Metabolomics of Escherichia coli for Disclosing Novel Metabolic Engineering Strategies for Enhancing Hydrogen and Ethanol Production.

The biological production of hydrogen is an appealing approach to mitigating the environmental problems caused by the diminishing supply of fossil fuels and the need for greener energy. Escherichia coli is one of the best-characterized microorganisms capable of consuming glycerol-a waste product of the biodiesel industry-and producing H2 and ethanol. However, the natural capacity of E. coli to generate these compounds is insufficient for commercial or industrial purposes. Metabolic engineering allows for the rewiring of the carbon source towards H2 production, although the strategies for achieving this aim are difficult to foresee. In this work, we use metabolomics platforms through GC-MS and FT-IR techniques to detect metabolic bottlenecks in the engineered ΔldhΔgndΔfrdBC::kan (M4) and ΔldhΔgndΔfrdBCΔtdcE::kan (M5) E. coli strains, previously reported as improved H2 and ethanol producers. In the M5 strain, increased intracellular citrate and malate were detected by GC-MS. These metabolites can be redirected towards acetyl-CoA and formate by the overexpression of the citrate lyase (CIT) enzyme and by co-overexpressing the anaplerotic human phosphoenol pyruvate carboxykinase (hPEPCK) or malic (MaeA) enzymes using inducible promoter vectors. These strategies enhanced specific H2 production by up to 1.25- and 1.49-fold, respectively, compared to the reference strains. Other parameters, such as ethanol and H2 yields, were also enhanced. However, these vectors may provoke metabolic burden in anaerobic conditions. Therefore, alternative strategies for a tighter control of protein expression should be addressed in order to avoid undesirable effects in the metabolic network.

Bioleaching of the α-alumina layer of spent three-way catalysts as a pretreatment for the recovery of platinum group metals.

Acid bioleaching of Al by Acidithiobacillus thiooxidans has been explored as an environmentally friendly pretreatment to facilitate the extraction of platinum group metals from spent three-way catalysts (TWC). Biogenic sulfur obtained from desulfurization bioreactors improved the production of acid by A. thiooxidans compared to commercially available elemental sulfur. The lixiviation abilities of bacteria-free biogenic acid and biogenic acid with exponential or stationary phase bacteria were compared against a control batch produced by commercial H2SO4. The maximum Al leaching percentage (54.5%) was achieved using biogenic acids with stationary-phase bacteria at a TWC pulp density of 5% w/v whereas bacteria-free biogenic acid (23.4%), biogenic acid with exponential phase bacteria (21.7%) and commercial H2SO4 (24.7%) showed lower leaching abilities. The effect of different pulp densities of ground TWC (5, 30, and 60% w/v) on Al leaching and bacterial growth was determined. While greater Al leaching yields were obtained at lower TWC pulp density solutions (54.5% at 5% w/v and 2.5% at 60% w/v), higher pulp densities enhanced microbial growth (2.3 × 109 cells/mL at 5% w/v and 9.5 × 1010 cells/mL at 60% w/v). The dissolution of the metal from the solid into the liquid phase triggered the production of biological polymeric substances that were able to absorb traces of both Al (up to 24.80% at 5% w/v) and Pt (up to 0.40% at 60% w/v).

Sustainable Recovery of Platinum Group Metals from Spent Automotive Three-Way Catalysts through a Biogenic Thiosulfate-Copper-Ammonia System.

