Gema Cabrera

Combined strategy for the precipitation of heavy metals and biodegradation of petroleum in industrial wastewaters.

The precipitation of chromium(III), copper(II), manganese(II) and zinc(II) by biogenic hydrogen sulfide generated by sulfate-reducing bacteria, Desulfovibrio sp., and the degradation of total petroleum hydrocarbons (TPH) in the presence of heavy metal by Pseudomonas aeruginosa AT18 have been carried out. An anaerobic stirred tank reactor was used to generate hydrogen sulfide with Desulfovibrio sp. culture and the precipitation of more than 95% of each metal was achieved in 24 h (metal solutions contained: 60, 49, 50 and 80 mg L(-1) of chromium, copper, manganese and zinc sulfates). A stirred tank reactor with P. aeruginosa AT18, in the presence of the heavy metal solution and 2% (v/v) of petroleum, led to the degradation of 60% of the total petroleum hydrocarbons and the removal of Cr(III) 99%, Cu(II) 93%, Zn(II) 46% and Mn(II) 88% in the medium through biosorption phenomena. These results enabled the development of an integrated system in which the two processes were combined. The overall aim of the study was achieved, with 84% of TPH degraded and all of the metals completely removed. Work is currently underway aimed at improving this system (decrease in operation time, culture of P. aeruginosa in anaerobic conditions) in an effort to apply this process in the bioremediation of natural media contaminated with heavy metals and petroleum.

Identification of enhanced hydrogen and ethanol Escherichia coli producer strains in a glycerol-based medium by screening in single-knock out mutant collections.

BackgroundEarth’s climate is warming as a result of anthropogenic emissions of greenhouse gases from fossil fuel combustion. Bioenergy, which includes biodiesel, biohydrogen and bioethanol, has emerged as a sustainable alternative fuel source. For this reason, in recent years biodiesel production has become widespread but this industry currently generates a huge amount of glycerol as a by-product, which has become an environmental problem in its own right. A feasible possibility to solve this problem is the use of waste glycerol as a carbon source for microbial transformation into biofuels such as hydrogen and ethanol. For instance, Escherichia coli is a microorganism that can synthesize these compounds under anaerobic conditions.ResultsIn this work an experimental procedure was established for screening E. coli single mutants to identify strains with enhanced ethanol and/or H2 productions compared to the wild type strain. In an initial screening of 150 single mutants, 12 novel strains (gnd, tdcE, rpiAnanE, tdcB, deoB, sucB, cpsG, frmA, glgC, fumA and gadB) were found to provide enhanced yields for at least one of the target products. The mutations, that improve most significantly the parameters evaluated (gnd and tdcE genes), were combined with other mutations in three engineered E. coli mutant strains in order to further redirect carbon flux towards the desired products.ConclusionsThis methodology can be a useful tool to disclose the metabolic pathways that are more susceptible to manipulation in order to obtain higher molar yields of hydrogen and ethanol using glycerol as main carbon source in multiple E. coli mutants.

Immobilization of Cells on Polyurethane Foam

In this chapter, protocols and details for the immobilization of a model cell onto polyurethane foam carriers are provided in order to facilitate the use of such systems in laboratory or industrial reactors. Polyurethane foam has recently acquired great relevance as a carrier for its good mechanical properties, high porosity, and large adsorption surface. In addition, it has a very low commercial cost. Two different immobilization protocols have been described, differing in the flow regime or the possibilities for the reactor: immobilization in a stirred tank reactor working in a discontinuous regime (by cycles) and immobilization in a packed column working in continuous operation mode. Protocols for carrier sterilization, analytical methodology, and immobilization are described.

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).

