Lisbeth Olsson

Fuel ethanol production from lignocellulose: a challenge for metabolic engineering and process integration

Abstract. With industrial development growing rapidly, there is a need for environmentally sustainable energy sources. Bioethanol (ethanol from biomass) is an attractive, sustainable energy source to fuel transportation. Based on the premise that fuel bioethanol can contribute to a cleaner environment and with the implementation of environmental protection laws in many countries, demand for this fuel is increasing. Efficient ethanol production processes and cheap substrates are needed. Current ethanol production processes using crops such as sugar cane and corn are well-established; however, utilization of a cheaper substrate such as lignocellulose could make bioethanol more competitive with fossil fuel. The processing and utilization of this substrate is complex, differing in many aspects from crop-based ethanol production. One important requirement is an efficient microorganism able to ferment a variety of sugars (pentoses, and hexoses) as well as to tolerate stress conditions. Through metabolic engineering, bacterial and yeast strains have been constructed which feature traits that are advantageous for ethanol production using lignocellulose sugars. After several rounds of modification/evaluation/modification, three main microbial platforms, Saccharomyces cerevisiae, Zymomonas mobilis, and Escherichia coli, have emerged and they have performed well in pilot studies. While there are ongoing efforts to further enhance their properties, improvement of the fermentation process is just one of several factorsthat needs to be fully optimized and integrated to generate a competitive lignocellulose ethanol plant.

Fermentation of lignocellulosic hydrolysates for ethanol production.

Ethanol production from lignocellulosic hydrolysates in an economically feasible process requires microorganisms that produce ethanol with a high yield from all sugars present (hexoses as well as pentoses) and have a high ethanol productivity in lignocellulosic hydrolysates, i.e., can withstand potential inhibitors. Different fermentation organisms among bacteria, yeasts, and fungi (natural as well as recombinant) are reviewed with emphasis on their performance in lignocellulosic hydrolysates. Depending on the type of lignocellulosic hydrolysate, the composition of inhibitors will differ and their influence on the microorganisms and the fermentation performance will consequently vary. The inhibition may be partly overcome by the removal of inhibitors, i.e., detoxification. Microbial constraints on parameters such as pH, temperature, and nutrient supplementation are discussed in relation to their implication on the process economy. Not only are the properties of the microorganism of importance in the process, but also the choice of fermentation strategies such as batch culture, continuous culture with cell recycling and in situ ethanol removal. For the realization of the ethanol production from lignocellulosic materials, the fermentation step has to be integrated with the rest of the process. These aspects are also discussed.

Metabolic Engineering of Saccharomyces cerevisiae

SUMMARY Comprehensive knowledge regarding Saccharomyces cerevisiae has accumulated over time, and today S. cerevisiae serves as a widley used biotechnological production organism as well as a eukaryotic model system. The high transformation efficiency, in addition to the availability of the complete yeast genome sequence, has facilitated genetic manipulation of this microorganism, and new approaches are constantly being taken to metabolicially engineer this organism in order to suit specific needs. In this paper, strategies and concepts for metabolic engineering are discussed and several examples based upon selected studies involving S. cerevisiae are reviewed. The many different studies of metabolic engineering using this organism illustrate all the categories of this multidisciplinary field: extension of substrate range, improvements of producitivity and yield, elimination of byproduct formation, improvement of process performance, improvements of cellular properties, and extension of product range including heterologous protein production.

Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae

Respiratory metabolism plays an important role in energy production in the form of ATP in all aerobically growing cells. However, a limitation in respiratory capacity results in overflow metabolism, leading to the formation of byproducts, a phenomenon known as “overflow metabolism” or “the Crabtree effect.” The yeast Saccharomyces cerevisiae has served as an important model organism for studying the Crabtree effect. When subjected to increasing glycolytic fluxes under aerobic conditions, there is a threshold value of the glucose uptake rate at which the metabolism shifts from purely respiratory to mixed respiratory and fermentative. It is well known that glucose repression of respiratory pathways occurs at high glycolytic fluxes, resulting in a decrease in respiratory capacity. Despite many years of detailed studies on this subject, it is not known whether the onset of the Crabtree effect is due to limited respiratory capacity or is caused by glucose-mediated repression of respiration. When respiration in S. cerevisiae was increased by introducing a heterologous alternative oxidase, we observed reduced aerobic ethanol formation. In contrast, increasing nonrespiratory NADH oxidation by overexpression of a water-forming NADH oxidase reduced aerobic glycerol formation. The metabolic response to elevated alternative oxidase occurred predominantly in the mitochondria, whereas NADH oxidase affected genes that catalyze cytosolic reactions. Moreover, NADH oxidase restored the deficiency of cytosolic NADH dehydrogenases in S. cerevisiae. These results indicate that NADH oxidase localizes in the cytosol, whereas alternative oxidase is directed to the mitochondria.

