BACKGROUND
as petroleum and other fossil resources are not sustainable, renewable alternatives are needed for production of fuels, chemicals, and materials.
lignocellulosic biomass is an abundant non-food renewable resource that can contribute to a shift towards a sustainable bio-based society  <cit> .
the three major constituents of lignocellulosic biomass are cellulose, hemicelluloses, and lignin.
cellulose and hemicelluloses are polysaccharides that can be hydrolyzed to monosaccharide sugars.
the monosaccharides obtained can then be utilized for production of ethanol or other commodities  <cit> .
saccharification is typically accomplished through enzymatic hydrolysis, as the high selectivity of enzymatic catalysis gives high sugar yields.
a pretreatment step is needed to achieve efficient bioconversion of lignocellulosic feedstocks, as the cellulose fraction is not easily accessible to enzymes  <cit> .
however, many pretreatment methods require harsh conditions and give rise to by-products, which decrease the sugar yields and inhibit enzymatic and microbial biocatalysts.
solvents that dissolve cellulose are of interest for pretreatment of lignocellulosic feedstocks prior to bioconversion.
in nature, cellulose occurs mainly as highly crystalline cellulose i  <cit> .
the crystallinity may prevent efficient enzymatic conversion and it is therefore desirable to disrupt the structure of the cellulose.
since cellulose is amphiphilic  <cit> , it has been suggested that both hydrophobic and hydrophilic interactions need to be broken to dissolve cellulose.
therefore, a suitable solvent system should contain a co-solvent that weakens the hydrophobic interactions in an aquatic environment  <cit> .
several solvent systems for cellulose are known  <cit> , but many of them suffer from problems like high toxicity and being difficult to recycle.
ionic liquids  are non-volatile solvents that can dissolve cellulose under gentle conditions.
several attempts have been made to utilize ils for pretreatment of cellulose  <cit> .
aspects that have been studied include the inhibitory effect on enzymes  <cit> , the influence of water content  <cit>  and cellulose load  <cit> , the particle size of the biomass  <cit> , the pretreatment temperature  <cit> , and the pretreatment time  <cit> .
however, an extensive study on the pretreatment effects of different ils on different types of cellulosic substrates is lacking.
comparisons have been made, but they are based on biomass from different sources and treatments performed under different conditions  <cit> .
thus, the aim of this study was to investigate the pretreatment effect of different ils on different types of substrates under identical conditions.
four ils  and seven substrates  were studied.
2
-
a
athe yields were calculated for the total synthesis  starting from the substituted imidazoles.
bnot applicable.
the four ils  were chosen since they have been used to dissolve cellulosic substrates and are recognized for their performance in such applications.
also, the set studied would allow for the detection of performance variations between different types of ils used for this purpose.
is one of the most studied ils when it comes to dissolving cellulose and serves as a good reference  <cit> .
is highly interesting because it has been reported to dissolve lignin in high water content , which is substantially more than -- and --based ils can handle  <cit> .
has the highest basicity among the selected -based ils, a property that is strongly linked to the capability of the il to dissolve cellulose  <cit> .
finally,  was selected because of its high reactivity, low viscosity, and ability to dissolve cellulose  <cit> .
it has been suggested that π-π interactions between the allyl side chain and lignin will improve lignin solubility  <cit> .
the set of ils chosen also gives the possibility to investigate the effect of the cation, by comparing the results from  with those from .
with regard to dissolution of wood saw dust and thermo-mechanical wood pulp, the performance of 1-allyl-3-methylimidazolium chloride  even surpassed that of   <cit> .
the set of ils studied  would also permit a comparison of ils that are capable of homogeneous dissolution of lignocellulose, i.e.
complete dissolution of all the main components of lignocellulose, and ils known to be restricted to heterogeneous dissolution of lignocellulose  <cit> .
,  and  should give rise to homogeneous dissolution of lignocellulose .
would effect heterogeneous dissolution of lignocellulose by selectively dissolving lignin and hemicellulose, but would not dissolve cellulose.
the study included cellulosic , hemicellulosic , as well as lignocellulosic  substrates .
the selected collection of substrates differ widely with regard to fundamental properties, such as crystallinity, degree of polymerization , and complexity .
the most complex substrates, aspen and spruce, represent hardwood and softwood, respectively.
softwood is typically the most recalcitrant type of lignocellulosic biomass  <cit> .
the sets of ils and substrates used in the investigation would allow a broad evaluation of the ils with regard to the effects of the anion and the cation as well as the importance of homogeneous and heterogeneous dissolution on the pretreatment efficiency.
to permit a comparison of il pretreatment with a conventional state-of-the-art pretreatment method, one of the lignocellulosic samples was also pretreated using classical sulfuric acid pretreatment.
using in-house prepared ils and advanced analytical methods such as high-performance anion-exchange chromatography  to quantify different monosaccharides released from the polymeric substrates by the hydrolytic enzymes, this investigation addresses the lack of direct comparisons of the effects of ils on cellulosic, hemicellulosic, and lignocellulosic samples.