This study explores an eco-friendly method for recovering platinum group metals from a synthetic automotive three-way catalyst (TWC). Bioleaching of palladium (Pd) using the thiosulfate-copper-ammonia leaching processes, with biogenic thiosulfate sourced from a bioreactor used for biogas biodesulfurization, is proposed as a sustainable alternative to conventional methods. Biogenic thiosulfate production was optimized in a gas-lift bioreactor by studying the pH (8-10) and operation modes (batch and continuous) under anoxic and microaerobic conditions for 35 d. The maximum concentration of 4.9 g S2O32- L-1 of biogenic thiosulfate was reached under optimal conditions (batch mode, pH = 10, and airflow rate 0.033 vvm). To optimize Pd bioleaching from a ground TWC, screening through a Plackett-Burman design determined that oxygen and temperature significantly affected the leaching yield negatively and positively, respectively. Based on these results, an optimization through an experimental design was performed, indicating the optimal conditions to be Na2S2O3 1.2 M, CuSO4 0.03 M, (NH4)2SO4 1.5 M, Na2SO3 0.2 M, pH 8, and 60 °C. A remarkable 96.2 and 93.2% of the total Pd was successfully extracted from the solid at 5% pulp density using both commercially available and biogenic thiosulfate, highlighting the method's versatility for Pd bioleaching from both thiosulfate sources.

Metabolomics for the design of new metabolic engineering strategies for improving aerobic succinic acid production in Escherichia coli.

INTRODUCTION: Glycerol is a byproduct from the biodiesel industry that can be biotransformed by Escherichia coli to high added-value products such as succinate under aerobic conditions. The main genetic engineering strategies to achieve this aim involve the mutation of succinate dehydrogenase (sdhA) gene and also those responsible for acetate synthesis including acetate kinase, phosphate acetyl transferase and pyruvate oxidase encoded by ackA, pta and pox genes respectively in the ΔsdhAΔack-ptaΔpox (M4) mutant. Other genetic manipulations to rewire the metabolism toward succinate consist on the activation of the glyoxylate shunt or blockage the pentose phosphate pathway (PPP) by deletion of isocitrate lyase repressor (iclR) or gluconate dehydrogenase (gnd) genes on M4-ΔiclR and M4-Δgnd mutants respectively. OBJECTIVE: To deeply understand the effect of the blocking of the pentose phosphate pathway (PPP) or the activation of the glyoxylate shunt, metabolite profiles were analyzed on M4-Δgnd, M4-ΔiclR and M4 mutants. METHODS: Metabolomics was performed by FT-IR and GC-MS for metabolite fingerprinting and HPLC for quantification of succinate and glycerol. RESULTS: Most of the 65 identified metabolites showed lower relative levels in the M4-ΔiclR and M4-Δgnd mutants than those of the M4. However, fructose 1,6-biphosphate, trehalose, isovaleric acid and mannitol relative concentrations were increased in M4-ΔiclR and M4-Δgnd mutants. To further improve succinate production, the synthesis of mannitol was suppressed by deletion of mannitol dehydrogenase (mtlD) on M4-ΔgndΔmtlD mutant that increase ~ 20% respect to M4-Δgnd. CONCLUSION: Metabolomics can serve as a holistic tool to identify bottlenecks in metabolic pathways by a non-rational design. Genetic manipulation to release these restrictions could increase the production of succinate.

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.

Effect of two different intermediate landfill leachates on the ammonium oxidation rate of non-adapted and adapted nitrifying biomass.

A widely employed approach to minimize the detrimental effect of landfill leachates (LL) on nitrifying biomass is to adapt it to these contaminated effluents prior to use. In the study reported here the impact of different intermediate landfill leachates (intermediate 1 (ILL1) and intermediate 2 (ILL2)) and synthetic medium (SM) on the nitritation rates of non-adapted and adapted nitrifying biomass were evaluated and modeled. The models, based on previously reported models (Haldane, Edwards and Aiba), considered the effect of three different heavy metals (Cu, Ni and Zn) present in both landfill leachates. The proposed models fitted well with the different biomasses. The highest specific substrate oxidation rate (qS) of the present study (41.85 ± 1.09 mg N-NH4+ g TSS-1 h-1) was obtained by the non-adapted biomass using SM. The non-adapted biomass was characterized by ~5- and ~28-fold higher nitritation rates on using the different ammonium sources tested (SM, ILL1 and ILL2) when compared to the other biomasses adapted to ILL1 (~9 mg N-NH4+ g TSS-1 h-1) and ILL2 (~1.3 mg N-NH4+ g TSS-1 h-1), respectively. The calculated inhibition constants indicate that the inhibitory effect of the heavy metals followed the order Ni>Zn>Cu. The results reported here bring into question the commonly accepted idea that an adaptation period of the biomass is required to treat landfill leachate.

