Escherichia coli and its application to biohydrogen production

"Hydrogen is an attractive energy carrier because of its high energy density, and used as a raw material in various chemical processes. Nowadays, hydrogen demand is supplied from non-renewable sources, and alternative sources are becoming mandatory. Hydrogen production by biological methods uses renewable resources as substrate and its production occurs at ambient temperature and atmospheric pressure. Thus, it is less energy intensive than the chemical and thermochemical methods used to produce hydrogen. This review is focused on fermentative hydrogen production by Escherichia coli. The hydrogen production pathway, the genetic manipulations, and expression of non-native pathways into this microorganism are reviewed. The hydrogen production using alternative substrates is a critical point to develop sustainable process by this reason the principal substrates for hydrogen production using E. coli are revised. Other strategies like two stages processes and immobilized cells are also discussed.

Maximizing hydrogen production and substrate consumption by Escherichia coli WDHL in cheese whey fermentation

"Fermentative hydrogen production is strongly affected by pH. In order to maximize hydrogen production and substrate consumption in Escherichia coli ΔhycA, ΔlacI (WDHL) cheese whey fermentation, the influence of pH control at values of 5.5, 6, and 6.5 was studied in batch stirred-tank bioreactors. From the conditions evaluated, pH 6.5 was the best condition, at which the highest cumulative hydrogen production and yield (1.78 mol H2/mol lactose) were obtained. Moreover, at this pH, all carbohydrates from the cheese whey were consumed, and a mix of ethanol and organic acids, mainly lactate, were produced from glucose, whereas galactose yielded acetate, ethanol, and succinate. Operating the reactor at pH 5.5 resulted in the highest maximum specific production rate, but smaller hydrogen yield because only glucose was metabolized and galactose was accumulated. At pH 6, not all cheese whey carbohydrates were consumed, and it was not favorable for hydrogen production. Lactose consumption and growth kinetics were not affected by the pH. The results show the importance of controlling pH to maximize hydrogen production and substrate consumption using cheese whey as substrate.

Hydrogen production by Escherichia coli ΔhycA ΔlacI using cheese whey as substrate

"This study reports a fermentative hydrogen production by Escherichia coli using cheese whey as substrate. To improve the biohydrogen production, an E. coli ΔhycA ΔlacI strain (WDHL) was constructed. The absence of hycA and lacI genes had a positive effect on the biohydrogen production. The strain produced 22% more biohydrogen in a shorter time than the wild-type (WT) strain. A Box-Behnken experimental design was used to optimize pH, temperature and substrate concentration. The optimal initial conditions for biohydrogen production by WDHL strain were pH 7.5, 37 °C and 20 g/L of cheese whey. The specific production rate was improved from 3.29 mL H2/optical density at 600 nm (OD600nm) unit-h produced by WDHL under non-optimal conditions to 5.88 mL H2/OD600nm unit-h under optimal conditions. Using optimal initial conditions, galactose can be metabolized by WDHL strain. The maximum yield obtained was 2.74 mol H2/mol lactose consumed, which is comparable with the yield reached in other hydrogen production processes with Clostridium sp. or mixed cultures.

Biological hydrogen production: trends and perspectives

"Biologically produced hydrogen (biohydrogen) is a valuable gas that is seen as a future energy carrier, since its utilization via combustion or fuel cells produces pure water. Heterotrophic fermentations for biohydrogen production are driven by a wide variety of microorganisms such as strict anaerobes, facultative anaerobes and aerobes kept under anoxic conditions. Substrates such as simple sugars, starch, cellulose, as well as diverse organic waste materials can be used for biohydrogen production. Various bioreactor types have been used and operated under batch and continuous conditions; substantial increases in hydrogen yields have been achieved through optimum design of the bioreactor and fermentation conditions. This review explores the research work carried out in fermentative hydrogen production using organic compounds as substrates. The review also presents the state of the art in novel molecular strategies to improve the hydrogen production.