RESULTS
ionic liquids
data on the properties and synthesis of the four ionic liquids synthesized as a part of the investigation are shown in table  <dig>  figure  <dig> shows the results of 1h and 13c nmr analysis of , , , and .
the figure shows the peak assignments for the 1h nmr spectra.
the clean spectra indicate that the ionic liquids were of high purity.
dialkylated imidazoles, such as ,  and  , are among the more common cations used for the dissolution of biomass.
in fact, 1-butyl-3-methylimidazolium chloride  was one of the first ils used to dissolve cellulose  <cit>  and lignocellulose  <cit> .
the focus has recently shifted from chloride-based to acetate-based ils  <cit> , which in this study are represented by  and  .
however, the stability of acetate-based ils is not as good as that of chloride-based ils, such as  .
the kamlet-taft polarity, or, more precisely, the hydrogen-bond basicity of the il, is believed to be strongly linked to its capability to dissolve or to swell lignocellulose  <cit> .
however, the basicity of an il is related to its hydrophilicity  <cit>  and poses a problem which has to do with the water content of the il.
in equilibrium with air, ils with acetate as anion can reach a water content of 27%   <cit> .
however, even a relatively low content of water  in a study of miscanthus <cit>  and 5-10%  in a study of maple-wood flour  <cit> ) has been shown to have a negative effect on the solubility of lignocellulose.
this is not optimal in a process, where the energy used for drying the biomass and the il needs to be taken in to account.
the water content of the ils  was determent by karl-fischer titration after the samples had been exposed to high vacuum  at 65°c overnight.
the water content of all ils was below  <dig>  wt%, except for , which on purpose had a high water content.
according to previous studies  <cit> , the sugar yield from lignocellulose pretreated with  was significantly higher when the water content was 10-40%.
the water content of the  used in our study, 20% , is thus within the desired range.
the yields of the ils  were calculated for the total synthesis starting from the corresponding 1-alkyl-imidazole .
this explains why the yields were sometimes relatively low .
and  were made from the corresponding saturated and unsaturated 1-alkyl-3-methylimidazolium chlorides.
the ion exchange was tracked using nmr to ensure that the acetate ion was duly present and that the ion exchange was successful.
the halide contamination was controlled by using hpaec and a sodium chloride standard.
the chloride contamination was <1% .
biomass samples
the amounts of structural carbohydrates and lignin were determined after extraction of the aspen and the spruce wood .
the values correspond well with those found in the literature  <cit> .
the glucan content of the aspen and spruce wood was rather similar, around 40% .
as expected, the aspen wood had a relatively high xylan content , while the spruce wood had relatively high mannan content .
the total lignin content of norway spruce  was higher than that of hybrid aspen .
the lignin content is highly relevant for enzymatic hydrolysis of cellulose  <cit> , as lignin can obstruct saccharification by forming a physical barrier, by catalytically unproductive binding of hydrolytic enzymes, and by formation of lignin-carbohydrate complexes .
a
b
areported as g of carbohydrate or lignin per g of wood  with standard deviations.
bklason lignin and acid-soluble lignin.
pretreatment and enzymatic saccharification
cellulosic, hemicellulosic and lignocellulosic samples  were pretreated with the four ils at 100°c for 20 h. the precipitation was carried out using a small amount of methanol and ultra-pure deionized water.
the precipitate was collected by centrifugation and washed three times with ultra-pure water and once with citrate buffer prior to the enzymatic saccharification.
the enzyme mixture used contains a wide variety of proteins, which are expected to degrade not only cellulose, but also xylan and mannan.
the use of a sensitive analytical method for determination of the yields of the monosaccharides  would allow detection of small quantities of sugars such as xylose and mannose.
the enzymatic saccharification was carried out analytically in a small scale , which made the processing of multiple parallel samples and the evaluation of the experiments using statistical analysis feasible.
crystalline samples
compared to the untreated control, the glucose production rate  of cotton increased dramatically after treatment with , , and  .
for cellulosic samples, pretreatment with  gave the best results .
the gpr increased significantly  also for the -pretreated sample compared to that of the control sample.
compared to the untreated control, the glucose yield  increased significantly after pretreatment with all of the ils except  .
pretreatment of cotton with ,  and  increased the yield with  <dig>   <dig> and 160%, respectively.
the difference can be attributed to that  causes heterogeneous dissolution of lignocellulose, while the other three ils cause homogeneous dissolution.
as cotton consists almost of pure cellulose , the capacity of  to dissolve lignin and hemicellulose would be of little or no help.
for ,  and , the increase in gpr  was higher than the increase in yield , which indicates that the effect of the pretreatment is most beneficial for the initial phase of the hydrolysis of the cellulose.
