Optimization of nickel and cobalt biosorption by native Serratia marcescens strains isolated from serpentine deposits using response surface methodology

The treatment of metal-polluted wastes is a challenging issue of environmental concern. Metals can be removed using microbial biomass, and this is an interesting approach towards the design of eco-friendly technologies for liquid waste treatment. The study reported here aimed to optimize nickel and cobalt biosorption from aqueous solutions using three native metal-resistant Serratia marcescens strains. Ni(II) and Co(II) biosorption by S. marcescens strains was found to fit better to Langmuir's model, with maximum uptake capacities of 13.5 mg g(-1) for Ni(II) ions and 19.9 mg g(-1) for Co(II) ions. Different experimental conditions of initial metal concentration, pH, initial biomass, and temperature were optimized using the Plackett-Burman method, and, finally, biomass and metal concentration were studied using the response surface methodology (RSM) to improve biosorption. The optimum uptake capacities for Co(II) ions by the three biosorbents used were obtained for initial metal concentrations of 35-40 mg L-1 and an initial biomass of 6 mg. For Ni(II) ions, the optimum uptake capacity was achieved with 1 mg of initial biomass for S. marcescens C-1 and C-19, and with 7 mg for S. marcescens C-16, with initial concentrations of 20-50 mg L-1. The results obtained demonstrate the viability of native S. marcescens strains as biosorbents for Ni(II) and Co(II) removal. This study also contributes to our understanding of the potential uses of serpentine microbial populations for the design of environmental cleanup technologies

Biosorption of nickel, cobalt, zinc and copper ions by Serratia marcescens strain 16 in mono and multimetallic systems

The metallurgical industry is one of the main sources of heavy metal pollution, which represents a severe threat to life. Metals can be removed from aqueous solutions by using microbial biomasses. This paper analyses the heavy metal biosorption capacity of Serratia marcescens strain 16 in single and multimetallic systems. The results obtained show that Co(II), Ni(II) and Zn(II) biosorption in monometallic systems is two to three times higher than in the presence of bi-metallic and multimetallic solutions. Fourier transform infrared spectroscopy confirmed that carbonyl, carboxyl and hydroxyl were the main functional groups, as well as the amide bands I and II involved in metal uptake, which are present in external structures of the bacterial cell. The results obtained demonstrated the viability of S. marcescens strain 16 as a biosorbent for the design of eco-friendly technologies for the treatment of waste liquor.The authors would like to acknowledge the financial support provided by the Iberoamerican PhD Program (UCA-UH), the AUIP and by the International Foundation of Science (Grant C/4078-2)

Study of the role played by NfsA, NfsB nitroreductase and NemA flavin reductase from Escherichia coli in the conversion of ethyl 2-(2′-nitrophenoxy)acetate to 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), a benzohydroxamic acid with interesting biological properties

Benzohydroxamic acids, such as 4-hydroxy-(2H)- 1,4-benzoxazin-3(4H)-one (D-DIBOA), exhibit interesting herbicidal, fungicidal and bactericidal properties. Recently, the chemical synthesis of D-DIBOA has been simplified to only two steps. In a previous paper, we demonstrated that the second step could be replaced by a biotransformation using Escherichia coli to reduce the nitro group of the precursor, ethyl 2-(2′-nitrophenoxy)acetate and obtain D-DIBOA. The NfsA and NfsB nitroreductases and the NemA xenobiotic reductase of E. coli have the capacity to reduce one or two nitro groups from a wide variety of nitroaromatic compounds, which are similar to the precursor. By this reason, we hypothesised that these three enzymes could be involved in this biotransformation. We have analysed the biotransformation yield (BY) of mutant strains in which one, two or three of these genes were knocked out, showing that only in the double nfsA/nfsB and in the triple nfsA/nfsB/nemA mutants, the BY was 0%. These results suggested that NfsA and NfsB are responsible for the biotransformation in the tested conditions. To confirm this, the nfsA and nfsB open reading frames were cloned into the pBAD expression vector and transformed into the nfsA and nfsB single mutants, respectively. In both cases, the biotransformation capacity of the strains was recovered (6.09±0.06% as in the wild-type strain) and incremented considerably when NfsA and NfsB were overexpressed (40.33%±9.42% and 59.68%±2.0% respectively)

Overexpression of the nitroreductase NfsB in an E. coli strain as a whole-cell biocatalyst for the production of chlorinated analogues of the natural herbicide DIBOA

