analysis showed that the crystalline structure of bentonite was destroyed when cyclohexane was used. The ability of bentonite to adsorb phenol from cyclohexane decreased as the water to cyclohexane ratio was increased. Furthermore, hysteresis was observed in phenol desorption from bentonite in aqueous solutions. The equilibrium data in aqueous solutions was well represented by the Langmuir and Freundlich isotherm models. The removal of phenol from aqueous solutions was observed without surface modification.     

Effect of cadmium on cytogenetic toxicity in hairy roots of Wedelia trilobata L. and their alleviation by exogenous CaCl2

Effects of cadmium (Cd) alone and in combination with calcium on mitosis and chromosomal aberration in the hairy root tips of Wedelia trilobata were investigated. The results showed that Cd concentrations below 50 μmol/L had a lesser or even a promoting effect on the mitotic index (MI) and the rate of chromosomal aberration in hairy root tips, while those higher than 100 μmol/L significantly decreased the MI and gradually stimulated the rate of chromosomal aberrations with prolonged time and increasing concentrations of Cd. Concentrations of 50 μmol/L Cd mainly induced C-mitosis, while more than 100 μmol/L Cd mainly caused chromosome breakage and chromosome adhesion in hairy root tip cells. When cultured with 300 μmol/L Cd, micronuclei were only observed in the interphase, middle, and late phase of hairy root tip cells. Compared with untreated controls, exogenous calcium had an alleviating effect on Cd-induced cytotoxicity by effectively enhancing the MI and reducing the rate of chromosomal aberration in root tip cells. The results presented here provide evidence that W. trilobata hairy roots with rapid autonomous growth could be used as a sensitive tool for monitoring and evaluation of Cd pollution in the environment.     

Phytoremediation of phenol using Vicia sativa L. plants and its antioxidative response

Common vetch (Vicia sativa L.) is a legume species with an extensive agricultural use. However, the phytoremediation potentiality of this species has not been sufficiently explored because little is known about its resistance to inorganic and organic pollutants. In the present work, phenol tolerance of common vetch was assayed at different stages of growth. Germination index and germination rate decreased only at high phenol concentrations (250 and 500 mg L(-1)), whereas 30-day-old plants were able to tolerate this pollutant, with high removal efficiencies. The activities of antioxidative enzymes, such as peroxidase (POD) and ascorbate peroxidase, increased significantly with the highest phenol concentration, whereas superoxide dismutase activity, malondialdehyde, and H(2)O(2) levels remained unaltered. Besides, an increase in two basic isoforms of POD was observed in plants treated with phenol. The results suggested that common vetch has an efficient protection mechanism against phenol-induced oxidative damage. Moreover, it could tolerate and remove high phenol concentrations, avoiding serious phytotoxic effects. Thus, V. sativa could be considered an interesting tool in the field of phytoremediation.     

Immobilization of aluminum with mucilage secreted by root cap and root border cells is related to aluminum resistance in Glycine max L

The root cap and root border cells (RBCs) of most plant species produced pectinaceous mucilage, which can bind metal cations. In order to evaluate the potential role of root mucilage on aluminum (Al) resistance, two soybean cultivars differing in Al resistance were aeroponic cultured, the effects of Al on root mucilage secretion, root growth, contents of mucilage-bound Al and root tip Al, and the capability of mucilage to bind Al were investigated. Increasing Al concentration and exposure time significantly enhanced mucilage excretion from both root caps and RBCs, decreased RBCs viability and relative root elongation except roots exposed to 400 μM Al for 48 h in Al-resistant cultivar. Removal of root mucilage from root tips resulted in a more severe inhibition of root elongation. Of the total Al accumulated in root, mucilage accounted 48–72 and 12–27 %, while root tip accounted 22–52 and 73–88 % in Al-resistant and Al-sensitive cultivars, respectively. A ²⁷Al nuclear magnetic resonance spectrum of the Al-adsorbed mucilage showed Al tightly bound to mucilage. Higher capacity to exclude Al in Al-resistant soybean cultivar is related to the immobilization and detoxification of Al by the mucilage secreted from root cap and RBCs.     