-1
after 72 h of enzymatic hydrolysis  of ionic-liquid-pretreated samples and non-pretreated control.
with regard to the microcrystalline sigmacell type  <dig> cellulose, the gprs of samples pretreated with ,  and  were significantly higher  than that of the control .
the glucose yields of pretreated and non-pretreated samples did not exhibit any significant differences .
treatment with  did not result in any improvement for either the gpr or the glucose yield, as could be expected since  does not dissolve cellulose.
when untreated sigmacell type  <dig> was used as substrate instead of untreated cotton, the gpr increased from  <dig>  g l-1 h- <dig> to  <dig>  g l-1 h- <dig> and the glucose yield increased from  <dig>  g g- <dig> to  <dig>  g g- <dig>  evidently, the sigmacell type  <dig> cellulose was more susceptible to enzymatic hydrolysis than cotton cellulose.
the difference can probably be attributed to the much lower degree of polymerization  of sigmacell type  <dig> compared to cotton , which would give the exo-acting cellobiohydrolases a larger number of cellulose chain ends to attack.
it is also possible that a lower degree of crystallinity of sigmacell type  <dig> cellulose  contributes to facilitating the enzymatic hydrolysis.
after pretreatment of cotton with ,  or , the gpr values were similar or slightly higher than the corresponding gpr values for sigmacell type  <dig> .
this was not due to that the substrate had been exhausted already after 2 h, as less than 35% of the substrate had been converted to glucose.
the yield values  from samples taken after 72 h were also lower than the theoretical maximum, which would be approx.
<dig>  g glucose per g cellulose.
the highest yield observed for the two cellulosic crystalline substrates,  <dig>  g g- <dig>  was obtained after pretreatment of cotton with  and corresponds to conversion of around 85% of the substrate to glucose.
thus, although the natural cotton was more recalcitrant than sigmacell type  <dig>  pretreatment with all of the ionic liquids except  removed that effect.
amorphous samples
the gpr values  and the glucose yield values  for regenerated amorphous cellulose  were rather similar to the values of the sigmacell type  <dig> cellulose that the rac was derived from.
this suggests that the crystallinity of the sigmacell type  <dig> cellulose  was not an obstacle for the enzymatic hydrolysis.
apart from conversion of crystalline cellulose to amorphous, treatment with acid may potentially also result in hydrolysis which could give decreased cellulose chain length, but evidently this did not lead to improved hydrolysis of rac compared to sigmacell type  <dig> under the experimental conditions studied.
enzymatic hydrolysis of untreated or il-pretreated hemicellulosic samples for 72 h resulted in very low yields  of glucose and arabinose.
enzymatic hydrolysis of untreated beech-wood xylan gave a xylose yield of  <dig>  ±  <dig>  g g- <dig>  while untreated locust bean gum galactomannan gave a galactose yield of  <dig>  ±  <dig>  g g- <dig> and a mannose yield of  <dig>  ±  <dig>  g g- <dig>  after pretreatment of the beech-wood xylan with ils, the xylose yields compared to that of the untreated control were 23% for , 25% for , 39% for , and 7% for .
for the locust bean gum galactomannan, the galactose yields compared to that of the untreated control were 33% for , 75% for , and 66% for .
no galactose was recovered from galactomannan pretreated with .
finally, the mannose yields compared to that of the untreated galactomannan control were 91% for , 73% for , and 3% for .
no mannose was recovered after pretreatment of galactomannan with .
thus, the recovery of hemicellulose sugars after pretreatment with ils was always poor.
a possible explanation for poor recovery of hemicellulosic sugars is that previous studies have shown that ils with a high basicity will hydrolyze hemicellulose to oligomeric sugars  <cit> .
it has also been shown that sulfate-based ils, especially -, will hydrolyze hemicellulose to monomeric sugars and even dehydrate pentose sugars to furfural  <cit> .
poor recovery of hemicellulose sugars has also been indicated in previous studies  <cit> .
wood samples
as expected considering the recalcitrance of softwood, untreated norway spruce exhibited lower gpr  and glucose yield  than untreated hybrid aspen .
pretreatment of the wood samples with the four ils resulted in significantly higher  gpr values and glucose yields .
the increase of the yields  varied between  <dig> and 950% compared to that of the untreated wood samples.
the highest yields were obtained upon use of  and  .
for aspen, the glucose yields with  and  were  <dig>  ±  <dig>  g g- <dig> and  <dig>  ±  <dig>  g g- <dig>  respectively.
these yields were slightly higher than that achieved in a control experiment with dilute sulfuric acid pretreatment and enzymatic hydrolysis of aspen, which resulted in a glucose yield of  <dig>  ±  <dig>  g g- <dig>  for aspen, the glucan conversion after pretreatment with  and  was 76% and 81%, respectively.