Los ácidos benzohidroxámicos, como el DIBOA (2,4-dihidroxi-2 H)-1,4-benzoxazin-3(4 H)-ona), son productos vegetales que presentan interesantes propiedades herbicidas, fungicidas y bactericidas. Una alternativa viable a su purificación a partir de fuentes naturales es la síntesis de compuestos análogos como el D-DIBOA (2-deoxi-DIBOA) y sus derivados clorados. Su síntesis química se ha simplificado en dos pasos. Sin embargo, el segundo paso es una reacción exotérmica e implica la liberación de hidrógeno, lo que hace que esta metodología sea cara y difícil de ampliar. En el presente estudio se estudia la posibilidad de producir los derivados clorados de las benzoxazinonas mediante un proceso biocatalítico in vivo que utiliza la capacidad de la cepa de E. coli nfsB-/pBAD-NfsB modificada para catalizar la síntesis de 6-Cl- D-DIBOA y 8-Cl- D-DIBOA a partir de sus respectivos precursores (PCs). Los resultados muestran que esta cepa es capaz de crecer en medios que contienen estos compuestos y producir el producto de biotransformación con unos rendimientos del 59,3% y el 46,7%. respectivamente. Además, la cepa es capaz de procesarel precursor (PC) no purificados del primer paso químico para obtener rendimientos similares a los obtenidos a partir del PC purificado. Se estudió la cinética de la reacción in in vitro con la nitrorreductasa NfsB recombinante purificada para caracterizar la catálisis y evaluar los efectos que varios componentes del precursor no purificado tiene durante el proceso de síntesis. Los resultados revelaron que la cinética es la de una enzima alostérica. El efecto inhibitorio del sustrato en el primer paso de la síntesis química, se encuentra también en algunas etapas previas a la purificación del precursor

A genetically engineered Escherichia coli strain overexpressing the nitroreductase NfsB is capable of producing the herbicide D‑DIBOA with 100% molar yield

Background The use of chemical herbicides has helped to improve agricultural production, although its intensive use has led to environmental damages. Plant allelochemicals are interesting alternatives due to their diversity and degradability in the environment. However, the main drawback of this option is their low natural production, which could be overcome by its chemical synthesis. In the case of the allelochemical DIBOA ((2,4-dihydroxy-2H)-1,4-benzoxazin-3(4H)-one), the synthesis of the analogous compound D-DIBOA (2-deoxy-DIBOA) has been achieved in two steps. However, the scale up of this synthesis is hindered by the second step, which uses an expensive catalyst and is an exothermic reaction, with hydrogen release and a relatively low molar yield (70%). We have previously explored the “Green Chemistry” alternative of using E. coli strains overexpressing the nitroreductase NfsB as a whole-cell-biocatalyst to replace this second step, although the molar yield in this case was lower than that of the chemical synthesis. Results In this work, we engineered an E. coli strain capable of carrying out this reaction with 100% molar yield and reaching a D-DIBOA concentration up to 379% respect to the highest biotransformation yield previously reported. This was achieved by a screening of 34 E. coli mutant strains in order to improve D-DIBOA production that led to the construction of the ΔlapAΔfliQ double mutant as an optimum genetic background for overexpression of the NfsB enzyme and D-DIBOA synthesis. Also, the use of a defined medium instead of a complex one, the optimization of the culture conditions and the development of processes with several substrate loads allowed obtaining maxima yields and concentrations. Conclusions The high yields and concentrations of D-DIBOA reached by the microbial-cell-factory approach developed in this work will facilitate its application to industrial scale. Also, the use of an optimized defined medium with only an organic molecule (glucose as carbon and energy source) in its composition will also facilitate the downstream processes

Identification of Enzymatic Bottlenecks for the Aerobic Production of Malate from Glycerol by the Systematic Gene Overexpression of Anaplerotic Enzymes in Escherichia coli

The biotechnological production of dicarboxylic acids (C4) from renewable carbon sources represents an attractive approach for the provision of these valuable compounds by green chemistry means. Glycerol has become a waste product of the biodiesel industry that serves as a highly reduced carbon source for some microorganisms. Escherichia coli is capable of consuming glycerol to produce succinate under anaerobic fermentation, but with the deletion of some tricarboxylic acid (TCA) cycle genes, it is also able to produce succinate and malate in aerobiosis. In this study, we investigate possible rate-limiting enzymes by overexpressing the C-feeding anaplerotic enzymes Ppc, MaeA, MaeB, and Pck in a mutant that lacks the succinate dehydrogenase (Sdh) enzyme. The overexpression of the TCA enzyme Mdh and the activation of the glyoxylate shunt was also examined. Using this unbiased approach, we found that phosphoenol pyruvate carboxylase (Ppc) overexpression enhances an oxidative pathway that leads to increasing succinate, while phosphoenol pyruvate carboxykinase (Pck) favors a more efficient reductive branch that produces mainly malate, at 57.5% of the theoretical maximum molar yield. The optimization of the culture medium revealed the importance of bicarbonate and pH in the production of malate. An additional mutation of the ppc gene highlights its central role in growth and C4 production

