Characterization of Bacterial and Archaeal Communities by DGGE and Next Generation Sequencing (NGS) of Nitrification Bioreactors Using Two Different Intermediate Landfill Leachates as Ammonium Substrate

Nitrification-denitrification is an environmentally friendly and cost-effective way to treat landfill leachates. Special attention has been given to the nitrification step, usually the limiting one due to its special sensitivity to environmental factors. Here, the effect of the acclimatization of the nitrifying biomass to two different intermediate landfill leachates with different salt concentrations, COD and BOD5 has been studied. Despite the complete nitrification being successfully performed, the specific nitritation rates were reduced after the biomass adaptation to both landfill leachates caused by the presence of heavy metals and the high salt concentration. NGS analysis of the biomass samples revealed that Proteobacteria (48.5%), Actinobacteriota (14.4%) and Chloroflexi (9.5%) were the dominant phyla in the non-adapted biomass. The leachate feeding led to a decrease in OTU diversity and favored the growth of the phyla Bacteroidetes (27.2%), Euryarchaeota (26.6%) and Proteobacteria (20.0%) accounting for more than 70% of relative abundance. Several OTUs capable of performing the nitritation belong to the Xanthobacteraceae and the Xanthomonadaceae families, the Saccharimonadales order, and the genus Nitrosomonas, Nitrosospira and Paracoccus. In the nitratation process, the Xanthobacteraceae family and Lautropia and Nitrolancea genera were found.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. The Spanish Government (Ministry of Economy and Competitiveness) and the Vice-rectorate for Research of the University of Cadiz provided financial support through the project CTM2016-79089-R "Enhancement of landfill gas by an integrated biological system (Biointegra3)" and UCA/REC01VI/2017 (Universidad de Cadiz) respectivel

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)

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

Application of a Microalgal Peptide-Enriched Extract as Media Component in E. coli Culture

This study evaluated the feasibility of applying a microalgal peptide-enriched extract, obtained through a sequential alkaline and enzymatic method, as a fundamental media component in Escherichia coli culture. The results demonstrated that the extract is viable as the primary nutrient source for E. coli cultures (wild type), even as a complete substitute for the widely used Luria-Bertani (LB) medium, using an extract dilution of 1:3. Its application in a proportion carbon limiting (dilution 1:7), with glycerol supplementation at the early stationary phase, improved the nutrient use efficiency and controlled the undesirable acetate production. Furthermore, this last strategy applied in an E. coli mutant (M4-Delta iclR) enhanced the production of succinate by 67.3% compared with the M9 medium. The innovative approach proposed in this study opens a wide range of applications and studies related to the production of bio-compounds through diverse microorganisms

ZNF330/NOA36 interacts with HSPA1 and HSPA8 and modulates cell cycle and proliferation in response to heat shock in HEK293 cells

Background: The human genome contains nearly 20.000 protein-coding genes, but there are still more than 6,000 proteins poorly characterized. Among them, ZNF330/NOA36 stand out because it is a highly evolutionarily conserved nucleolar zinc-finger protein found in the genome of ancient animal phyla like sponges or cnidarians, up to humans. Firstly described as a human autoantigen, NOA36 is expressed in all tissues and human cell lines, and it has been related to apoptosis in human cells as well as in muscle morphogenesis and hematopoiesis in Drosophila. Nevertheless, further research is required to better understand the roles of this highly conserved protein. Results: Here, we have investigated possible interactors of human ZNF330/NOA36 through affinity-purification mass spectrometry (AP-MS). Among them, NOA36 interaction with HSPA1 and HSPA8 heat shock proteins was disclosed and further validated by co-immunoprecipitation. Also, “Enhancer of Rudimentary Homolog” (ERH), a protein involved in cell cycle regulation, was detected in the AP-MS approach. Furthermore, we developed a NOA36 knockout cell line using CRISPR/Cas9n in HEK293, and we found that the cell cycle profile was modified, and proliferation decreased after heat shock in the knocked-out cells. These differences were not due to a different expression of the HSPs genes detected in the AP-MS after inducing stress. Conclusions: Our results indicate that NOA36 is necessary for proliferation recovery in response to thermal stress to achieve a regular cell cycle profile, likely by interaction with HSPA1 and HSPA8. Further studies would be required to disclose the relevance of NOA36-EHR interaction in this context.14 página

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

Síntesis y caracterización de las aleaciones cuaternarias CuTaAlSe3 Y CuTaGaSe3

The ingots of CuTaAlSe3 and CuTaGaSe3 were synthesized using the melt and annealing technique. X-Ray Diffraction (XRD) and Differential Thermal Analysis (DTA) techniques were used for characterization of the alloy. The analysis of the diffraction pattern indicates that both samples are composed by two phases; the mean phase indexes as a hexagonal structure, whereas the secondary phase indexes as a tetragonal chalcopyrite-like structure. From the shape of the DTA peaks for the CuTaAlSe3 and CuTaGaSe3 alloys, was deduced that the melting is incongruent for both materials, value of ~1348K and ~1235K, respectively.  Los lingotes de CuTaAlSe3 y CuTaGaSe3 se sintetizaron usando la técnica de fusión y recocido. Se usaron técnicas de difracción de Rayos X (DRX) y Análisis Térmico Diferencial (ATD) para la caracterización de las aleaciones. El análisis del patrón de difracción, indica que ambas muestras están compuestas por dos fases; la fase promedio indexada como una estructura Hexagonal, mientras que la fase secundaria indexa como una estructura tetragonal de tipo calcopirita. De la forma de los picos del ATD para las aleaciones CuTaAlSe3 y CuTaGaSe3, se deduce que la fusión es incongruente para ambos materiales, de valor aproximado 1348K y 1235K, respectivamente

Aproximación a la checklist de los gipsófitos ibéricos

Approximation to the Iberian gypsophytes checklist. The close relationship between some kinds of soils and the flora growing on them has been long known. Gypsum soils stand as a prominent example of this relationship, since some plants exclusively grow on gypsum outcrops. Nowadays this close relationship is known as gypsophily and the plant species exhibiting it are called gypsophytes. As Cavanilles already found out in the 18th century, the Iberian flora is rich in gypsophytes. From then on many botanists have considerably enlarged the list of gypsophytes. However we still lack a more or less conclusive catalogue of the Iberian gypsophytes. Only some publications dealing with the gypsum flora or vegetation may serve as references for the drafting of a preliminary catalogue. This idea has recently inspired a preliminary list of gypsophytes which has been later enlarged with the addition of new species recorded now and then in a number of specific Floras and other works of a general scope. On the basis of this list, 12 botanists adept at gypsum flora have been asked to rank the liking of these species for gypsum soils. The gypsophilous character of 140 species have been thus ranked on a scale ranging from 1 to 5 (with 5 representing an absolute liking for gypsum soils). According to the median values, some 50 species can be considered as either absolute or preferent gypsophytes