when spruce was pretreated using  or , more than 90% of the glucan was converted to glucose.
that is a higher value than achieved with the pure cellulosic crystalline and amorphous substrates, which indicates the potential of using ils for pretreatment of lignocellulose.
the glucose yields after pretreatment of aspen with the ils that did not have acetate as anion were lower and corresponded to  <dig>  ±  <dig>  g g- <dig> for  and  <dig>  ±  <dig>  g g- <dig> for .
the glucan conversions after pretreatment of aspen with  and  were 28% and 62%, respectively.
the corresponding values for spruce were 57% and 20%.
while  did not work very well for pretreatment of the recalcitrant spruce wood, the results obtained with aspen were not far behind the results achieved with acetate-based ils .
there were no significant differences between  and  in terms of pretreatment of any of the samples in this study.
this indicates that the choice of cation is often not decisive in terms of the efficiency of the pretreatment process.
the higher yields obtained upon use of ionic liquids with an acetate anion can most likely be explained by the higher basicity of the acetate compared to the chloride and hydrogen sulfate .
the comparatively low yields found for the hydrogen sulfate anion compared to the acetates can be attributed to the fact that it does not dissolve cellulose.
however, it does dissolve lignin and hemicelluloses, which would explain the higher yields achieved compared to non-pretreated wood samples.
CONCLUSIONS
four ils were evaluated for pretreatment of seven cellulosic, hemicellulosic and lignocellulosic substrates prior to enzymatic saccharification.
the wide variety of substrates investigated and the use of the same pretreatment conditions for all ils studied facilitated comparisons of the performance of the different ils.
although they were not the most efficient pretreatment agents for simple cellulosic substrates, ils with acetate as anion gave the best results for complex lignocellulosic substrates from wood.
however, among the ils used in this study the ones with acetate as anion are among the least stable.
moreover, they are cumbersome to synthesize, and they are sensitive to moisture.
the halide-based il  performed very well for simple cellulosic substrates, but was less efficient for pretreatment of wood.
possesses advantages from the process perspective, since it is easy to synthesize, relatively stable, and less sensitive to moisture than the acetate-based ils.
disadvantages with  include its high melting point and high viscosity.
the difference between homogeneous and heterogeneous dissolution of lignocellulose was studied by the inclusion of  in the set of ils studied.
homogeneous dissolution was not always better, as  gave better results than  with hybrid aspen, the less recalcitrant type of lignocellulose included in the study.
this warrants further attention in future studies, for example by optimization of the pretreatment process.
the best choices, as judged by the saccharification efficiency, appeared to be  for cellulosic substrates, acetate-based ils or  for hybrid aspen, and acetate-based ils for norway spruce.
pretreatment can, however, involve high water content, something that has always been a problem with homogeneous dissolution of lignocellulose.
the possibility to include water and the stability of  have potential to decrease the cost for the regeneration process.
therefore other factors than the capacity to dissolve cellulose, such as il stability, the possibility to include water, and regeneration of the il, need to be considered when choosing ils that are most suited for industrial pretreatment.
conversion of crystalline sigmacell type  <dig> cellulose to rac did not improve enzymatic hydrolysis.
this was shown for both non-pretreated cellulose and cellulose that was pretreated with different ils.
ils that enhanced hydrolysis of the crystalline form also enhanced hydrolysis of the rac.
pretreatment with ils gave sugar yields comparable with those obtained with acidic pretreatment technique.
pretreatment with ils can be performed using gentle conditions, while acidic pretreatment is typically performed at high temperature and high pressure, and in a corrosive environment, factors that increase the demands on the quality of the process equipment and contributes to lower yields due to the formation of by-products from the lignocellulose feed.
high yields are critical in the production of biofuels and other bulk chemicals.
ils also have low vapor pressure which is advantageous with regard to safety and environmental aspects.
thus, provided that ils used in industrial processes can be reused, there are distinct advantages that make further research on the use of ils for conversion of lignocellulose worthwhile.
future areas of research include recycling of ils, up-scaling, process integration, and techno-economical assessment.

