Unraveling the molecular response of Brassica napus hairy roots in the active Naphthol blue-black removal: Insights from proteomic analysis.

In vitro plant cultures are able to remove and metabolise xenobiotics, making them promising tools for decontamination strategies. In this work, we evaluated Brassica napus hairy roots (HRs) to tolerate and remove high concentrations of the azo dye Naphthol Blue-Black (NBB). Experiments were performed using both growing and resting culture systems at different pHs. Reuse of HRs biomass was evaluated in successive decolourisation cycles. Proteomics was applied to understand the molecular responses likely to be involved in the tolerance and removal of NBB. The HRs tolerated up to 480 µg mL-1 NBB, and 100 % removal was achieved at 180 µg mL-1 NBB after 10 days using both culture systems. Interestingly, the HRs are robust enough to be reused, showing 55-60 % removal even after three reuse cycles. The highest dye removal rates were achieved during the first 2 days of incubation, as initial removal is mainly driven by passive processes. Active mechanisms are triggered later by regulating the expression of proteins with different biological functions, mainly those related to xenobiotic metabolism, such as hydrolytic and redox enzymes. These results suggest that B. napus HRs are a robust tool that could make a significant contribution to textile wastewater treatment.

Remediation of endosulfan-contaminated water by hairy roots: removal and phytometabolization assessment.

Although many countries banned the insecticide endosulfan, it is still an environmental pollutant. Plants metabolize the two diastereomers of the formulations known as technical grade endosulfan (TGE) by two phase I pathways: hydrolysis leading to less toxic derivatives and oxidation giving endosulfan sulfate which is as toxic as endosulfan itself. We assessed the removal, bioaccumulation and phase I metabolization of TGE from water matrices using hairy root clones (HRs) of three edible species, Brassica napus, Raphanus sativus and Capsicum annuum. B. napus and C. annuum HRs removed 86% of TGE from the bioreaction media in 2 and 96 h, respectively, whereas R. sativus HRs removed 91% of TGE within 6 h of biotreatment. In the experiments with B. napus, only endosulfan sulfate was detected in both biomass and medium, whereas R. sativus and C. annuum accumulated endosulfan sulfate and endosulfan alcohol. Besides, endosulfan lactone was detected in C. annuum reaction medium. Acute ichthyotoxicity assays toward Poecilia reticulata showed that media contaminated with TGE lethal levels did not produce mortality after the phytotreatments. This research highlights the feasibility of using HRs to evaluate plant enzymatic abilities toward xenobiotics and their potential for the design of ex situ decontamination processes.

Natural trypanocidal product produced by endophytic fungi through co-culturing.

Endophytic fungi live inside vegetal tissues without causing damage to the host plant and may provide lead compounds for drug discovery. The co-culture of two or more endophytic fungi can trigger silent gene clusters, which could lead to the isolation of bioactive compounds. In this study, two endophytic strains isolated from Handroanthus impetiginosus leaves, identified as Talaromyces purpurogenus H4 and Phanerochaete sp. H2, were grown in mixed and axenic cultures. The meroterpenoid austin was detected only in the extracts from the mixed culture. Once isolated, austin displayed very interesting trypanocidal activity, with an IC50 value of 36.6 ± 1.2 µg/mL against Trypanosoma cruzi in the epimastigote form. The results obtained highlight the importance of the co-culturing of endophytic fungi to obtain natural bioactive products. The findings also enhance our understanding of the ecological relationships between endophytic fungi.

Effects of P limitation and molecules from peanut root exudates on pqqE gene expression and pqq promoter activity in the phosphate-solubilizing strain Serratia sp. S119.