Metabolites of 2,2'-dichlorobiphenyl and 2,6-dichlorobiphenyl in hairy root culture of black nightshade Solanum nigrum SNC-9O

The hairy root culture of black nightshade (Solanum nigrum) SNC-9O was exposed to 2,2′-dichlorobiphenyl (PCB 4) and 2,6-dichlorobiphenyl (PCB 10) to follow the metabolites produced. The analytical standards of 4-hydroxy-2,2′-dichlorobiphenyl, 5′-hydroxy-2,2′-dichlorobiphenyl, 4-hydroxy-2,6-dichlorobiphenyl, 2-hydroxy-2′,6′-dichlorobiphenyl, 3-hydroxy-2′,6′-dichlorobiphenyl and 4-hydroxy-2′,6′-dichlorobiphenyl have been synthesized. Hydroxy-metabolites of both PCB 4 and PCB 10 were present in the biomass. These appeared mainly as conjugates rather than as free hydroxy-PCBs, both maintained in plant cells. The concentrations of non-conjugated hydroxy-PCBs ranged between 0.9 and 35.2 μg kg⁻¹ of biomass fresh weight and the concentration of the conjugated ones ranged between 2.0 and 113.0 μg kg⁻¹ depending on the position of hydroxyl. The para- position of biphenyl (4 or 4′) seems to be preferred for hydroxylation. Methoxy-PCBs and hydroxy-methoxy-PCBs have also been identified in plant cells. Hydroxyl in the meta-position (3, 3′, 5 or 5′) appears to be preferred for methylation in hydroxy-PCBs. Hydroxy-methoxy-PCBs have occurred in the conjugated form as well.     

Plant metabolites of polychlorinated biphenyls in hairy root culture of black nightshade Solanum nigrum SNC-9O

The present study is intended to determine metabolites of 12 dichlorinated, seven trichlorinated, five tetrachlorinated and one pentachlorinated PCB congener transformed by black nightshade (Solanum nigrum) hairy root culture SNC-9O. Free hydroxylated PCB metabolites were identified based on the mass spectra characteristics after gas chromatography separation. The number of metabolites decreases with an increasing number of chlorine atoms per molecule of PCB. Dichlorinated PCBs lead always to at least two metabolites. In the case of PCB 9 some metabolites could be identified by comparing their RF values due to available standards. The 2',5'-dichloro-2-biphenylol, 2',5'-dichloro-3-biphenylol and 2',5'-dichloro-4-biphenylol, present as the main metabolite, were found in biomass of SNC-9O hairy root culture. Two monochlorinated biphenylols were found in biomass of SNC-9O degrading PCB 9 congener. It was the only case when metabolites with decreased number of chlorine atoms compared to parent PCB were found. Trichlorinated PCBs mostly lead to a lower number of metabolites but tetrachlorinated and pentachlorinated PCBs mostly did not give any metabolites. In the media, only traces of metabolites were found in sporadic cases, so exudation of unbound biphenylols from the cells is not expected.     

黄菖蒲和狭叶香蒲根系对氮磷的吸收动力学

采用改进的常规耗竭法,研究了黄菖蒲（Iris pseudacorus L.）和狭叶香蒲（Typha angustifolia L.）根系对NH₄⁺、NO₃^-和H₂PO₄^-的吸收特征及差异。结果表明,这2种植物根系对NH₄⁺、NO₃^-和H₂PO₄^-的吸收动力学特征均可采用Michaelis-Menten方程描述。2种植物根系对NH₄⁺、NO₃^-和H₂PO₄^-的亲和力（Km）和最大吸收速率（Vmax）有显著差异。吸收H₂PO₄^-时,黄菖蒲根系具有较高的Vmax值和较低的Km值,说明黄菖蒲具有嗜磷特性,并能够适应广范围浓度的H₂PO₄^-环境,适宜用于污染水体磷的去除;吸收NO₃^-时,狭叶香蒲根系具有较高的Vmax值和较低的Km值,表明狭叶香蒲可用于广范围浓度NO₃^-污染的水体修复;吸收NH₄⁺时,黄菖蒲根系具有较低的Vmax值和Km值,而狭叶香蒲根系具有较高的Vmax值和Km值,说明黄菖蒲适宜用于NH₄⁺污染较轻水体的修复,而在NH₄⁺污染较重水体中宜选用狭叶香蒲作为先锋植物。     