 as petroleum and other fossil resources are not sustainable, renewable alternatives are needed for production of fuels, chemicals, and materials. lignocellulosic biomass is an abundant non-food renewable resource that can contribute to a shift towards a sustainable bio-based society. the three major constituents of lignocellulosic biomass are cellulose, hemicelluloses, and lignin. cellulose and hemicelluloses are polysaccharides that can be hydrolyzed to monosaccharide sugars. the monosaccharides obtained can then be utilized for production of ethanol or other commodities. saccharification is typically accomplished through enzymatic hydrolysis, as the high selectivity of enzymatic catalysis gives high sugar yields. a pretreatment step is needed to achieve efficient bioconversion of lignocellulosic feedstocks, as the cellulose fraction is not easily accessible to enzymes. however, many pretreatment methods require harsh conditions and give rise to by-products, which decrease the sugar yields and inhibit enzymatic and microbial biocatalysts. solvents that dissolve cellulose are of interest for pretreatment of lignocellulosic feedstocks prior to bioconversion. in nature, cellulose occurs mainly as highly crystalline cellulose i. the crystallinity may prevent efficient enzymatic conversion and it is therefore desirable to disrupt the structure of the cellulose. since cellulose is amphiphilic, it has been suggested that both hydrophobic and hydrophilic interactions need to be broken to dissolve cellulose. therefore, a suitable solvent system should contain a co-solvent that weakens the hydrophobic interactions in an aquatic environment. several solvent systems for cellulose are known, but many of them suffer from problems like high toxicity and being difficult to recycle. ionic liquids are non-volatile solvents that can dissolve cellulose under gentle conditions. several attempts have been made to utilize ils for pretreatment of cellulose. aspects that have been studied include the inhibitory effect on enzymes, the influence of water content and cellulose load, the particle size of the biomass, the pretreatment temperature, and the pretreatment time. however, an extensive study on the pretreatment effects of different ils on different types of cellulosic substrates is lacking. comparisons have been made, but they are based on biomass from different sources and treatments performed under different conditions. thus, the aim of this study was to investigate the pretreatment effect of different ils on different types of substrates under identical conditions. four ils and seven substrates were studied. 2 - a athe yields were calculated for the total synthesis starting from the substituted imidazoles. bnot applicable. the four ils were chosen since they have been used to dissolve cellulosic substrates and are recognized for their performance in such applications. also, the set studied would allow for the detection of performance variations between different types of ils used for this purpose. is one of the most studied ils when it comes to dissolving cellulose and serves as a good reference. is highly interesting because it has been reported to dissolve lignin in high water content, which is substantially more than -- and --based ils can handle. has the highest basicity among the selected -based ils, a property that is strongly linked to the capability of the il to dissolve cellulose. finally, was selected because of its high reactivity, low viscosity, and ability to dissolve cellulose. it has been suggested that π-π interactions between the allyl side chain and lignin will improve lignin solubility. the set of ils chosen also gives the possibility to investigate the effect of the cation, by comparing the results from with those from. with regard to dissolution of wood saw dust and thermo-mechanical wood pulp, the performance of 1-allyl-3-methylimidazolium chloride even surpassed that of. the set of ils studied would also permit a comparison of ils that are capable of homogeneous dissolution of lignocellulose, i.e. complete dissolution of all the main components of lignocellulose, and ils known to be restricted to heterogeneous dissolution of lignocellulose., and should give rise to homogeneous dissolution of lignocellulose. would effect heterogeneous dissolution of lignocellulose by selectively dissolving lignin and hemicellulose, but would not dissolve cellulose. the study included cellulosic, hemicellulosic, as well as lignocellulosic substrates. the selected collection of substrates differ widely with regard to fundamental properties, such as crystallinity, degree of polymerization, and complexity. the most complex substrates, aspen and spruce, represent hardwood and softwood, respectively. softwood is typically the most recalcitrant type of lignocellulosic biomass. the sets of ils and substrates used in the investigation would allow a broad evaluation of the ils with regard to the effects of the anion and the cation as well as the importance of homogeneous and heterogeneous dissolution on the pretreatment efficiency. to permit a comparison of il pretreatment with a conventional state-of-the-art pretreatment method, one of the lignocellulosic samples was also pretreated using classical sulfuric acid pretreatment. using in-house prepared ils and advanced analytical methods such as high-performance anion-exchange chromatography to quantify different monosaccharides released from the polymeric substrates by the hydrolytic enzymes, this investigation addresses the lack of direct comparisons of the effects of ils on cellulosic, hemicellulosic, and lignocellulosic samples.  