Optimization of the Biocatalysis for D-DIBOA Synthesis Using a Quick and Sensitive New Spectrophotometric Quantification Method

D-DIBOA (4-hydroxy-(2H)-1,4-benzoxazin-3-(4H)-one) is an allelopathic-derived compound with interesting herbicidal, fungicidal, and insecticide properties whose production has been successfully achieved by biocatalysis using a genetically engineered Escherichia coli strain. However, improvement and scaling-up of this process are hampered by the current methodology for D-DIBOA quantification, which is based on high-performance liquid chromatographic (HPLC), a time-consuming technique that requires expensive equipment and the use of environmentally unsafe solvents. In this work, we established and validated a rapid, simple, and sensitive spectrophotometric method for the quantification of the D-DIBOA produced by whole-cell biocatalysis, with limits of detection and quantification of 0.0165 and 0.0501 mu mol center dot mL(-1) respectively. This analysis takes place in only a few seconds and can be carried out using 100 mu L of the sample in a microtiter plate reader. We performed several whole-cell biocatalysis strategies to optimize the process by monitoring D-DIBOA production every hour to keep control of both precursor and D-DIBOA concentrations in the bioreactor. These experiments allowed increasing the D-DIBOA production from the previously reported 5.01 mM up to 7.17 mM (43% increase). This methodology will facilitate processes such as the optimization of the biocatalyst, the scaling up, and the downstream purification

A systematic analysis of TCA Escherichia coli mutants reveals suitable genetic backgrounds for enhanced hydrogen and ethanol production using glycerol as main carbon source

Biodiesel has emerged as an environmentally friendly alternative to fossil fuels; however, the low price of glycerol feed-stocks generated from the biodiesel industry has become a burden to this industry. A feasible alternative is the microbial biotransformation of waste glycerol to hydrogen and ethanol. Escherichia coli, a microorganism commonly used for metabolic engineering, is able to biotransform glycerol into these products. Nevertheless, the wild type strain yields can be improved by rewiring the carbon flux to the desired products by genetic engineering. Due to the importance of the central carbon metabolism in hydrogen and ethanol synthesis, E. coli single null mutant strains for enzymes of the TCA cycle and other related reactions were studied in this work. These strains were grown anaerobically in a glycerol-based medium and the concentrations of ethanol, glycerol, succinate and hydrogen were analysed by HPLC and GC. It was found that the reductive branch is the more relevant pathway for the aim of this work, with malate playing a central role. It was also found that the putative C4-transporter dcuD mutant improved the target product yields. These results will contribute to reveal novel metabolic engineering strategies for improving hydrogen and ethanol production by E. coli

Automatable downstream purification of the benzohydroxamic acid D-DIBOA from a biocatalytic synthesis

Herbicides play a vital role in agriculture, contributing to increased crop productivity by minimizing weed growth, but their low degradability presents a threat to the environment and human health. Allelochemicals, such as DIBOA (2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4 H)-one), are secondary metabolites released by certain plants that affect the survival or growth of other organisms. Although these metabolites have an attractive po-tential for use as herbicides, their low natural production is a critical hurdle. Previously, the synthesis of the biologically active analog D-DIBOA (4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one) was achieved, using an engi-neered E. coli strain as a whole-cell biocatalyst, capable of transforming a precursor compound into D-DIBOA and exporting it into the culture medium, although it cannot be directly applied to crops. Here a chromatographic method to purify D-DIBOA from this cell culture medium without producing organic solvent wastes is described. The purification of D-DIBOA from a filtered culture medium to the pure compound could also be automated. Biological tests with the purified compound on weed models showed that it has virtually the same activity than the chemically synthesized D-DIBOA

Bottom up ethics - neuroenhancement in education and employment

Neuroenhancement involves the use of neurotechnologies to improve cognitive, affective or behavioural functioning, where these are not judged to be clinically impaired. Questions about enhancement have become one of the key topics of neuroethics over the past decade. The current study draws on in-depth public engagement activities in ten European countries giving a bottom-up perspective on the ethics and desirability of enhancement. This informed the design of an online contrastive vignette experiment that was administered to representative samples of 1000 respondents in the ten countries and the United States. The experiment investigated how the gender of the protagonist, his or her level of performance, the efficacy of the enhancer and the mode of enhancement affected support for neuroenhancement in both educational and employment contexts. Of these, higher efficacy and lower performance were found to increase willingness to support enhancement. A series of commonly articulated claims about the individual and societal dimensions of neuroenhancement were derived from the public engagement activities. Underlying these claims, multivariate analysis identified two social values. The Societal/Protective highlights counter normative consequences and opposes the use enhancers. The Individual/Proactionary highlights opportunities and supports use. For most respondents these values are not mutually exclusive. This suggests that for many neuroenhancement is viewed simultaneously as a source of both promise and concern