Alfredo Martinez: Escherichia coli (E. coli) as Microbial Cell Factory for the Biorefinery Concept
After this Alfredo Martinez from the Institute of Biotechnology at UNAM in Mexico (Department of Cell engineering and Biocatalysis) introduced the participants in the topic of Escherichia coli as Microbial Cell Factory for the Biorefinery Concept. Mr Martinez started with an introduction about his working field: cell engineering, biocatalysis and fermentative pathways for the production of biofuels and biochemicals. He conducts metabolic engineering, synthetic biology and bioprocess development with E. coli for biofuels (fuel ethanol, butanol and long chain alcohols), and biopolymer precursors production (lactate, butyrate, and R-3-hydroxybutyrate), as well as physiological studies with oleaginous microalgae under heterotrophic conditions. Mr Martinez then gave some facts about E. coli, including that 33% of all therapeutic proteins for human use are currently produced using E. coli in industrial fermenters.
Regarding the challenge of biofuels Mr Martinez then showed how to generate ethanol (agro-fuels) and (agro-) chemicals from lignocellulose with the use of E. coli. The main objective of these projects is to design microorganisms and processes to transform all sugars contained in lignocellulose (cellulose and hemicellulose: pentoses, hexoses, and disaccharides) into ethanol or other biochemicals, mainly organic acids. The potential primary fermentation products that can be obtained using E. coli include D-lactate, acetate, pyruvate, succinate, hydrogen and ethanol.
Mr Martinez showed then that the price for the produced lactic acid is around 1 US$/kg meanwhile the price for PLA (poly lactic acid) is more than 4 US$/kg. However, the price for bioethanol is only 0.5 US$/L and the theoretical yields of both products from different sugars are 100% for lactic acid and only 51% for ethanol. These biochemical and price facts demonstrate the basic relevance to integrate other biochemical commodities into the production of biofuels, aiming to improve product outputs and economic feasibility in the concept of small-scale biorefineries. So, the next objective was to design microorganisms and processes to transform lignocellulose to optically pure lactates (D or L) to be used as biochemical building blocks for the production of PLA. A set of six scientific papers has been reported during the first period of the SMIBIO project, where the procedures for metabolic engineering of E. coli have been reported during the development of the ERANet-LAC cooperative project. Such papers also include results using agave bagasse, corn stover and sugarcane bagasse for the production of D-lactate with lactogenic E. coli and corn stover and corn cobs for the production of ethanol. Up to 60 g/L of D-lactate has been produced with volumetric productivities above 1 g/L/h and yields above 95% of the theoretical. These metabolically engineered strains have the ability to ferment C5 and C6 sugars found in hemicellulosic hydrolysates.
Most of the results to produce lactic acid were generated at laboratory scale. However, a fermentation scale-up procedure has been established from 0.2 liters to 11- and 110-liter fermenters. The oxygen transfer rate, at an oxygen limiting condition, was set up as a scale-up criterion to keep the same titer, yield and productivity attained at laboratory scale. Furthermore, a derivative strain has also been generated that produces only L-lactic acid; this strain has similar capacities to the one that produces only D-lactic acid.
According to Mr Martinez, the worldwide market demand for the products that can be obtained from biodegradable biopolymers, such as PLA, are in the range of the current polypropylene market: 80 million tons (2014). Specifically, for PLAs a rising demand has been detected, from 500.000 tons in 2010 the market is expected to grow to 1.000.000 tons in 2020.
Another biochemical platform that shall be developed to produce biopolymer building blocks with E. coli is succinic acid. From this molecule bio-polymers, bio-surfactants, bio-solvents, bio-detergents, bio-flavorings and bio-fragrances can be generated. A global market of more than 15 billion US$ exists for such products. Mr Martinez concluded his presentation pointing out the sufficient knowledge generated with metabolic engineered E. coli from scientific research groups around the globe. Within the framework of biorefineries, such studies include the production of ethanol, butanol, propanol, biodiesel and biofuels as well as bioplastics, biopolymers, solvents, phenolics, fatty acids, organic acids, and pigments. Unfortunately, most of these studies are reaching only the proof of concept. Hence, this knowledge has an opportunity if several metabolic engineered strains of E. coli are used for the sustainable production of energy and biofuels, as well as for the production of biochemical building blocks for biopolymer and biochemicals, with the purpose to increase the economic feasibility of small-scale biorefineries in European and Latin American countries.