Evaluation of various pretreatment conditions and the effect on key variables, such as the overall yield of sugars or ethanol, needs to be assessed in an easy way to be able to judge the result. In several studies on pretreatment of biomass the “severity factor” has been used for comparing pretreatment results. Although it does not provide an accurate measure of the severity it can be used for rough estimates [8,9]. The severity correlation describes the severity of the pretreatment as a function of treatment time (minutes) and temperature (°C), Tref = 100 °C.

log(Ro) = log (t exp ^ ^ef) j j. (1)

When pretreatment is performed under acidic conditions (e. g. by impregna­tion with H2SO4), the effect of pH can be taken into consideration by the combined severity [10] defined as:

Combined severity (CS) = log(Ro) — pH (2)

It is well known that more severe conditions during pretreatment will cause greater degradation of hemicellulose sugars [11-13]. A high severity in the pretreatment is nevertheless required to enhance the enzymatic digestibility of cellulose [14]. The ideal pretreatment would hydrolyse the hemicellulose to its monomer sugars without further degradation. It would also cause an increase of the pore size and reduce cellulose crystallinity to enhance the en­zymatic digestibility of the cellulose fibres. However, these two effects are not reached at the same pretreatment severity, at least not using current technologies.

Assessment of pretreatment is usually done by using some of (or a combi­nation of) the following methods:

1. Analysis of the content of sugars liberated during pretreatment to the liquid as a combination of monomer and oligomer sugars, as well as an­alysis of the carbohydrate content of the water-insoluble solids (WIS). This gives the total recovery of carbohydrates in the pretreatment step.

2. Enzymatic hydrolysis (EH) of the WIS, either washed or non-washed.

3. Fermentation of the pretreatment liquid to assess inhibition of the fer­menting microorganism.

4. Simultaneous saccharification and fermentation (SSF) of either the whole slurry or the washed WIS.

The enzymatic hydrolysis (in 1 and 4) is performed using cellulases, i. e. a mixture of various cellobiohydrolases and endoglucanases supplemented with yd-glucosidase. The latter is not a cellulase as it only cleaves cellobiose into two glucose molecules. It has, however, a very important role in hydro­lysis since cellobiose is an end-product inhibitor of many cellulases [15,16]. On the other hand, d-glucosidase is also inhibited by glucose [17]. Since the enzymes are inhibited by the end products, the build-up of any of these products affects the cellulose hydrolysis negatively. The maximum cellulase activity for most fungus-derived cellulases and d-glucosidase occurs at 50 ± 5 °C and a pH of 4.0-5.0. However, the optimal conditions for enzymatic hydrolysis change with the hydrolysis residence time [18] and are also depen­dent on the source of the enzymes.

The enzymatic hydrolysis for assessment of pretreatment can be per­formed using various conditions (substrate concentration, enzyme dosage, temperature, stirring speed etc.). A common way is to use washed material at 2 wt % WIS, or alternatively at 1 wt % cellulose, to avoid end-product in­hibition [19]. This could be seen as the maximum achievable digestibility or glucose yield. However, it does not reflect the pretreatment efficiency in terms of avoiding formation of compounds that are inhibitory to the cellulases. In a full-scale process it is crucial to reach high sugar and ethanol concentrations in order to decrease the energy demand in the downstream processes. To in­crease the sugar concentration during large-scale operation, it is assumed that the whole slurry after pretreatment would be used without introducing sepa­ration steps, which would dilute the process stream. Furthermore, the overall substrate loading in enzymatic hydrolysis would probably need to be above 10 wt % WIS to meet the energy requirement for ethanol recovery. To mimic a situation that will be more similar to final process conditions, the enzymatic hydrolysis can be performed using the whole slurry from the pretreatment di­luted to various WIS concentrations, e. g. 10 wt %. In this case also the effect of inhibitors is assessed. However, due to the higher concentration of sugars the enzymes will also suffer from end-product inhibition.

To assess the effect of possible inhibitors acting on the microorganism used for fermentation of the sugars released in the enzymatic hydrolysis, method 2 is most often combined with method 3. The overall ethanol yield depends not only on the sugar yield, but also on the fermentability of the solution. Inhibition is influenced by the concentration of the soluble sub­stances released during pretreatment, present in the original raw material, e. g. acetic acid, or formed in the pretreatment step. Some of the substances present in the slurry are furfural and 5-hydroxymethylfurfural (HMF), which are the result of degradation of pentoses and hexoses, respectively. Furfural may react further to yield formic acid, or it may polymerize. HMF can be converted to formic acid and levulinic acid. In some pretreatments lignin degradation products are also formed. The concentrations of these and all other inhibitory substances in the fermentation step depend on the con­figuration of the preceding process steps. At ethanol concentrations below 4 to 5 wt % the energy demand increases rapidly with decreasing ethanol concentration. It is thus important to evaluate the fermentability of the con­centrated pretreatment hydrolysates. The fermentability test is usually per­formed on the liquid obtained from the pretreatment, either directly or di­luted to a concentration corresponding to what is expected to be suitable in a final process.

Another option for evaluation of the pretreatment step is to perform SSF either on the whole slurry diluted to a suitable WIS concentration or on the washed water-insoluble solid material, in both cases at a WIS around 5% or higher. In this case the glucose produced is immediately consumed by the fermenting microorganism, e. g. Saccharomyces cerevisiae, which removes the end-product inhibition of glucose and cellobiose. SSF adds information about the pretreatment efficiency, since SSF usually gives a higher overall ethanol yield than separate enzymatic hydrolysis and fermentation (SHF) due to con­version by the microorganism of some compounds that are inhibitory to the enzymes to less inhibitory compounds [20]. Also in the assessment by SSF the conditions may vary, e. g. substrate concentration, enzyme dosage, concentra­tion of microorganism etc.

It has to be added that variations between different laboratories in con­figurations and conditions used for assessment of the pretreatment make it very difficult to compare various pretreatment methods unless they are assessed in exactly the same way. Even so, the conclusions may be incor­rect as the conditions used may be unfavourable to a specific method. For instance, the use of hemicellulases in the enzymatic hydrolysis, instead of only cellulases, will be beneficial to pretreatment methods that result in large amounts of oligomer hemicellulose sugars, as will be discussed in the results section.

It is our opinion that the assessment of pretreatment has to be performed in a more rigorous way. The standard enzymatic hydrolysis at low substrate concentration may well be used to assess the maximum digestibility. However, in this case both cellulases and hemicellulases are needed. The “real” assess­ment should be performed by optimizing the conditions for all subsequent process steps under more realistic process conditions, taking into account the special features of the pretreated material, and then comparing the produc­tion cost for the various alternatives.

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