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 µmol·mL-1 respectively. This analysis takes place in only a few seconds and can be carried out using 100 µL 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 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.

A comparative study of eubacterial communities by PCR-DGGE fingerprints in anoxic and aerobic biotrickling filters used for biogas desulfurization.

Biological desulfurization has proven to be a process that is technically and economically feasible on using biotrickling filters that can be performed under aerobic and anoxic conditions. However, microbial communities are different mainly due to the use of different final electron acceptors. The analysis of microbial communities in these systems has not been addressed with regard to the anoxic process. The aim of the work reported here was to analyse the eubacterial community in the two types of bioreactor along the packed bed and during the operation time. The analysis was carried out using the 16S PCR-DGGE molecular fingerprint technique. The microbial profile analysis in the aerobic bioreactor revealed that the community was more diverse and stratified compared to those obtained in the two anoxic bioreactors, influenced by environmental factors. The main OTU involved in this process is genus Thiobacillus, although different species were detected depending on each operational condition.

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

BACKGROUND: Earth'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. RESULTS: In 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, rpiA nanE, 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. CONCLUSIONS: This 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.

Kinetic and stoichiometric characterization of anoxic sulfide oxidation by SO-NR mixed cultures from anoxic biotrickling filters.

Monitoring the biological activity in biotrickling filters is difficult since it implies estimating biomass concentration and its growth yield, which can hardly be measured in immobilized biomass systems. In this study, the characterization of a sulfide-oxidizing nitrate-reducing biomass obtained from an anoxic biotrickling filter was performed through the application of respirometric and titrimetric techniques. Previously, the biomass was maintained in a continuous stirred tank reactor under steady-state conditions resulting in a growth yield of 0.328 ± 0.045 g VSS/g S. To properly assess biological activity in respirometric tests, abiotic assays were conducted to characterize the stripping of CO2 and sulfide. The global mass transfer coefficient for both processes was estimated. Subsequently, different respirometric tests were performed: (1) to solve the stoichiometry related to the autotrophic denitrification of sulfide using either nitrate or nitrite as electron acceptors, (2) to evaluate the inhibition caused by nitrite and sulfide on sulfide oxidation, and (3) to propose, calibrate, and validate a kinetic model considering both electron acceptors in the overall anoxic biodesulfurization process. The kinetic model considered a Haldane-type equation to describe sulfide and nitrite inhibitions, a non-competitive inhibition to reflect the effect of sulfide on the elemental sulfur oxidation besides single-step denitrification since no nitrite was produced during the biological assays.

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).

Strategies for pH control in a biofilter packed with sugarcane bagasse for hydrogen sulfide removal.

The biological removal of hydrogen sulfide at low concentration (<120 ppmv) was studied in a laboratory-scale biofilter packed with sugarcane bagasse and inoculated with a sulfur-oxidizing bacterial consortium isolated from activated sludge from a wastewater treatment plant (WWTP). Inlet loads from 1.31 to 20.2 g Sm(-3) h(-1) were supplied to the biofilter, and empty bed residence times (EBRTs) of 30, 20 and 10 s were tested. In all cases, the removal efficiency was greater than 99%. Two methods for the pH control were tested: increasing the phosphate buffer capacity of the mineral medium (method I), and a new method, which involves the addition of solid CaCO(3) to the bagasse at the upper inlet of the biofilter (method II). For method I, pH increased gradually along the bed (from the bottom to the top), from a constant value of 3.0 to 7.0. For method II, pH was constant (2.4 ± 0.8) along the bed, and then a steep increase of pH was observed at the top to 7.1. We suggest the use of CaCO(3) instead of phosphate buffer because the former is less expensive, it is a simple method and the results obtained with the two methods are similar.

