AGRE-0063 High Temperature Ethanol Fermentation of Lignocellulosic Waste

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Biological Conversion : Biotechnology : ECLAIR Cluster II - Lignocellulose : Liquid Biofuels and Biogas : Process Engineering : Straw : Wood (Lignocellulose)

SUMMARY

The cost of raw materials continues to be a limiting factor in the production of bioethanol (fuel alcohol) from traditional raw materials (such as sugar and starch) which can be used as substrate for yeast-based processes. At the same time, there are large amounts of agricultural residues (such as straw) as well as agro-industrial process waste streams (such as from production of maize syrups) and liquors (from wood pulping) that are of low or negative (due to costs of current effluent disposal methods) value. Many of these possible substrates contain sugars (five-carbon) which are not fermented by conventional yeast. In many cases, the major sugar available is xylose. A number of different approaches have been taken to solve the problems (inhibition, by-product formation, low product concentrations) associated with xylose fermentation. These include genetic engineering to modify the metabolism of bacteria or common yeast and screening of organisms capable of using five carbon sugars. One such organism is Bacillus stearothermophilus which uses a wide range of sugars at temperatures of up to 75°C. A mutant strain of this (strain LLD-R) has been investigated in detail and whilst some limitations in terms of microbiology were encountered, results have led to the establishment of the company Agrol Ltd (UK) to develop and exploit the technology.

INTRODUCTION

The economics of fermentation of lignocellulosic materials to produce ethanol can be improved if both the hexose (mainly glucose) and pentose (mainly xylose) sugars derived from cellulose and hemicellulose respectively can be used. This project continued previous work using a mutant strain of the thermophilic (heat tolerant) bacteria known as Bacillus stearothermophilus. The project was led by the Centre for Biotechnology, Imperial College of Science, Technology and Medicine, London (UK) who had carried out extensive fermentation and microbiological work in the past, including that funded by other EU sources. At the time the work was carried out, the Institut Francais du Petrole (IFP) had access to potential hemicellulosic hydrolysates from their pilot plant in Soustons, France and suitable fermentation facilities in their Paris laboratories. However, staff and interests within these organisations have now changed, with further development being in the hands of a development company, as detailed on the front of this leaflet. The clear advantage of the mutant thermophile was the ability to ferment xylose, which may represent 20% of the dry weight of many agricultural residues, such as straws, and are the main component of polluting paper pulping wastes.

OBJECTIVES

A two-stage aerobic/anaerobic continuous "Closed System" fermentation had been conceived as a suitable model for a commercial process because it was, in principle, self-optimising, self-regulating and essentially non-polluting. Steps towards the fermentation of pure xylose were compared with that of sucrose in an "Open" fermentation system before testing the "Closed System" concept. Similar studies were carried out using realistic feedstocks, derived from waste hydrolysis. The possibility of using membranes for separation was explored, together with computer model studies. The main problem encountered was a deficiency in the then current ICL mutant strain. This reverted readily and the resulting wild type grew better under conditions that are optimal for ethanol production, producing lactate instead. Hence an important task was to isolate a deletion mutant of the relevant L-lactate dehydrogenase gene that would be incapable of reversion. A secondary task was to isolate mutants that might show higher ethanol productivity.

ACTIVITIES

Studies of the microbial physiology in continuous cultures showed that sucrose was utilised very rapidly and xylose only slightly less so. Ethanol yields at pH 7, 70ºC, were about 75% of theoretical but rose in non-growing cells at higher sugar concentrations, lower pH and higher temperature. To achieve optimum conditions, an anaerobic membrane cell-recycle reactor was used and proved useful even with the original strain because take-overs by revertants are infrequent when growth is minimal, as in this system. The "Open System" was tested on xylose, again progress was hampered by revertant take-overs at higher sugar concentrations, limiting the commercial relevance of the results. This also occurred when the "Closed System" itself was tested. Even so, encouraging fermentation results were obtained with dilute acid hydrolysates of corn cobs or wheatstraw, with the latter proving to be the best. The organism also utilised all of the pectin and hemicellulose sugars derived from enzymatic hydrolysis of sugar beet pulp. All these studies were hampered by the tendency of the organism to sporulate, leading to take-overs by wild type revertants that produce lactate instead of ethanol. Extensive efforts to isolate a non-reverting mutant failed. The reason was found by mapping the LLD gene region in question. This revealed that spontaneous mutants arise by intrinsic transposon insertion in the promoter region and that these revert with high frequency. Failure to obtain a non-reverting and non-sporulating strain hampered the work of partners. Nevertheless it was possible to predict the performance of a theoretical non-reverting and non-sporulating strain and to model both a "Vacuum Closed System" process and a simpler "Vacuum Fed Batch" process based on these data. Both show considerable economic potential.

COMMERCIALISATION

A development company, Agrol Ltd (UK), has been formed as detailed on the front of this Item.

PARTICIPANTS

Institut Francais du Petrole (Paris, France)

Department of Microbiology, University College (Galway, Ireland)

Centrale Recherche Laboratoire GIC, Ecole Centrale (Paris, France).