Final Report Summary - NILE (New Improvements for Ligno-cellulosic Ethanol)
The purpose of the NILE project was to develop cost effective production of clean bioethanol from lignocellulosic biomass (LCB) enabling its use as a transport biofuel. The project investigated and evaluated innovative systems and technologies, which were applied in a fully integrated pilot plant. Its integrated structure allowed for the research outcomes to be connected to the industrial process, evaluated, assessed and disseminated, thus increasing European competiveness.
The objectives of the NILE concerned the following issues:
1. decrease of the cost of enzymatic hydrolysis of lignocellulose through the use of new enzyme systems;
2. removal of current intrinsic limitations in the conversion of fermentable sugars to ethanol;
3. validation of the engineered enzyme systems and yeast strains in the model pilot plant using softwood as a model feedstock and wheat straw as an alternative feedstock;
4. analysis of socioeconomic and environmental impacts of the use of bioethanol produced from LCB based on the data obtained;
5. dissemination of the acquired knowledge and training of target groups.
The NILE project aimed to reduce by five times the cost of enzymatic hydrolysis, which currently accounts for almost half of the cost of the complete ethanol production process. The project focused to the fungi which are used for producing cellulaces and tried to improve their performance, through extensive analysis of the produced enzymes, attempts to engineer some of them and test of other organisms' cellulaces. The fungi generated enzymes were defined and enzymes that were not present in the process but could act as helpers were searched and tested for their suitability. In addition, enzymes that could represent a limiting step in the reaction were identified. The analysis performed showed that the enzyme CBH2 limits the hydrolysis rate. This led to the engineering of two CBH2 variants, whose improved performance was verified in the laboratory. Moreover, new artificial cellulaces were built, composed by two smaller enzymes which could act synergistically, and so faster, than if they acted independently in two separate reactions. Fungi with the capacity to secrete larger enzyme quantities than a reference strain were developed through mutagenesis and innovative screening methods.
The extensive research on hydrolysis produced important outcomes, which were presented in articles and resulted in patent filings. In terms of cost reduction though the maximum predicted potential loading reduction is lower than the initial target set. It appears that the difficulty of enhancing hydrolysis reaction was underestimated and that improvements with helper enzymes cannot be easily obtained in all cases.
Another target of the NILE project was to develop yeast strains which could result in improved fermentation of all sugars in lignocellulose hydrolysates. For fermentation to occur pentoses were brought into the cell, so that enzymes could convert them to ethanol. Codon optimisation carried out on genes during the project significantly improved the reaction. In addition, site-directed mutagenesis was used to create a new enzyme of high performance. Evolutionary adaptation, achieved by letting the strain grow in sequential batches in media containing inhibitors, resulted in clones of improved behaviour.
NILE also developed strategies for the fermentation of all sugars in non-detoxified lignocellulose hydrolysates. The challenges of the process were to obtain high conversion in the enzymatic hydrolysis, high conversion of the pentose sugars and robustness of the yeast towards the inhibitors of the hydrolysis. The selected principal approach was simultaneous saccharification and fermentation (SSF). The attempt resulted in significant increases in the ethanol yield and the conversion of xylose for both wheat straw and spruce feedstock. In order to reach the target value of the ethanol yield in the fermentation, a more complete xylose conversion was needed for pretreated wheat straw, whereas for spruce it was necessary to have a more complete hydrolysis.
Two pilot plants were employed in order to investigate the new yeast and enzymes both in laboratory and near industrial conditions, using either softwood or wheat straw as raw materials. For the overall costs to be reduced it was critical to determine process bottlenecks and investigate different scenarios of integration. In addition, combustion and granulation properties of the lignin hydrolysis residues (LHR) were evaluated, since lignin is important in determining the overall economic viability of the process due to its energy content. Lignin application in non-energy end uses was also addressed. Various alternatives were examined in order to identify cost. Flow sheeting programs were used to allow for sensitivity analyses of the various process steps.
The enzyme mixes developed during the project were found equally as efficient for hydrolysis as today commercial enzymes, thus by further adapting the pre-treatment conditions efficiency could be further increased. From laboratory evaluation using wheat straw as row material, hydrolysis turned out being equally efficient to that of the commercial reference, while a significant increase of the ethanol yield was achieved during fermentation. LHR material appeared to have bulk densities and mechanical properties comparable to those of commercial solid biofuel; however, supplementary attention should be given on by-products formed during combustion. LHR also appeared to have the potential to serve as a sustainable alternative for various commercial uses.
The likely social and economic impact of cellulosic ethanol production was examined through adequate assessments, with the following aspects being taken into consideration:
1. the supply-chain cost modelling. A spreadsheet based tool was developed for this purpose, which revealed that the relationship between the cost of feedstock and the value obtained for ethanol is important for the overall commercial viability determination. The results of the modelling suggested that ethanol produced from softwood could be cost competitive with gasoline; however, production from straw would generally be less competitive, owing to the smaller proportion of easily fermentable pentose sugars.
2. the elaboration of a life cycle analysis (LCA), which aimed to define the environmental impacts and to identify the most favourable environmental scenario.
