(470f) An Integrated Approach for the Production of Bioethanol and High Value Bio-Products From Lignocellulosic Biomass

In the last centuries, people in the developed countries became almost totally dependent on fossil sources, in order to produce energy fuels and other basic chemicals required for maintaining a high life quality. However, with the depletion of fossil oil-resources, the ever increasing oil-price and the negative environmental impact due to the gaseous emissions associated with fossil fuels consumption, the world is actively searching for alternative energy sources. Plant biomass has the potential to partially satisfy the global needs in basic fuels and chemicals, while minimizing environmental impact and increasing sustainability.

Lignocellulosic materials (i.e., dedicated energy crops, such as perennial grasses, forestry co-products, agricultural residuals and vegetable waste etc.) are excellent examples of alternative carbon and energy sources with a large potential for flexible production of bio-based products. Lignocellulose, the most abundant polysaccharide-containing biomass available in the world, is an extremely complex and widely varying nano-scale composite, combining cellulose, hemi-cellulose, and lignin, along with a variable level of extractives. The presence of cellulose and hemi-cellulose, therefore, makes lignocellulosic feedstock a potential candidate for biochemical conversion processes, representing, at the same time, a promising outlet to security of supply issues for future production of bio-based products.

Among the various lignocellulosic feedstocks, the dedicated perennial herbaceous species with a C₃ photosynthesis system (e.g., Phalaris aquatica L. - Harding Grass, HG) represent a potential solution that can improve further the robustness and reliability of this renewable, lignocellulosic-based source. Characteristics of these types of dedicated perennial plants are their fast growth, their high biomass yields, the low input costs, the region-specificity, and the annual or, in many cases, less-frequent harvests. Moreover, they can grow on non-prime agricultural land, such as under-utilized and abandoned farmland, and do not compete for prime agricultural land required by commodity crops, grown for the food and feed markets.

The sugar monomers in lignocellulosic biomass, existing in the form of polysaccharides, are not readily available for bioconversion, due to the recalcitrance of plant cell wall structure. As a result, a multi-step process is required in order to liberate the carbohydrates for bioconversion to biofuels (e.g., ethanol), biochemicals (e.g., succinic acid), biopolymers (e.g., polyhydroxybutyrate, PHB) and other high value bio-based products. Dilute acid pretreatment followed by enzymatic hydrolysis is a typical process used to convert lignocellulosic biomass to fermentable sugars. The fermentation of both hexoses and pentoses via specific microbial factories into the desired bio-products follows the above treatment steps. Additionally, the separation of the desired products from the fermentation broth is of highly importance.

In the present work, the above-described integrated biochemical process was pursued with respect to the production of bioethanol from lignocellulosic biomass, involving three major stages, namely i) pretreatment, ii) enzymatic hydrolysis and iii) fermentation, which were studied and optimized separately: The optimization of the biomass pretreatment process was experimentally investigated following a statistical experimental design (Taguchi), via the maximization of hemi-cellulose solubilization and the recovery of pentose sugar monomers. The three principal variables studied were the reaction temperature, the concentration of sulfuric acid and the hydrolysis time. The parameters were controlled in 3 different levels (high, low and intermediate). From a series of experiments, the optimal conditions were identified. More specifically, the experimental run using 1.5% sulfuric acid at 120 ^oC for 45 min, resulted in approximately 80% conversion of the total available hemi-cellulose into pentose sugar monomers. Moreover, the results revealed that the combination of factors with mild conditions yield the maximum amount of pentoses, while enhancing the hydrolysis of cellulose in the following process step.

The enzymatic hydrolysis of the solid residue of the pretreatment stage was also optimized, by using a statistical experimental design (Box Denken), including the addition of surfactants. For the experimental study of the saccharification of cellulose four principal variables were selected and tested (biomass content, amount of enzyme, hydrolysis time and surfactant concentration-PEG 4000) at three different levels (high, low and intermediate). The mixture of commercial enzymes comprised cellulase (Celluclast 1.5L) and beta-glucosidase (Novozyme 188), derived from the fungi Trichoderma reseei and Aspergillus niger, respectively. From the experimental results, it was concluded that the optimal saccharification conditions were: 4% solids concentration, 20 FPU enzymes, 0.04 g/g solid surfactant concentration and a 72 h hydrolysis time. Furthermore, an empirical regression model was developed for the prediction of the maximum glucose production in the range of the selected variables. The maximum value of the concentration of glucose was equal to 12.9 g/l. Experiments were also carried out with increased amounts of initial solids content, in order to increase the final concentration of glucose in the hydrolyzate solution, a prerequisite for the subsequent fermentation stage.

The fermentation (cultivation of the yeast Saccharomyces cerevisiae) of the recovered glucose in the hydrolyzate solution, derived from the enzymatic hydrolysis stage, was held both in flask and bioreactor units. Initially, the optimum conditions (i.e., agitation speed, pH, inoculum size, and medium composition in terms of glucose and nitrogen sources concentrations, yeast strain) were identified under batch conditions in flask experiments. Experiments were also carried out under fed-batch mode using similar cultivation conditions. The optimal fed-batch policy identified in the flask experiments, resulted in an enhanced ethanol production: maximum ethanol concentration equal to 18.84 g/l), simultaneously with an overall productivity 0.82 g/(l∙h). Subsequently, the optimal fermentation fed-batch policy was transferred to a two-liter bioreactor unit, where four different fed-batch runs were evaluated. It was found that a simple continuous feeding policy can significantly increase the bioethanol concentration and the overall productivity. The maximum ethanol productivity was equal to 1.97 g/(l∙h).

Beyond the fermentative production of bioethanol from the available hexose monomers (mainly glucose), in the present research program, the synthesis of additional high value products was pursued. More specifically: i) the production of succinic acid by the fermentation of the available pentose monomers, liberated from the pretreatment stage, by Actinobacillus succinogenes, ii) the synthesis and recovery of biodegradable polyhydroxybutyrate (PHB) via the cultivation of Azohydromonas lata, and iii) the thermo-chemical production of plant nanocellulose directly from the dried biomass feedstock. These production lines could be part of an integrated biorefinery, combining the production of bioethanol with the production of high value-added materials, thus, significantly contributing to the economic viability of the biorefinery plant.