ionic liquids data on the properties and synthesis of the four ionic liquids synthesized as a part of the investigation are shown in table  figure  shows the results of 1h and 13c nmr analysis of,,, and. the figure shows the peak assignments for the 1h nmr spectra. the clean spectra indicate that the ionic liquids were of high purity. dialkylated imidazoles, such as, and, are among the more common cations used for the dissolution of biomass. in fact, 1-butyl-3-methylimidazolium chloride was one of the first ils used to dissolve cellulose and lignocellulose. the focus has recently shifted from chloride-based to acetate-based ils, which in this study are represented by and. however, the stability of acetate-based ils is not as good as that of chloride-based ils, such as. the kamlet-taft polarity, or, more precisely, the hydrogen-bond basicity of the il, is believed to be strongly linked to its capability to dissolve or to swell lignocellulose. however, the basicity of an il is related to its hydrophilicity and poses a problem which has to do with the water content of the il. in equilibrium with air, ils with acetate as anion can reach a water content of 27%. however, even a relatively low content of water in a study of miscanthus and 5-10% in a study of maple-wood flour ) has been shown to have a negative effect on the solubility of lignocellulose. this is not optimal in a process, where the energy used for drying the biomass and the il needs to be taken in to account. the water content of the ils was determent by karl-fischer titration after the samples had been exposed to high vacuum at 65°c overnight. the water content of all ils was below  wt%, except for, which on purpose had a high water content. according to previous studies, the sugar yield from lignocellulose pretreated with was significantly higher when the water content was 10-40%. the water content of the used in our study, 20%, is thus within the desired range. the yields of the ils were calculated for the total synthesis starting from the corresponding 1-alkyl-imidazole. this explains why the yields were sometimes relatively low. and were made from the corresponding saturated and unsaturated 1-alkyl-3-methylimidazolium chlorides. the ion exchange was tracked using nmr to ensure that the acetate ion was duly present and that the ion exchange was successful. the halide contamination was controlled by using hpaec and a sodium chloride standard. the chloride contamination was <1%. biomass samples the amounts of structural carbohydrates and lignin were determined after extraction of the aspen and the spruce wood. the values correspond well with those found in the literature. the glucan content of the aspen and spruce wood was rather similar, around 40%. as expected, the aspen wood had a relatively high xylan content, while the spruce wood had relatively high mannan content. the total lignin content of norway spruce was higher than that of hybrid aspen. the lignin content is highly relevant for enzymatic hydrolysis of cellulose, as lignin can obstruct saccharification by forming a physical barrier, by catalytically unproductive binding of hydrolytic enzymes, and by formation of lignin-carbohydrate complexes. a b areported as g of carbohydrate or lignin per g of wood with standard deviations. bklason lignin and acid-soluble lignin. pretreatment and enzymatic saccharification cellulosic, hemicellulosic and lignocellulosic samples were pretreated with the four ils at 100°c for 20 h. the precipitation was carried out using a small amount of methanol and ultra-pure deionized water. the precipitate was collected by centrifugation and washed three times with ultra-pure water and once with citrate buffer prior to the enzymatic saccharification. the enzyme mixture used contains a wide variety of proteins, which are expected to degrade not only cellulose, but also xylan and mannan. the use of a sensitive analytical method for determination of the yields of the monosaccharides would allow detection of small quantities of sugars such as xylose and mannose. the enzymatic saccharification was carried out analytically in a small scale, which made the processing of multiple parallel samples and the evaluation of the experiments using statistical analysis feasible. crystalline samples compared to the untreated control, the glucose production rate of cotton increased dramatically after treatment with,, and. for cellulosic samples, pretreatment with gave the best results. the gpr increased significantly also for the -pretreated sample compared to that of the control sample. compared to the untreated control, the glucose yield increased significantly after pretreatment with all of the ils except. pretreatment of cotton with, and increased the yield with and 160%, respectively. the difference can be attributed to that causes heterogeneous dissolution of lignocellulose, while the other three ils cause homogeneous dissolution. as cotton consists almost of pure cellulose, the capacity of to dissolve lignin and hemicellulose would be of little or no help. for, and, the increase in gpr was higher than the increase in yield, which indicates that the effect of the pretreatment is most beneficial for the initial phase of the hydrolysis of the cellulose. -1 after 72 h of enzymatic hydrolysis of ionic-liquid-pretreated samples and non-pretreated control. with regard to the microcrystalline sigmacell type cellulose, the gprs of samples pretreated with, and were significantly higher than that of the control. the glucose yields of pretreated and non-pretreated samples did not exhibit any significant differences. treatment with did not result in any improvement for either the gpr or the glucose yield, as could be expected since does not dissolve cellulose. when untreated sigmacell type was used as substrate instead of untreated cotton, the gpr increased from  g l-1 h- to  g l-1 h- and the glucose yield increased from  g g- to  g g- evidently, the sigmacell type cellulose was more susceptible to enzymatic hydrolysis than cotton cellulose. the difference can probably be attributed to the much lower degree of polymerization of sigmacell type compared to cotton, which would give the exo-acting cellobiohydrolases a larger number of cellulose chain ends to attack. it is also possible that a lower degree of crystallinity of sigmacell type cellulose contributes to facilitating the enzymatic hydrolysis. after pretreatment of cotton with, or, the gpr values were similar or slightly higher than the corresponding gpr values for sigmacell type. this was not due to that the substrate had been exhausted already after 2 h, as less than 35% of the substrate had been converted to glucose. the yield values from samples taken after 72 h were also lower than the theoretical maximum, which would be approx.  