Hydrogen production by Escherichia coli genetically modified

Tesis (Doctorado en Ciencias en Biología Molecular)"El hidrógeno es considerado como el acarreador energético del futuro debido a su alto contenido energético y a que sólo genera agua como subproducto. Entre los métodos de producción de este gas, los procesos biológicos son una alternativa atractiva ya que requieren menos energía y se pueden utilizar subproductos agroalimentarios como sustratos. El suero de leche es el principal subproducto de la industria quesera y por lo tanto se usó como sustrato para la producción de hidrógeno por medio de Escherichia coli. La deleción de los genes hycA y lacI en la cepa WDHL, permitió un rápido consumo de lactosa y un incremento del 22% en la producción de hidrógeno comparado con la cepa silvestre. La velocidad específica de producción de hidrógeno se incrementó 78.7 % al utilizar las condiciones óptimas de pH, temperatura y concentración de suero de leche. De acuerdo a los resultados obtenidos, el pH mostró ser unos de los factores más influyentes en la producción de hidrógeno. Al controlar el pH a 6.5 se alcanzó la mayor producción de hidrógeno y el rendimiento más alto. Lactosa, glucosa y galactosa son fuentes de carbono que se encuentran comúnmente en desechos de la industria agroalimentaria, por lo tanto la producción de hidrógeno a partir de estos sustratos también fue evaluada. El rendimiento de hidrógeno a partir de glucosa (0.19 mol de H2/ mol de glucosa) fue menor que el que se obtiene a partir de galactosa (1.15 mol de H2/ mol de galactosa). El bajo rendimiento de hidrógeno a partir de glucosa se debe a que el metabolismo se desvía hacia la producción de lactato, en comparación con la fermentación de galactosa donde se favorece la producción de formiato, que es convertido rápidamente en hidrógeno. Además se desarrolló una red neuronal artificial que predice satisfactoriamente la producción de hidrógeno a partir exclusivamente de parámetros en línea.""Due to its high energy content and because its use only result in water as subproduct, hydrogen is seen as the energetic carrier for the future. Among the methods of production of this gas, the biological processes are an attractive choice since are less energy intensive and can use agro-food by-products as substrates. The cheese whey is the main by-product of the cheese manufacturing industry, and by this reason it was used in this work for hydrogen production by Escherichia coli. The deletion of hycA and lacI genes in the strain WDHL resulted in a faster consumption of lactose and an increase of 22% in hydrogen production compared with the wild type strain. The hydrogen specific production rate was increased by 78.7% when optimized conditions of pH, temperature and cheese whey concentration. According to the results the pH is one of the most important factors is hydrogen production. Controlling the pH at 6.5 resulted in the highest hydrogen production and yield. Lactose, glucose and galactose are carbon sources commonly present in wastes of the agro-food industry, the hydrogen production from these substrates was also evaluated. The hydrogen yield from glucose (0.19 mol of H2/ mol of glucose) was lower than that from galactose (0.19 mol of H2/ mol of galactose). The low hydrogen yield from glucose is due to the large production of lactate, whereas in the galactose fermentation the formate production is increased and it is converted to hydrogen. Moreover an artificial neural network to estimate the hydrogen production using only on-line parameters was developed.

Obtención de cepas mutantes de Escherichia coli sobreproductoras de hidrógeno a partir de lactosuero

Tesis (Maestría en Ciencias en Biología Molecular)"Los problemas causados por la contaminación ambiental y la disminución de las reservas de petróleo han conducido a la búsqueda de nuevas fuentes energéticas que permitan un desarrollo sustentable. El hidrógeno es una alternativa con amplio potencial ya que se puede utilizar en celdas de combustible y solo genera calor y vapor de agua como subproducto. La producción biológica de hidrógeno (biohidrógeno) presenta algunas ventajas respecto a las formas convencionales de obtención, debido a que se lleva a cabo a temperatura ambiente, bajas presiones y se puede acoplar a la utilización de residuos orgánicos. El objetivo de este trabajo es la construcción y uso de cepas de E. coli manipuladas genéticamente para realizar la fermentación anaerobia de lactosuero (subproducto de la industria de lácteos) y obtener como producto principal hidrógeno molecular. Para obtener cepas mutantes sobreproductoras de hidrógeno se realizó la remoción de los genes hycA y tatC. El gen hycA, codifica para el regulador negativo del operón del formiato y el producto del gen tatC, tiene un papel fundamental en la vía Tat del transporte de formiato deshidrogenasas e hidrogenasas que compiten por los electrones que darían origen a hidrógeno molecular. También se obtuvo una cepa doble mutante (delta)hycA, (delta) lacI, éste último gen codifica para la proteína represora del operón de lactosa, por lo tanto, en una mutante que carece de este gen, la expresión del operón lac se vuelve constitutiva. La ausencia de los genes hycA y lacI, permitió que se incrementara la cantidad y la velocidad de producción de hidrógeno a partir de lactosuero.""Problems caused by enviromental pollution and the decrease of the fossil fuels reserves have led to the search of new sustainable energy sources. Hydrogen is a promising option since its utilization in fuel cells produces pure water and energy. Biological hydrogen production (biohydrogen) is a more advantageus alternative than the conventional methods. This is because the biological processes are carried out at ambient temperature and pressure, and organic residues can be used for hydrogen production. The aim of this work is the construction of E. coli mutant strains that can use the cheese whey (by-product of the cheese production) as sustrate to produce hydrogen. In order to obtain overproducer mutant strains, we knocked-out the hycA and tatC genes. The hycA gene encodes for the repressor of the formate operon. The tatC gene encodes for a protein that is involved in the transport of respiratory hydrogenases and formate dehydrogenases by Tat translocation pathway. We also constructed a double mutant strain that had hycA and lacI genes deleted. The lacI gene encodes the lac operon repressor and its deletion leads to the constitutive expression of the lac operon. Both the hydrogen production and rate were increased in the mutant strain ΔhycA ΔlacI using cheese whey.

