Paola S. González

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.

Lethal and teratogenic effects of phenol on Bufo arenarum embryos.

Phenol and their derivatives are used in several industries and they have a high potential toxicity for animal and plant species. They were found in variable concentrations, as high as 1000 mg/L, in industrial wastewater and, they are often discharged into the environment. Amphibian embryos are useful indicators of environmental pollution. However, to our knowledge, there are not studies focussed on the toxic effects of phenol on Bufo arenarum, which is an anuran widely distributed in South America. Therefore, the effect of phenol on the survival and morphogenesis of these amphibian embryos was evaluated by means of AMPHITOX test. Embryos at 25 stage of development (acute test) and embryos at 2-4 blastomers stage (early life stage test), were exposed to phenol solutions in concentrations ranging from 25 to 250 mg/L, which were frequently found in the environment. Mortality and malformations were registered each 24h. LC(50), LC(99), NOEC, TC(50) and TI(50) values were 183.70, 250, 60, 113 mg/L and 1.62, respectively, at 96 h of treatment. Mortality and the percentage of malformations increased with increasing phenol concentrations. Teratogenic effects more frequently produced by phenol were: axial flexure, persistent yolk plug and different abnormalities which caused death of blastulae. Moreover, other malformations were registered, such as irregular form, acephalism, edema, axial shortening and underdevelopment of gills, among others. Larvae of B. arenarum, at early embryonic stages (blastulae), showed higher sensitivity to phenol than tadpoles at stage 25. Results confirm high susceptibility of amphibians to phenol and that environmental concentrations of this pollutant might be harmful to these populations.

Removal of 2,4-diclorophenol from aqueous solutions using tobacco hairy root cultures

2,4-Dichlorophenol (2,4-DCP) is harmful for aquatic life and human health, so many attempts have focused on removing it through innocuous technologies. Hairy roots (HR) represent an interesting plant system to study the process and to remove efficiently this compound. In the present work, tobacco HR clones were obtained and one of them was selected for 2,4-DCP phytoremediation assays. These cultures removed 2,4-DCP in short time and with high efficiency (98%, 88% and 83%) for solutions initially containing 250, 500 and 1000 mg/L, respectively. Removal process was mainly associated with peroxidase activity. The highest efficiency for 2,4-DCP (500 mg/L) removal was reached at 60 min and using 10 mM H(2)O(2). Moreover, HR could be re-used, almost for three consecutive cycles. The diminution of pH and the increase of chloride ions in post-removal solutions suggested that 2,4-DCP dehalogenation was mediated by peroxidases. Moreover, changes in deposition pattern of lignin in HR exposed to 2,4-DCP suggested that cell walls of xylem and phloem elements would be the site of deposition of some products formed and they would be a lignin-type polymer. These findings contribute to understand 2,4-DCP removal process with tobacco HR and it might have implications in the use of this system for decontamination of polluted waters.

Hairy Roots, their Multiple Applications and Recent Patents

In the last years, hairy root (HR) cultures are gaining attention in the biotechnology industry. This particular plant cell culture derives from explants infected with Agrobacterium rhizogenes. They constitute a relatively new approach to in vitro plant biotechnology and modern HR cultures are far away from the valuables findings performed by Philip R. White in the 1930s, who obtained indefinite growth of excised root tips. HR cultures are characterized by genetic and biochemical stability and high growth rate without expensive exogenous hormones source. HR cultures have allowed a deep study of plant metabolic pathways and the production of valuable secondary metabolites and enzymes, with therapeutic or industrial application. Furthermore, the potential of HR cultures is increasing continuously since different biotechnological strategies such as genetic engineering, elicitation and metabolic traps are currently being explored for discovery of new metabolites and pathways, as well as for increasing metabolites biosynthesis and/or secretion. Advances in design of proper bioreactors for HR growth are being of great interest, since scale up of metabolite production will allow the integration of this technology to industrial processes. Another application of HR cultures is related to their capabilities to biotransform and to degrade different xenobiotics. In this context, removal assays using this plant model system are useful tools for phytoremediation assays, previous to the application in the field. This review highlights the more recent application of HRs and those new patents which show their multiple utilities.

Application of hairy roots for phytoremediation: what makes them an interesting tool for this purpose?

In recent years, hairy roots (HRs) have been successfully used as research tools for screening the potentialities of different plant species to tolerate, accumulate, and/or remove environmental pollutants, such as PCBs, TNT, pharmaceuticals, textile dyes, phenolics, heavy metals, and radionuclides. This is in part due to several advantages of this plant model system and the fact that roots have evolved specific mechanisms to deal with pollutants because they are the first organs to have contact with them. In addition, by using HRs some metabolic pathways and enzymatic catalyzed reactions involved in pollutants detoxification can be elucidated as well as the mechanisms of uptake, transformation, conjugation, and compartmentation of pollutants in vacuoles and/or cell walls, which are important detoxification sites in plants. Plant roots also stimulate the degradation of contaminants by the release of root exudates and oxido-reductive enzymes, such as peroxidases (Px) and laccases, that are associated with the removal of some organic pollutants. HRs are also considered good alternatives as enzyme sources for remediation purposes. Furthermore, application of genetic engineering methods and development of microbe-assisted phytoremediation are feasible strategies to enhance plant capabilities to tolerate, accumulate, and/or metabolize pollutants and, hence, to create or find an appropriate plant system for environmental cleanup. The present review highlights current knowledge, recent progress, areas which need to be explored, and future perspectives related to the application and improvement of the efficiency of HRs for phytoremediation research.