Development and validation of a sampling and analysis method to determine biogenic sulfur in a desulfurization bioreactor by gas chromatography coupled with a pulsed flame photometric detector (GC-PFPD).

Suspended biomass bioreactors can be operated to remove H2S from biogas under anoxic conditions and produce elemental sulfur, the commercial value of which has been demonstrated. In the present paper, a novel methodology comprising the optimization of a determination method performed in a gas chromatograph equipped with a pulsed flame photometric detector (GC-PFPD), combined with a simple preparation based on filtration and extraction with toluene, is proposed. The injector temperature and carrier gas flow rate (QHe) values were optimized using a response surface methodology based on a face-centred composite central design. This optimization revealed that the optimum conditions were an injector temperature and carrier gas flow rate of 222 °C and 7 mL min-1, respectively. The chromatographic method shows an analysis time of 48 min, a detection limit of more than 5.9 mg L-1, a relative standard deviation of less than 3.71%, and a sulfur recovery percentage of more than 98%. These values provide excellent linearity and a reasonable concentration range (10-200 mg L-1). Finally, a measurement error of 4.45% was obtained when using the present method in a selectivity test.

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 potential 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 engineered 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.

Integration of a nitrification bioreactor and an anoxic biotrickling filter for simultaneous ammonium-rich water treatment and biogas desulfurization.

A preliminary assessment has been carried out on the integration of an anoxic biotrickling filter and a nitrification bioreactor for the simultaneous treatment of ammonium-rich water and H2S contained in a biogas stream. The nutrient consumption in the biotrickling filter was as follows (mol-1 NO3--N): 6.3·10-4 ± 1.2·10-4 mol PO43--P, 0.04 ± 0.05 mol NH4+-N and 0.04 ± 0.03 mol K+-K. Furthermore, it was possible to supply a mixture of biogenic NO3- and NO2- into the biotrickling filter from the nitrification bioreactor to obtain a maximum elimination capacity of 152 gH2S-S m-3 h-1. The equivalence between the two compounds was 1 mol NO3--N equal to 1.6 mol NO2--N. The biotrickling filter was also operated under a stepped variable inlet load (30-100 gH2S-S m-3 h-1) and outlet H2S concentrations of less than 150 ppmV were obtained. It was also possible to maintain the outlet H2S concentration close to 15 ppmV with a feedback controller by manipulating the feed flow (in the nitrification bioreactor). Two stepped variable inlet loads were tested (60-111 and 16-102 gH2S-S m-3 h-1) under this type of control. The implementation of feedback control could enable the exploitation of biogas in a fuel cell, since the H2S concentrations were 15.1 ± 4.3 and 15.0 ± 3.4 ppmV. Finally, the anoxic biotrickling filter experienced partial denitrification and this implied a loss of the desulfurization effectiveness related to SO42- production.

Anoxic biogas biodesulfurization promoting elemental sulfur production in a Continuous Stirred Tank Bioreactor.

Biological desulfurization of biogas has been extensively studied using biotrickling filters (BTFs). However, the accumulation of elemental sulfur (S°) on the packing material limits the use of this technology. To overcome this issue, the use of a continuous stirred tank bioreactor (CSTBR) under anoxic conditions for biogas desulfurization and S° production is proposed in the present study. The effect of the main parameters (stirring speed, N/S molar ratio, hydraulic residence time (HRT) and gas residence time (GRT)) on the bioreactor performance was studied. Under an inlet load (IL) of 100 g S-H2S m-3 h-1 and a GRT of 119 s, the CSTBR optimal operating conditions were 60 rpm, N/S molar ratio of 1.1 and a HRT of 42 h, in which a removal efficiency (RE) and S° production of 98.6 ± 0.4 % and 88 % were obtained, respectively. Under a GRT of 41 s and an IL of 232 g S-H2S m-3 h-1 the maximum elimination capacity (EC) of 166.0 ± 7.2 g S-H2S m-3 h-1 (RE = 71.7 ± 3.1 %) was obtained. A proportional-integral feedback control strategy was successfully applied to the bioreactor operated under a stepped variable IL.