Different strategies for recovering metals from CARON process residue

The capacity of Acidithiobacillus thiooxidans DMS 11478 to recover the heavy metals contained in the residue obtained from the CARON process has been evaluated. Different bioreactor configurations were studied: a two-stage batch system and two semi-continuous systems (stirred-tank reactor leaching and column leaching). In the two-stage system, 46.8% Co, 36.0% Mg, 26.3% Mn and 22.3% Ni were solubilised after 6h of contact between the residue and the bacteria-free bioacid. The results obtained with the stirred-tank reactor and the column were similar: 50% of the Mg and Co and 40% of the Mn and Ni were solubilised after thirty one days. The operation in the column reactor allowed the solid-liquid ratio to be increased and the pH to be kept at low values (<1.0). Recirculation of the leachate in the column had a positive effect on metal removal; at sixty five days (optimum time) the solubilisation levels were as follows: 86% Co, 83% Mg, 72% Mn and Ni, 62% Fe and 23% Cr. The results corroborate the feasibility of the systems studied for the leaching of metals from CARON process residue and these methodologies can be considered viable for the recovery of valuable metals.

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.

2.22 – Biofilters

Current environmental legislation is focused in removal and/or reducing emissions of pollutants. Biological treatments of wastes are considered as an alternative opposite traditional physicochemical methods. In recent decades, the use of biofilters to removal of contaminants from wastewater and waste gases is being developed. Biofilters use microorganisms, which are capable of degrading many compounds, fixed to an inorganic/organic medium (carrier) to break down pollutants present in a fluid stream. In this article, basic aspects about biofilter configuration, filter media, microorganisms involved, and properties that affect the biofilter performance are presented. Once these aspects are clarified, work deals with the biofilter design. Efforts are focused to summarize the terminology, operational features, and design equations that are essential for this kind of bioreactors. In the design, treatments of liquid and gaseous waste streams, due to the singular differentiating characteristics of these equipments, are distinguished.

Heterologous expression of the human Phosphoenol Pyruvate Carboxykinase (hPEPCK-M) improves hydrogen and ethanol synthesis in the Escherichia coli dcuD mutant when grown in a glycerol-based medium.

The production of biodiesel has emerged as an alternative to fossil fuels. However, this industry generates glycerol as a by-product in such large quantities that it has become an environmental problem. The biotransformation of this excess glycerol into other renewable bio-energy sources, like H2 and ethanol, by microorganisms such as Escherichia coli is an interesting possibility that warrants investigation. In this work we hypothesized that the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) could be improved by a controlled expression of the human mitochondrial GTP-dependent PEP carboxykinase. This heterologous expression was tested in several E. coli mutant backgrounds with increased availability of C4 intermediates. It was found that this metabolic rewiring improved the synthesis of the target products in several mutants, with the dcuD mutant being the most suitable background for hydrogen and ethanol specific productions and glycerol consumption. These factors increased by 2.46, 1.73 and 1.95 times, respectively, when compared to those obtained for the wild-type strain.

Integrated System to Biological Solubilization and Precipitation of Heavy Metals

The work consists on the study of a sulphur–oxidizing bacteria (At. thiooxidans) immobilisation over polyurethane foam and the integration of two continuous processes: the solubilization of heavy metals by acidic medium generated by sulphur-oxidizing bacteria and the subsequent precipitation of metals as sulphides with H2S biologically generated by sulphate-reducing bacteria (Desulfovibrio sp). At. thiooxidans was satisfactory immobilised over polyurethane foam and added to a column reactor. Acidic medium generated was added to a column with 50 g of an artificial contaminated sand (85 mg Cr(III), 20 mg Ni(II), 200 mg Zn(II)). The effluent of this step was collected in a reservoir tank, in which H2S from sulphate-reducing reactor was included to carry out the precipitation of metals. After 2.4 l of acid medium was passed through the column, it was observed that 14.6% of Cr(III), 26.7% of Ni(II) and 90.5% of Zn(II) were solubilized. The leachate was treated with 2.2 l of reducing medium, and 2.2% Cr(III),54% Ni(II) and 28% Zn(II) were precipitated.