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.

Characterization of a phenol-degrading bacterium isolated from an industrial effluent and its potential application for bioremediation

The use of native microorganisms is a useful strategy for phenol bioremediation. In the present work, a bacterial strain, named RTE1.4, was isolated from effluents of a chemical industry. The strain was able to grow at high concentrations of phenol and its derivatives, such as guaiacol, 2,4-dichlorophenol and pentachlorophenol, as well as in a medium containing industrial effluents. This bacterium was identified as Acinetobacter sp. using morphological, physiological, biochemical and 16S rRNA gene analysis. Acinetobacter sp. RTE1.4 degraded phenol (200 to 600 mg/L) at wide pH range and temperature (5–9 and 25–37°C, respectively) demonstrating high adaptation ability to different conditions. The strain would metabolize phenol by the ortho-pathway since catechol 1,2-dioxygenase activity was detected. When bacteria were grown in medium containing phenol, an altered whole-cell protein pattern was observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), with the lack of some low-molecular mass polypeptides and an increase in the relative abundance of high-molecular mass proteins after treatment. Considering that the use of native strains in bioremediation studies shows several ecological advantages and that the studied bacterium showed high tolerance and biodegradation capabilities, Acinetobacter sp. RTE1.4 could be an appropriate microorganism for improving bioremediation and biotreatment of areas polluted with phenol and/or some of its derivatives. Moreover, the establishment of the optimal growth conditions (pH, temperature, concentration of the pollutant) would provide baseline data for bulk production of the strain and its use in bioremediation processes.

Application of two bacterial strains for wastewater bioremediation and assessment of phenolics biodegradation

The use of native bacteria is a useful strategy to decontaminate industrial effluents. In this work, two bacterial strains isolated from polluted environments constitutes a promising alternative since they were able to remove several phenolic compounds not only from synthetic solutions but also from effluents derived from a chemical industry and a tannery which are complex matrices. Acinetobacter sp. RTE1.4 showed ability to completely remove 2-methoxyphenol (1000 mg/L) while Rhodococcus sp. CS1 not only degrade the same concentration of this compound but also removed 4- chlorophenol, 2,4-dichlorophenol and pentachlorophenol with high efficiency. Moreover, both bacteria degraded phenols naturally present or even exogenously added at high concentrations in effluents from the chemical industry and a tannery in short time (up to 5 d). In addition, a significant reduction of biological oxygen demand and chemical oxygen demand values was achieved after 7 d of treatment for both effluents using Acinetobacter sp. RTE1.4 and Rhodococcus sp. CS1, respectively. These results showed that Acinetobacter sp. RTE1.4 and Rhodococcus sp. CS1 might be considered as useful biotechnological tools for an efficient treatment of different effluents, since they showed wide versatility to detoxify these complex matrices, even supplemented with high phenol concentrations.

Biochemical and molecular mechanisms involved in simultaneous phenol and Cr(VI) removal by Acinetobacter guillouiae SFC 500-1A

Bioremediation has emerged as an environmental friendly strategy to deal with environmental pollution. Since the majority of polluted sites contain complex mixtures of inorganic and organic pollutants, it is important to find bacterial strains that can cope with multiple contaminants. In this work, a bacterial strain isolated from tannery sediments was identified as Acinetobacter guillouiae SFC 500-1A. This strain was able to simultaneously remove high phenol and Cr(VI) concentrations, and the mechanisms involved in such process were evaluated. The phenol biodegradation was catalized by a phenol-induced catechol 1,2-dioxygenase through an ortho-cleavage pathway. Also, NADH-dependent chromate reductase activity was measured in the cytosolic fraction. The ability of this strain to reduce Cr(VI) to Cr(III) was corroborated by detection of Cr(III) in cellular biomass after the removal process. While phenol did not affect significantly the chromate reductase activity, Cr(VI) was a major disruptor of catechol dioxygenase activity. Nevertheless, this activity was high even in presence of high Cr(VI) concentrations. Our results suggest the potential application of A. guillouiae SFC 500-1A for wastewaters treatment, and the obtained data provide the insights into the removal mechanisms, dynamics, and possible limitations of the bioremediation.

Improvement of simultaneous Cr(VI) and phenol removal by an immobilised bacterial consortium and characterisation of biodegradation products

Microbial bioremediation emerged some decades ago as an eco-friendly technology to restore polluted sites. Traditionally, the search for microorganisms suitable for bioremediation has been based on the selection of isolated strains able to remove a specific type of pollutant. However, this strategy has now become obsolete, since co-pollution is a global reality. Thus, current studies attempt to find bacterial cultures capable of coping with a mixture of organic and inorganic compounds. In this sense, the bacterial consortium SFC 500-1 has demonstrated efficiency for Cr(VI) and phenol removal, both of which are found in many industrial wastewaters. In the present study, the ability of SFC 500-1 for simultaneous removal was improved through its entrapment in a Ca-alginate matrix. This strategy led to an increased removal of Cr(VI), which was partially reduced to Cr(III). Immobilised cells were able to tolerate and degrade phenol up to 1,500mg/l at high rates, forming catechol and cis,cis-muconate as oxidation intermediates. Successful removal potential through 5 cycles of reuse, as well as after long-term storage, was another important advantage of the immobilised consortium. These characteristics make SFC 500-1 an interesting system for potential application in the biotreatment of co-polluted effluents.