麦芽糖假丝酵母降解苯酚和4-氯酚的特性

考察麦芽糖假丝酵母对单底物4-氯酚的降解特性,研究苯酚与4-氯酚在双底物降解体系中的相互作用,并对比分析该菌株对苯酚和4-氯酚的降解能力。研究发现,麦芽糖假丝酵母能以4-氯酚为唯一碳源和能源,可在93 h内降解350 mg/L 4-氯酚,其降解4-氯酚的能力低于苯酚。酶活分析表明,4-氯酚降解遵循邻位裂解途径。细胞生长动力学过程符合Haldane方程,动力学参数为:μmax=0.252 h-1,Ks=33.58 mg/L,Ki=147.44 mg/L,相关系数R2=0.99。在苯酚/4-氯酚双底物降解体系中,较低浓度苯酚(200~400 mg/L)可以促进4-氯酚的降解,而较高浓度苯酚会产生抑制作用,当苯酚初始浓度为300 mg/L时,4-氯酚降解速率最大;4-氯酚的存在会抑制苯酚的降解。采用Abuhamed等人提出的动力学方程可以准确描述苯酚/4-氯酚双底物降解体系中细胞生长过程,得到两物质之间的相互抑制参数: I1,2=1.71,I2,1=3.25,相关系数R2=0.93。     

Kinetics of phenol and chlorophenol utilization by Acinetobacter species

Although microbial transformations via cometabolism have been widely observed, the few available kinetic models of cometabolism have not adequately addressed the case of inhibition from both the growth and nongrowth substrates. The present study investigated the degradation kinetics of self-inhibitory growth (phenol) and nongrowth (4-chlorophenol, 4-CP) substrates, present individually and in combination. Specifically, batch experiments were performed using an Acinetobacter isolate growing on phenol alone and with 4-CP present. In addition, batch experiments were also performed to evaluate the transformation of 4-CP by resting, phenol-induced Acinetobacter cultures. The Haldane kinetic model adequately predicted the biodegradation of phenol alone, although a slight discrepancy was noted in cases of higher initial phenol concentrations. Similarly, a Haldane model for substrate utilization was also able to describe the trends in 4-CP transformation by the resting cell cultures. The 4-CP transformation by the Acinetobacter species growing on phenol was modeled using a competitive kinetic model of cometabolism, which included growth and nongrowth substrate inhibition and cross-inhibition terms. Excellent agreement was obtained between the model predictions using experimentally estimated parameter values and the experimental data for the synchronous disappearance of phenol and 4-CP.     

Antioxidant metabolism of coffee cell suspension cultures in response to cadmium

The antioxidant responses of coffee (Coffea arabica L.) cell suspension cultures to cadmium (Cd) were investigated. Cd accumulated very rapidly in the cells and this accumulation was directly correlated with an increase in applied CdCl(2) concentration in the external medium. At 0.05mM CdCl(2), growth was stimulated, but at 0.5mM CdCl(2), the growth rate was reduced. An alteration in activated oxygen metabolism was detected by visual analysis as well as by an increase in lipid peroxidation at the higher CdCl(2) concentration. Catalase (CAT; EC 1.11.1.6), glutathione reductase (GR; EC 1.6.4.2) and superoxide dismutase (SOD; EC 1.15.1.1) activity increased, particularly at the higher concentration of CdCl(2). Ascorbate peroxidase (APX; EC 1.11.1.11) activity was increased at the lower CdCl(2) concentration used, but could not be detected in cells growing in the higher CdCl(2) concentration after 24h of growth, whilst guaiacol peroxidase (GOPX; EC 1.11.1.7) did not show a clear response to Cd treatment. An analysis by non-denaturing PAGE followed by staining for enzyme activity, revealed one CAT isoenzyme, nine SOD isoenzymes and four GR isoenzymes. The SOD isoenzymes were differently affected by CdCl(2) treatment and one GR isoenzyme was shown to specifically respond to CdCl(2). The results suggest that the higher concentrations of CdCl(2) may lead to oxidative stress. The main response appears to be via the induction of SOD and CAT activities for the removal of reactive oxygen species (ROS), and by the induction of GR to ensure the availability of reduced glutathione for the synthesis of Cd-binding peptides, which may also be related to the inhibition of APX activity probably due to glutathione and ascorbate depletion.