g glucose per g cellulose. the highest yield observed for the two cellulosic crystalline substrates,  g g- was obtained after pretreatment of cotton with and corresponds to conversion of around 85% of the substrate to glucose. thus, although the natural cotton was more recalcitrant than sigmacell type pretreatment with all of the ionic liquids except removed that effect. amorphous samples the gpr values and the glucose yield values for regenerated amorphous cellulose were rather similar to the values of the sigmacell type cellulose that the rac was derived from. this suggests that the crystallinity of the sigmacell type cellulose was not an obstacle for the enzymatic hydrolysis. apart from conversion of crystalline cellulose to amorphous, treatment with acid may potentially also result in hydrolysis which could give decreased cellulose chain length, but evidently this did not lead to improved hydrolysis of rac compared to sigmacell type under the experimental conditions studied. enzymatic hydrolysis of untreated or il-pretreated hemicellulosic samples for 72 h resulted in very low yields of glucose and arabinose. enzymatic hydrolysis of untreated beech-wood xylan gave a xylose yield of  ±   g g- while untreated locust bean gum galactomannan gave a galactose yield of  ±   g g- and a mannose yield of  ±   g g- after pretreatment of the beech-wood xylan with ils, the xylose yields compared to that of the untreated control were 23% for, 25% for, 39% for, and 7% for. for the locust bean gum galactomannan, the galactose yields compared to that of the untreated control were 33% for, 75% for, and 66% for. no galactose was recovered from galactomannan pretreated with. finally, the mannose yields compared to that of the untreated galactomannan control were 91% for, 73% for, and 3% for. no mannose was recovered after pretreatment of galactomannan with. thus, the recovery of hemicellulose sugars after pretreatment with ils was always poor. a possible explanation for poor recovery of hemicellulosic sugars is that previous studies have shown that ils with a high basicity will hydrolyze hemicellulose to oligomeric sugars. it has also been shown that sulfate-based ils, especially -, will hydrolyze hemicellulose to monomeric sugars and even dehydrate pentose sugars to furfural. poor recovery of hemicellulose sugars has also been indicated in previous studies. wood samples as expected considering the recalcitrance of softwood, untreated norway spruce exhibited lower gpr and glucose yield than untreated hybrid aspen. pretreatment of the wood samples with the four ils resulted in significantly higher gpr values and glucose yields. the increase of the yields varied between and 950% compared to that of the untreated wood samples. the highest yields were obtained upon use of and. for aspen, the glucose yields with and were  ±   g g- and  ±   g g- respectively. these yields were slightly higher than that achieved in a control experiment with dilute sulfuric acid pretreatment and enzymatic hydrolysis of aspen, which resulted in a glucose yield of  ±   g g- for aspen, the glucan conversion after pretreatment with and was 76% and 81%, respectively. when spruce was pretreated using or, more than 90% of the glucan was converted to glucose. that is a higher value than achieved with the pure cellulosic crystalline and amorphous substrates, which indicates the potential of using ils for pretreatment of lignocellulose. the glucose yields after pretreatment of aspen with the ils that did not have acetate as anion were lower and corresponded to  ±   g g- for and  ±   g g- for. the glucan conversions after pretreatment of aspen with and were 28% and 62%, respectively. the corresponding values for spruce were 57% and 20%. while did not work very well for pretreatment of the recalcitrant spruce wood, the results obtained with aspen were not far behind the results achieved with acetate-based ils. there were no significant differences between and in terms of pretreatment of any of the samples in this study. this indicates that the choice of cation is often not decisive in terms of the efficiency of the pretreatment process. the higher yields obtained upon use of ionic liquids with an acetate anion can most likely be explained by the higher basicity of the acetate compared to the chloride and hydrogen sulfate. the comparatively low yields found for the hydrogen sulfate anion compared to the acetates can be attributed to the fact that it does not dissolve cellulose. however, it does dissolve lignin and hemicelluloses, which would explain the higher yields achieved compared to non-pretreated wood samples.  four ils were evaluated for pretreatment of seven cellulosic, hemicellulosic and lignocellulosic substrates prior to enzymatic saccharification. the wide variety of substrates investigated and the use of the same pretreatment conditions for all ils studied facilitated comparisons of the performance of the different ils. although they were not the most efficient pretreatment agents for simple cellulosic substrates, ils with acetate as anion gave the best results for complex lignocellulosic substrates from wood. however, among the ils used in this study the ones with acetate as anion are among the least stable. moreover, they are cumbersome to synthesize, and they are sensitive to moisture. the halide-based il performed very well for simple cellulosic substrates, but was less efficient for pretreatment of wood. possesses advantages from the process perspective, since it is easy to synthesize, relatively stable, and less sensitive to moisture than the acetate-based ils. disadvantages with include its high melting point and