The mineral phosphate-solubilizing phenotype in bacteria is attributed predominantly to secretion of gluconic acid produced by oxidation of glucose by the glucose dehydrogenase enzyme and its cofactor, pyrroloquinoline quinone. This study analyzes pqqE gene expression and pqq promoter activity in the native phosphate-solubilizing bacterium Serratia sp S119 growing under P-limitation, and in the presence of root exudates obtained from peanut plants, also growing under P-limitation. Results indicated that Serratia sp. S119 contains a pqq operon composed of six genes (pqqA,B,C,D,E,F) and two promoters, one upstream of pqqA and other between pqqA and pqqB. PqqE gene expression and pqq promoter activity increased under P-limiting growth conditions and not under N-deficient conditions. In the plant-bacteria interaction assay, the activity of the bacterial pqq promoter region varied depending on the concentration and type of root exudates and on the bacterial growth phase. Root exudates from peanut plants growing under P-available and P-limiting conditions showed differences in their composition. It is concluded from this study that the response of Serratia sp. S119 to phosphorus limitation involves an increase in expression of pqq genes, and that molecules exuded by peanut roots modify expression of these phosphate-solubilizing bacterial genes during plant-bacteria interactions.

Plant tissue cultures as sources of new ene- and ketoreductase activities.

While many redox enzymes are nowadays available for synthetic applications, the toolbox of ene-reductases is still limited. Consequently, the screening for these enzymes from diverse sources in the search of new biocatalyst suitable for green chemistry approaches is needed. Among 13 plant tissue cultures, Medicago sativa and Tessaria absinthioides calli, as well as Capsicum annuum hairy roots, were selected due to their ability to hydrogenate the CC double bond of the model substrate 2-cyclohexene-1-one. The three axenic plant cultures showed more preference toward highly activated molecules such as nitrostyrene and maleimide rather than the classical substrates of the well-known Old Yellow Enzymes, resembling the skills of the NAD(P)H-dependent flavin-independent enzymes. When the three biocatalytic systems were applied in the reduction of chalcones, T. absinthioides showed high chemoselectivity toward the CC double bond whereas the other two demonstrated abilities to biohydrogenate the CC double bounds and the carbonyl groups in a sequential fashion.

Biotechnological tools to improve bioremediation of phenol by Acinetobacter sp. RTE1.4.

The use of native bacteria is a useful strategy to decontaminate industrial effluents as well as the environment. Acinetobacter sp. RTE1.4 was previously isolated from polluted environments and constitutes a promising alternative for this purpose due to its capability to remove phenol from synthetic solutions and industrial effluents. In this work, this strain was identified at species level as A. tandoii RTE1.4. Phenol degradation pathway was studied and some reaction intermediates were detected, confirming that this strain degraded phenol through ortho-cleavage of the aromatic ring. Phenol removal assays were carried out in a stirred tank bioreactor and a complete degradation of the contaminant was achieved after only 7 h, at an aeration rate of 3 vvm and at agitation of 600 rpm. Moreover, this bacterium was immobilized into calcium alginate beads and an increase in phenol biodegradation with respect to free cells was observed. The immobilized cells were reused for four consecutive cycles and stored at 4°C for 9 months, during which phenol removal efficiency was maintained. Post-removal solutions were evaluated by Microtox® test, showing a toxicity reduction after bacterial treatment. These findings demonstrated that A. tandoii RTE1.4 might be considered as a useful biotechnological tool for an efficient treatment of different solutions contaminated with phenol in bioreactors, using either free or immobilized cells.

Deracemization of secondary alcohols by chemo-enzymatic sequence with plant cells.

A screening based on undifferentiated plant cells allowed identifying Gardenia jasminoides as the best biocatalyst to perform the kinetic resolution of 1-phenylethanol. This species was further tested for its ability to oxidize stereoselectively the (S)-isomers from racemic mixtures of secondary alcohols leaving their antipodes unaffected in Tris-HCl buffer. Those substrates which afforded the best results in the kinetic resolution were subjected to a chemo-enzymatic sequence of deracemization. G. jasminoides immobilized cells in calcium alginate were used for the oxidation of the (S)-enantiomers and, in a second step, NaBH(4) was added to the same vessel for the reduction of the corresponding ketone. The sequential repetition of these two steps allowed obtaining the R-alcohols in 82-90% yield in high optical purity (71-96% ee). Despite the viability of the cells is affected by the chemical reagent, their enzymes remain active due to the protective environment of the calcium alginate beads.