Fermentation of lactose and its constituent sugars by Escherichia coli WDHL: impact on hydrogen production

"Fermentations of lactose, glucose and galactose using Escherichia coli WDHL, a hydrogen over producer strain, were performed. With glucose as substrate pyruvate was mainly routed to the lactate pathway, resulting in hydrogen production and yield of 1037 mL and 0.30 mol H2/mol of glucose, respectively. When galactose was the substrate, the pyruvate formate lyase pathway was the main route for pyruvate and a fermentation yield of 1.12 mol H2/mol of galactose and a hydrogen production of 2080 mL were obtained. The fermentation of lactose or glucose plus galactose showed a similar yield of 1.02 mol H2/mol of hexose consumed. This work clearly demonstrated that the kinetics of hydrogen and metabolites production as well as the hydrogen yield were affected by the type of sugar used as substrate as reflected by the deviations from the metabolic hydrogen-production pathway.

Estimation of hydrogen production in genetically modified E. coli fermentations using an artificial neural network

"Biological hydrogen production is an active research area due to the importance of this gas as an energy carrier and the advantages of using biological systems to produce it. A cheap and practical on-line hydrogen determination is desired in those processes. In this study, an artificial neural network (ANN) was developed to estimate the hydrogen production in fermentative processes. A back propagation neural network (BPNN) of one hidden layer with 12 nodes was selected. The BPNN training was done using the conjugated gradient algorithm and on-line measurements of dissolved CO2, pH and oxidation-reduction potential during the fermentations of cheese whey by Escherichia coli ΔhycA ΔlacI (WDHL) strain with or without pH control. The correlation coefficient between the hydrogen production determined by gas chromatography and the hydrogen production estimated by the BPNN was 0.955. Results showed that the BPNN successfully estimated the hydrogen production using only on-line parameters in genetically modified E. coli fermentations either with or without pH control. This approach could be used for other hydrogen production systems.

Analysis of the Propionate Metabolism in <em>Bacillus subtilis</em> during 3-Indolacetic Production

The genera Bacillus belongs to the group of microorganisms that are known as plant growth-promoting bacteria, their metabolism has evolved to produce molecules that benefit the growth of the plant, and the production of 3-indole acetic acid (IAA) is part of its secondary metabolism. In this work, Bacillus subtilis was cultivated in a bioreactor to produce IAA using propionate and glucose as carbon sources in an M9-modified media; in both cases, tryptophan was added as a co-substrate. The yield of IAA using propionate is 17% higher compared to glucose. After 48 h of cultivation, the final concentration was 310 mg IAA/L using propionate and 230 mg IAA/L using glucose, with a concentration of 500 mg Trp/L. To gain more insight into propionate metabolism and its advantages, the genome-scale metabolic model of B. subtilis (iBSU 1147) and computational analysis were used to calculate flux distribution and evaluate the metabolic capabilities to produce IAA using propionate. The metabolic fluxes demonstrate that propionate uptake favors the production of precursors needed for the synthesis of the hormone, and the sensitivity analysis shows that the control of a specific growth rate has a positive impact on the production of IAA