Ensiling as pretreatment of grass for lignocellulosic biomass conversion

Abstract

Development of sound technologies of biomass conversion will be increasingly important for many years to come as planetary bounderies drive the development towards a biobased society. Pretreatment of lignocellulosic biomass is, in this regard, an essential technology. Current pretreatment methods, based on severe physio-chemical processes, are effective, however, they are also costly and energy demanding. An alternative biological pretreatment method, based on the well-known biomass preservation of ensiling, has been proposed. Ensiling holds potential as an integrated storage and pretreatment method with low cost and low energy requirements, plus brings about multiple advantages with regards to agricultural management. However, the pretreatment effect of ensiling, and the overall effects for further conversion are limited. In this study, ensiling was evaluated as a method of pretreatment for subsequent enzymatic saccharification of cellulose and hemicellulose, by using the temperate grass Festulolium Hykor. The method was additionally combined with hydrothermal treatment, in order to decrease the required severity of an industrial applied pretreatment method. The first part of the project was devoted to method development. This resulted in the development of a simple and flexible standard method forlaboratory ensilingwith a high reproducibility,which is well suited for high-throughput experiments.   A comprehensive study on important parameters in ensiling was conducted to find optimal conditions providing the best possible pretreatment effect. The parameters were biomass composition, varied by ensiling of four seasonal cuts of grass, different dry matter (DM) content at ensiling, and an addition of different lactic acid bacteria species. First of all, the study confirmed that ensiling can act as a method of pretreatment and improve the enzymatic cellulose convertibility of grass. Furthermore, low DM ensiling was found to improve the effects of pretreatment due to a higher production of organic acids in the silage. The effect of applied lactic acid bacteria species was, however, insignificant. Cellulose conversion was noted to be largely determined by the stage of maturity of the four different cuts of grass. Less mature grass had high convertibility but less amount of cellulose and vice versa. This led to the conclusion that an optimal maturity of grass can be found, which gives an optimal glucose release. However, limitations of the method were also noted. The ensiling of grass came with a considerable loss of water soluble carbohydrates (WSC), which was in fact higher than the improved glucose release. Furthermore, the amount of released glucose was not adequate to support an efficient production of ethanol. Lastly, the conversion of xylan was extremely low in both grass and grass silage. Optimization of the enzymatic saccharification of grass was attempted through improvement of the hemicellulase content in the enzyme blend. However, neither additional xylanases (Cellic HTec2® and ß-xylosidase) nor hemicellulose degrading esterases (acetyl xylan esterase and ferulic acid esterase) showed any improvements of xylan or glucan convertibility. Furthermore, hemicellulases were added before ensiling in order to assist and improve the pretreatment effect. This resulted in, however, the undesired effect that additionally released monosaccharides were utilized during storage and had a negative impact on sugar release after enzymatic saccharification. In both of the above mentioned experiments on optimization ofsugar release by means of enzymes, it was noted that the hemicellulose structure of Festulolium Hykor appeared unusually resistant to enzymatic degradation. Due to the low conversion results on Festulolium Hykor, the last part of the project was based on a new tenet: Ensiling can not provide sufficient pretreatment effect to be a stand-alone pretreatment method. Ensiling was therefore combined with hydrothermal treatment (HTT), and the pretreatment combination was applied to both grass (Festulolium  Hykor) and wheat straw, in order to compare the effect upon two categorically different biomasses. For wheat straw, it was found that ensiling in combination with HTT increased the severity of HTT and facilitated a reduction in optimum HTT temperature of 10 to 20 °C. This could, however, not be proven for grass, since the overall release of mono- and oligosaccharides for the combined pretreatment of grass did not exceed HTT of grass alone. This was due to a combination of high loss of WSC during silage storage of grass and only minor improvements of HTT induced by ensiling. In comparison, the ensiling of wheat straw improved cellulose convertibility by a maximum factor of 1.9 at 170 °C, where the ensiling of grass only improved cellulose convertibility by a maximum factor of 1.3. Furthermore, the HTT pretreatment of both grass and grass silage gave considerably lower xylan convertibility than HTT of wheat straw and wheat straw silage. The reason for the inaccessible xylan in grass is believed to be found in a high complexity of branching and cross linkages creating a heterogeneous and resistant grass hemicellulose. However, further studies are necessary. The study concludes that ensiling may provide a pretreatment effect in itself, depending on the silage conditions and the recalcitrance of the biomass. However, ensiling will always be at the expense of an amount of WSC; and the significance of the gain from the pretreatment effect versus the loss of WSC will again depend on the silage conditions and the nature of the biomass. Ensiling was proven not to be a stand-alone pretreatment of Festulolium Hykor and should instead be considered as a sound method for biomass storage with possible benefits to biomass conversion. On the other hand, ensiling provided significant improvements to a combined pretreatment of ensiling and HTT. However, the improvements largely depends on the loss of WSC and the type of biomass in question. In this regard, it should be duly stressed that ensiling is not merely a pretreatment method, but an integrated storage and pretreatment method with effects on both agricultural management, biomass feedstock logistics, and biomass conversion. This thesis aimed to study only the last issue of biomass conversion.