Comparison of SHF and SSF processes from steam‐exploded wheat straw for ethanol production by xylose‐fermenting and robust glucose‐fermenting Saccharomyces cerevisiae strains

In this study, bioethanol production from steam‐exploded wheat straw using different process configurations was evaluated using two Saccharomyces cerevisiae strains, F12 and Red Star. The strain F12 has been engineerically modified to allow xylose consumption as cereal straw contain considerable amounts of pentoses. Red Star is a robust hexose‐fermenting strain used for industrial fuel ethanol fermentations and it was used for comparative purposes. The highest ethanol concentration, 23.7 g/L, was reached using the whole slurry (10%, w/v) and the recombinant strain (F12) in an SSF process, it showed an ethanol yield on consumed sugars of 0.43 g/g and a volumetric ethanol productivity of 0.7 g/L h for the first 3 h. Ethanol concentrations obtained in SSF processes were in all cases higher than those from SHF at the same conditions. Furthermore, using the whole slurry, final ethanol concentration was improved in all tests due to the increase of potential fermentable sugars in the fermentation broth. Inhibitory compounds present in the pretreated wheat straw caused a significantly negative effect on the fermentation rate. However, it was found that the inhibitors furfural and HMF were completely metabolized by the yeast during SSF by metabolic redox reactions. An often encountered problem during xylose fermentation is considerable xylitol production that occurs due to metabolic redox imbalance. However, in our work this redox imbalance was counteracted by the detoxification reactions and no xylitol was produced. Biotechnol. Bioeng. 2008;100: 1122–1131.

Fermentative performance of bacteria and yeasts in lignocellulose hydrolysates

The sugar consumption rates and the product formation of yeasts (Saccharomyces cidri NCYC 775, S. cerevisiae NCYC 1047, S. cerevisiae ATCC 4132) and bacteria (Lactobacillus brevis DSM 20054, Lactococcus lactis ssp. lactis ATCC 19435, Escherichia coli ATCC 11303, Zymomonas mobilis ATCC 31821) were investigated in spent sulphite liquor and an enzymatic hydrolysate of steam-pretreated Salix caprea at different pH values in order to elucidate the suitability of the organisms with respect to future genetic engineering approaches. The possible inhibitory action of the two substrates on the investigated microorganisms was also considered. S. cerevisiae emerged as one of the better candidates, owing to its fast sugar consumption rate and efficient ethanol production.

Increasing galactose consumption by Saccharomyces cerevisiae through metabolic engineering of the GAL gene regulatory network.

Increasing the flux through central carbon metabolism is difficult because of rigidity in regulatory structures, at both the genetic and the enzymatic levels. Here we describe metabolic engineering of a regulatory network to obtain a balanced increase in the activity of all the enzymes in the pathway, and ultimately, increasing metabolic flux through the pathway of interest. By manipulating the GAL gene regulatory network of Saccharomyces cerevisiae, which is a tightly regulated system, we produced prototroph mutant strains, which increased the flux through the galactose utilization pathway by eliminating three known negative regulators of the GAL system: Gal6, Gal80, and Mig1. This led to a 41% increase in flux through the galactose utilization pathway compared with the wild-type strain. This is of significant interest within the field of biotechnology since galactose is present in many industrial media. The improved galactose consumption of the gal mutants did not favor biomass formation, but rather caused excessive respiro-fermentative metabolism, with the ethanol production rate increasing linearly with glycolytic flux.

Glucose control in Saccharomyces cerevisiae : the role of MIG1 in metabolic functions

Industrial cultivation media, such as molasses, wort, agricultural waste and lignocellulose hydrolysates, contain a mixture of metabolizable carbohydrates. These carbohydrates are taken up by cells in a certain order with intermittent lag phases due to a set of mechanisms controlled by glucose, referred to hereafter as glucose control. Thus, the presence or uptake of glucose has a negative impact upon the metabolism of other sugars. Glucose repression reduces the transcription rate of repressible genes, and is the most thoroughly investigated mechanism of glucose control (Fig. 1).

Lignocellulosic ethanol production at high-gravity: challenges and perspectives

In brewing and ethanol-based biofuel industries, high-gravity fermentation produces 10-15% (v/v) ethanol, resulting in improved overall productivity, reduced capital cost, and reduced energy input compared to processing at normal gravity. High-gravity technology ensures a successful implementation of cellulose to ethanol conversion as a cost-competitive process. Implementation of such technologies is possible if all process steps can be performed at high biomass concentrations. This review focuses on challenges and technological efforts in processing at high-gravity conditions and how these conditions influence the physiology and metabolism of fermenting microorganisms, the action of enzymes, and other process-related factors. Lignocellulosic materials add challenges compared to implemented processes due to high inhibitors content and the physical properties of these materials at high gravity.

Influence of the carbon source on production of cellulases, hemicellulases and pectinases by Trichoderma reesei Rut C-30

The growth and enzyme production by Trichoderma reesei Rut C-30 using different lignocellulosic materials as carbon source were investigated. Cellulose, sugar beet pulp and alkaline extracted sugar beet pulp (resulting in partial removal of hemicellulose, lignin and pectin) or mixtures thereof were used as carbon sources. It was found that endoglucanase and endoxylanse activities were produced throughout the cultivations, whereas α-arabinosidase was induced late during the cultivation. The highest amounts of endoglucanse, could be measured when T. reesei Rut C-30 was grown on cellulose or cellulose containing mixtures. Endoxylanase was produced on all substrates, but the presence of cellulose was favourable for the production. Polygalacturonase activity could be measured at high varying levels throughout the cultivations, except during growth on cellulose. The varying levels might originate from the production of different isoenzymes of polygalacturonase.