high viscosity. the difference between homogeneous and heterogeneous dissolution of lignocellulose was studied by the inclusion of in the set of ils studied. homogeneous dissolution was not always better, as gave better results than with hybrid aspen, the less recalcitrant type of lignocellulose included in the study. this warrants further attention in future studies, for example by optimization of the pretreatment process. the best choices, as judged by the saccharification efficiency, appeared to be for cellulosic substrates, acetate-based ils or for hybrid aspen, and acetate-based ils for norway spruce. pretreatment can, however, involve high water content, something that has always been a problem with homogeneous dissolution of lignocellulose. the possibility to include water and the stability of have potential to decrease the cost for the regeneration process. therefore other factors than the capacity to dissolve cellulose, such as il stability, the possibility to include water, and regeneration of the il, need to be considered when choosing ils that are most suited for industrial pretreatment. conversion of crystalline sigmacell type cellulose to rac did not improve enzymatic hydrolysis. this was shown for both non-pretreated cellulose and cellulose that was pretreated with different ils. ils that enhanced hydrolysis of the crystalline form also enhanced hydrolysis of the rac. pretreatment with ils gave sugar yields comparable with those obtained with acidic pretreatment technique. pretreatment with ils can be performed using gentle conditions, while acidic pretreatment is typically performed at high temperature and high pressure, and in a corrosive environment, factors that increase the demands on the quality of the process equipment and contributes to lower yields due to the formation of by-products from the lignocellulose feed. high yields are critical in the production of biofuels and other bulk chemicals. ils also have low vapor pressure which is advantageous with regard to safety and environmental aspects. thus, provided that ils used in industrial processes can be reused, there are distinct advantages that make further research on the use of ils for conversion of lignocellulose worthwhile. future areas of research include recycling of ils, up-scaling, process integration, and techno-economical assessment. 
the monosaccharides in the enzymatic hydrolysate were determined using high-performance anion-exchange chromatography. the best results, as judged by the saccharification efficiency, were achieved with for cellulosic substrates and with the acetate-based ils for hybrid aspen and norway spruce. lignocellulosic biomass is highly recalcitrant and various pretreatment techniques are needed to facilitate its effective enzymatic hydrolysis to produce sugars for further conversion to bio-based chemicals. the ability of ils to dissolve lignocellulosic biomass under gentle conditions and with little or no by-product formation contributes to making them highly interesting alternatives for pretreatment in processes where high product yields are of critical importance. in order to achieve a broad evaluation, seven cellulosic, hemicellulosic and lignocellulosic substrates, crystalline as well as amorphous, were selected. the lignocellulosic substrates included hybrid aspen and norway spruce. after pretreatment with acetate-based ils, the conversion to glucose of glucan in recalcitrant softwood lignocellulose reached similar levels as obtained with pure crystalline and amorphous cellulosic substrates. heterogeneous dissolution with gave promising results with aspen, the less recalcitrant of the two types of lignocellulose included in the investigation. ionic liquids are of interest in pretreatment because of their potential to dissolve lignocellulosic materials including crystalline cellulose. four imidazolium-based ionic liquids well known for their capability to dissolve lignocellulosic species were synthesized and then used for pretreatment of substrates prior to enzymatic hydrolysis. il pretreatment of lignocellulose resulted in sugar yields comparable with that obtained with acidic pretreatment.
 lignocellulosic biomass is highly recalcitrant and various pretreatment techniques are needed to facilitate its effective enzymatic hydrolysis to produce sugars for further conversion to bio-based chemicals. ionic liquids are of interest in pretreatment because of their potential to dissolve lignocellulosic materials including crystalline cellulose.  four imidazolium-based ionic liquids well known for their capability to dissolve lignocellulosic species were synthesized and then used for pretreatment of substrates prior to enzymatic hydrolysis. in order to achieve a broad evaluation, seven cellulosic, hemicellulosic and lignocellulosic substrates, crystalline as well as amorphous, were selected. the lignocellulosic substrates included hybrid aspen and norway spruce. the monosaccharides in the enzymatic hydrolysate were determined using high-performance anion-exchange chromatography. the best results, as judged by the saccharification efficiency, were achieved with for cellulosic substrates and with the acetate-based ils for hybrid aspen and norway spruce. after pretreatment with acetate-based ils, the conversion to glucose of glucan in recalcitrant softwood lignocellulose reached similar levels as obtained with pure crystalline and amorphous cellulosic substrates. il pretreatment of lignocellulose resulted in sugar yields comparable with that obtained with acidic pretreatment. heterogeneous dissolution with gave promising results with aspen, the less recalcitrant of the two types of lignocellulose included in the investigation.  the ability of ils to dissolve lignocellulosic biomass under gentle conditions and with little or no by-product formation contributes to making them highly interesting alternatives for pretreatment in processes where high product yields are of critical importance. ionic liquidpretreatmentlignocelluloseenzymatic saccharification 