Characterization of eubacterial communities by Denaturing Gradient Gel Electrophoresis (DGGE) and Next Generation Sequencing (NGS) in a desulfurization biotrickling filter using progressive changes of nitrate and nitrite as final electron acceptors.

Anoxic biotrickling filters (BTFs) represent a technology with high H2S elimination capacity and removal efficiencies widely studied for biogas desulfurization. Three changes in the final electron acceptors were made using nitrate and nitrite during an operating period of 520 days. The stability and performance of the anoxic BTF were maintained when a significant perturbation was applied to the system that involved the progressive change of nitrate to nitrite and vice versa. Here the impact of electron acceptor changes on the microbial community was characterized by denaturing gel gradient electrophoresis (DGGE) and next generation sequencing (NGS). Both platforms revealed that the community underwent changes during the perturbations but was resilient because the removal capacity did not significantly change. Proteobacteria and Bacteroidetes were the main Phyla and Sulfurimonas and Thiobacillus the main nitrate-reducing sulfide-oxidizing bacteria (NR-SOB) genera involved in the biodesulfurization process.

Metabolic engineering for the optimization of hydrogen production in Escherichia coli: A review.

Hydrogen is a potential sustainable energy source and it could become an alternative to fossil fuel combustion, thus helping to reduce greenhouse gas emissions. The biological production of hydrogen, instead of its chemical synthesis, is a promising possibility since this process requires less energy and is more sustainable and eco-friendly. Several microorganisms have been used for this purpose, but Escherichia coli is one of the most widely used in this field. The literature in this area has increased exponentially in the last 10 years and several strategies have been reported in an effort to improve hydrogen production. In this work, the stay of the art of hydrogen biosynthesis by E. coli and metabolic engineering strategies to enhance hydrogen production are reviewed. This work includes a discussion about the hydrogenase complexes responsible for the hydrogen synthesis in this microorganism and the central carbon metabolism pathways connected to this process. The main metabolic engineering strategies applied are discussed, including heterologous gene expression, adaptive evolution and metabolic and protein engineering. On the other hand, culture conditions, including the use of carbon sources such as glycerol, glucose or organic wastes, have also been considered. Yields and productivities of the most relevant engineered strains reported using several carbon sources are also compared.

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.

Benzohydroxamic acids, such as DIBOA (2,4-dihydroxy-2 H)-1,4-benzoxazin-3(4 H)-one), are plant products that exhibit interesting herbicidal, fungicidal and bactericidal properties. A feasible alternative to their purification from natural sources is the synthesis of analogous compounds such as D-DIBOA (2-deoxy-DIBOA) and their chlorinated derivatives. Their chemical synthesis has been simplified into two steps. However, the second step is an exothermic reaction and involves hydrogen release, which makes this methodology expensive and difficult to scale up. The study reported here concerns the possibility of producing chlorobenzoxazinones by a whole-cell biocatalytic process using the ability of the engineered E. coli nfsB-/pBAD-NfsB to catalyse the synthesis of 6-Cl-D-DIBOA and 8-Cl-D-DIBOA from their respective precursors (PCs). The results show that this strain is able to grow in media that contain these compounds and to produce the target molecules with 59.3% and 46.7% biotransformation yields, respectively. Moreover, the strain is capable of processing non-purified PCs from the first chemical step to give similar yields to those obtained from the purified PCs. The kinetics of the reaction in vitro with purified recombinant NfsB nitroreductase were studied to characterise the catalysis further and evaluate the effects that several components of the non-purified PCs have on the process. The results revealed that the kinetics are that of an allosteric enzyme. The inhibitory effect of the substrate of the first step of the chemical synthesis, which is present in some non-purified PCs, was also demonstrated.