3. the evaluation of stakeholders and barriers to development, caused by the fact that, with the exception of Sweden, ethanol is a new fuel for the European market. The study focused primarily on the non technical barriers, with areas such as political impediments, market breakthrough barriers, legislation, regulations and standards, awareness and education and other practical problems being considered. The report on stakeholders included all different types of organisations, from research institutes to private companies.
4. the determination of the policy for the technology to be commercially exploited. Work on this field considered the path to market for cellulosic ethanol, the contribution of policy to the successful technology development and the potential contribution of cellulosic ethanol to discrete policy objectives. Best practice guidelines for demonstration and scale-up of the technology were revealed.
5. the examination of a case study for the integration of the production of lignocellulosic ethanol into an existing paper mill, which resulted in conclusions for all the different processes of the technology application.
European targets related to reducing emissions and dependence on petrol, to increasing the application of renewable energy sources and to improving the energy efficiency, along with the development of new engine platforms by car manufacturers, turn bioethanol into a promising automotive fuel.
The NILE project team experimented using both high and low ethanol content blends. Performance and emissions of the test vehicle have been measured using both first and second generation process bioethanol in order to demonstrate that engine calibration could not change between the two fuels. The experiment aimed to evaluate bioethanol with regard to turbocharged and downsized engines and proved that the optimisation of the engine compression ratio could be done based on bioethanol properties and, when operating with gasoline, knocking could be avoided, thus reducing the boosting pressure at the intake.
High blends caused reduced deposits on the cold parts of the engine in comparison to gasoline, whereas this was not the case for low blends. On the other hand, deposits on hottest parts such as piston tops and cylinder head showed a linear reduction with increase in the ethanol content. Under full load conditions the combination of the high octane number with the high latent heat of vaporisation allowed for a more efficient engine regulation, with even a slight increase in maximum performance. Because of the possible optimisation of the spark advance timing due to the knocking resistance of bioethanol, high ethanol content blends proved to be beneficial in terms of energy efficiency.
The results of the NILE project could be summarised as follows:
1. among the various approaches aiming to improve the enzymatic hydrolysis of lignocellulosic raw materials, some proved to be successful whereas others require further investigation;
2. addition of helper enzymes had no significant effect in diluted acid conditions;
3. the potential improvements of enzymes were greatly dependent upon the efficacy of the pre-treatment, whose up-scaling proved to be of major importance;
4. four new industrial strains were successfully developed and it was shown that an adapted fermentation strategy was necessary for the full exploitation of those strains;
5. optimised supply chains, integrated facilities and a favourable market were required for the profitable technology application;
6. from tests carried out related to the use of ethanol in downsized engines and engines coupled with turbocharger it occurred that, apart from acetaldehyde emissions, ethanol was beneficial. In addition, cellulose-based ethanol could, with normal distillation and dewatering, meet the fuel sector specifications.
The objectives of the NILE concerned the following issues:
1. decrease of the cost of enzymatic hydrolysis of lignocellulose through the use of new enzyme systems;
2. removal of current intrinsic limitations in the conversion of fermentable sugars to ethanol;
3. validation of the engineered enzyme systems and yeast strains in the model pilot plant using softwood as a model feedstock and wheat straw as an alternative feedstock;
4. analysis of socioeconomic and environmental impacts of the use of bioethanol produced from LCB based on the data obtained;
5. dissemination of the acquired knowledge and training of target groups.
The NILE project aimed to reduce by five times the cost of enzymatic hydrolysis, which currently accounts for almost half of the cost of the complete ethanol production process. The project focused to the fungi which are used for producing cellulaces and tried to improve their performance, through extensive analysis of the produced enzymes, attempts to engineer some of them and test of other organisms' cellulaces. The fungi generated enzymes were defined and enzymes that were not present in the process but could act as helpers were searched and tested for their suitability. In addition, enzymes that could represent a limiting step in the reaction were identified. The analysis performed showed that the enzyme CBH2 limits the hydrolysis rate. This led to the engineering of two CBH2 variants, whose improved performance was verified in the laboratory. Moreover, new artificial cellulaces were built, composed by two smaller enzymes which could act synergistically, and so faster, than if they acted independently in two separate reactions. Fungi with the capacity to secrete larger enzyme quantities than a reference strain were developed through mutagenesis and innovative screening methods.
The extensive research on hydrolysis produced important outcomes, which were presented in articles and resulted in patent filings. In terms of cost reduction though the maximum predicted potential loading reduction is lower than the initial target set. It appears that the difficulty of enhancing hydrolysis reaction was underestimated and that improvements with helper enzymes cannot be easily obtained in all cases.
Another target of the NILE project was to develop yeast strains which could result in improved fermentation of all sugars in lignocellulose hydrolysates. For fermentation to occur pentoses were brought into the cell, so that enzymes could convert them to ethanol. Codon optimisation carried out on genes during the project significantly improved the reaction. In addition, site-directed mutagenesis was used to create a new enzyme of high performance. Evolutionary adaptation, achieved by letting the strain grow in sequential batches in media containing inhibitors, resulted in clones of improved behaviour.
NILE also developed strategies for the fermentation of all sugars in non-detoxified lignocellulose hydrolysates. The challenges of the process were to obtain high conversion in the enzymatic hydrolysis, high conversion of the pentose sugars and robustness of the yeast towards the inhibitors of the hydrolysis. The selected principal approach was simultaneous saccharification and fermentation (SSF). The attempt resulted in significant increases in the ethanol yield and the conversion of xylose for both wheat straw and spruce feedstock. In order to reach the target value of the ethanol yield in the fermentation, a more complete xylose conversion was needed for pretreated wheat straw, whereas for spruce it was necessary to have a more complete hydrolysis.
Two pilot plants were employed in order to investigate the new yeast and enzymes both in laboratory and near industrial conditions, using either softwood or wheat straw as raw materials. For the overall costs to be reduced it was critical to determine process bottlenecks and investigate different scenarios of integration. In addition, combustion and granulation properties of the lignin hydrolysis residues (LHR) were evaluated, since lignin is important in determining the overall economic viability of the process due to its energy content. Lignin application in non-energy end uses was also addressed. Various alternatives were examined in order to identify cost. Flow sheeting programs were used to allow for sensitivity analyses of the various process steps.
The enzyme mixes developed during the project were found equally as efficient for hydrolysis as today commercial enzymes, thus by further adapting the pre-treatment conditions efficiency could be further increased. From laboratory evaluation using wheat straw as row material, hydrolysis turned out being equally efficient to that of the commercial reference, while a significant increase of the ethanol yield was achieved during fermentation. LHR material appeared to have bulk densities and mechanical properties comparable to those of commercial solid biofuel; however, supplementary attention should be given on by-products formed during combustion. LHR also appeared to have the potential to serve as a sustainable alternative for various commercial uses.
The likely social and economic impact of cellulosic ethanol production was examined through adequate assessments, with the following aspects being taken into consideration:
1. the supply-chain cost modelling. A spreadsheet based tool was developed for this purpose, which revealed that the relationship between the cost of feedstock and the value obtained for ethanol is important for the overall commercial viability determination. The results of the modelling suggested that ethanol produced from softwood could be cost competitive with gasoline; however, production from straw would generally be less competitive, owing to the smaller proportion of easily fermentable pentose sugars.
2. the elaboration of a life cycle analysis (LCA), which aimed to define the environmental impacts and to identify the most favourable environmental scenario.
3. the evaluation of stakeholders and barriers to development, caused by the fact that, with the exception of Sweden, ethanol is a new fuel for the European market. The study focused primarily on the non technical barriers, with areas such as political impediments, market breakthrough barriers, legislation, regulations and standards, awareness and education and other practical problems being considered. The report on stakeholders included all different types of organisations, from research institutes to private companies.
4. the determination of the policy for the technology to be commercially exploited. Work on this field considered the path to market for cellulosic ethanol, the contribution of policy to the successful technology development and the potential contribution of cellulosic ethanol to discrete policy objectives. Best practice guidelines for demonstration and scale-up of the technology were revealed.
5. the examination of a case study for the integration of the production of lignocellulosic ethanol into an existing paper mill, which resulted in conclusions for all the different processes of the technology application.
European targets related to reducing emissions and dependence on petrol, to increasing the application of renewable energy sources and to improving the energy efficiency, along with the development of new engine platforms by car manufacturers, turn bioethanol into a promising automotive fuel.
The NILE project team experimented using both high and low ethanol content blends. Performance and emissions of the test vehicle have been measured using both first and second generation process bioethanol in order to demonstrate that engine calibration could not change between the two fuels. The experiment aimed to evaluate bioethanol with regard to turbocharged and downsized engines and proved that the optimisation of the engine compression ratio could be done based on bioethanol properties and, when operating with gasoline, knocking could be avoided, thus reducing the boosting pressure at the intake.
High blends caused reduced deposits on the cold parts of the engine in comparison to gasoline, whereas this was not the case for low blends. On the other hand, deposits on hottest parts such as piston tops and cylinder head showed a linear reduction with increase in the ethanol content. Under full load conditions the combination of the high octane number with the high latent heat of vaporisation allowed for a more efficient engine regulation, with even a slight increase in maximum performance. Because of the possible optimisation of the spark advance timing due to the knocking resistance of bioethanol, high ethanol content blends proved to be beneficial in terms of energy efficiency.
The results of the NILE project could be summarised as follows:
1. among the various approaches aiming to improve the enzymatic hydrolysis of lignocellulosic raw materials, some proved to be successful whereas others require further investigation;
2. addition of helper enzymes had no significant effect in diluted acid conditions;
3. the potential improvements of enzymes were greatly dependent upon the efficacy of the pre-treatment, whose up-scaling proved to be of major importance;
4. four new industrial strains were successfully developed and it was shown that an adapted fermentation strategy was necessary for the full exploitation of those strains;
5. optimised supply chains, integrated facilities and a favourable market were required for the profitable technology application;
6. from tests carried out related to the use of ethanol in downsized engines and engines coupled with turbocharger it occurred that, apart from acetaldehyde emissions, ethanol was beneficial. In addition, cellulose-based ethanol could, with normal distillation and dewatering, meet the fuel sector specifications.
