Lignocellulose, the structural material in the cell wall of all woody plants, is the most abundant renewable raw material. In 2008, the worldwide availability of raw wood was 3.4 billion cubic meters. Lignocellulose is the main component of residues such as straw or wood which, as so-called second-generation raw materials (not based on starch), do not conflict with food production.
Lignocellulose consists essentially of polymeric C6 and C5 sugars (cellulose, hemicellulose) and the biopolymer lignin (phenolic macromolecule). These components can serve as starting materials for biobased platform chemicals and materials after appropriate digestion and subsequent decomposition into their monomer building blocks. The Fraunhofer IGB deals with the optimization and scaling of the individual process steps of lignocellulose treatment in order to provide the main components of various lignocellulose-containing raw materials as holistically as possible for further material utilization and thus make a significant contribution to the establishment of a lignocellulose biorefinery.
Due to its compact structure and high lignin content, lignocellulose is very resistant to enzymatic attack, in contrast to sugar- and starch-based raw materials such as sugar beet and potatoes. For this reason, suitable pulping methods are required for lignocellulose-based raw materials in order to ensure optimum coupling utilization of these biogenic raw materials. The digestion effectively separates the components lignin (aromatic compounds), cellulose (C6 sugar) and hemicellulose (C5 sugar) and makes them available for industrial use.
There are many different digestion principles that can be divided into physical, physicochemical, chemical and biological processes. Of particular interest here are the so-called organosolv processes, which comprise a large number of processes that use either organic acids, alcohols or aromatics as solvents in combination with water. Alcohols, especially low-molecular alcohols, are the organic solvents most frequently used in organosolv processes, as they ensure a more energy-efficient recovery. Which conditions and chemicals are used in the organosolv process depends on the regenerative raw material source used and the desired target products.
The digestion of lignocellulose usually produces two streams of valuable substances: firstly, the cellulose-containing fibre, which can subsequently be hydrolyzed to glucose by cellulolytic enzymes (fibre hydrolysate), and secondly, a liquid digestion solution. This digestion solution contains dissolved hemicellulose sugar and dissolved lignin. Once the lignin has been separated by precipitation, these components can also be recycled separately.
By digestion, lignocellulose, as described above, is converted into the material streams cellulose fiber and the supernatant of the digestion solution (hemicellulose). In the next step, the monomeric sugars are obtained from cellulose or hemicellulose by enzymatic treatment. Enzyme complexes are used for this purpose, which work together synergistically in order to achieve an optimum sugar yield. Cellulases break down cellulose into the glucose units it contains, hemicellulases release the sugars contained in hemicellulose, mainly xylose.
Another effect of the digestion is the change in the crystallinity of the remaining cellulose fraction and an increase in the specific surface area of the fiber particles. This facilitates the adsorption of the enzymes and thus increases the yield. Furthermore, the even distribution of the hydrolytic enzymes by sufficient mixing of the pulp mass is of great importance for an optimal space-time yield of the hydrolysis process. The Fraunhofer IGB has developed a powerful stirred reactor system that is suitable for mixing fiber pulp with up to 30 percent solids content due to its special stirrer geometry. Yields of > 100 g/kg glucose were achieved. In this way a complete hydrolysis and thus a holistic use of the cellulose fraction can be guaranteed.
The fractions obtained are suitable for various applications. The glucose-containing fiber hydrolysate can be used by a large number of microorganisms as a substrate in fermentations. After lignin precipitation and enzymatic cleavage of the contained sugar oligomers, the hemicellulose-rich digestion solution can be used for the fermentation of microorganisms in addition to the fiber hydrolysate. Through the chosen digestion method, the fractions may contain toxic degradation products of lignocellulose such as furfural or hydroxymethyl-furfural, which can impair the growth of microorganisms.
Detoxification methods can be used to increase fermentability for such organisms. To this end, we have established an enzymatic detoxification process at the Fraunhofer IGB. A laccase of Trametes versicolor was immobilized and the minimum enzyme concentration required to remove > 80 percent of the aromatic substances from the solution was determined. It has also been shown that no laccase mediator is required for this process. The remaining toxic substances can then be removed from the fractions, e.g. by adsorption on carrier materials.
Lignin is the largest natural, regenerative source of raw materials for aromatic compounds. A material-industrial use of lignin requires a conversion of the raw material to suitable biosynthetic building blocks, which in turn can be polymerized to polyurethane or other plastics.
Various organisms are known for the natural degradation of wood and lignin-containing biomass. These are insects, white rot fungi and bacteria. These organisms produce different enzyme cocktails which are needed to degrade the complex structure of lignin. So far there is no large-scale biotechnological process for lignin degradation and modification. An essential prerequisite for the future use of biotechnological, ecological processes for the efficient utilisation of lignin is the availability of efficient and stable biocatalysts.
In addition to improving the enzymatic hydrolysability, a suitable biomass digestion should lead to a minimisation of sugar and lignin degradation products in order to guarantee the fermentability of the resulting sugar streams and to minimise the loss of yield of the sugar types. However, harsh physical-chemical digestion processes lead to loss and a reduction in the quality of individual fractions. In addition, a suitable pretreatment process should make the removed lignin and the dissolved hemicellulose usable to be able to produce secondary products such as xylitol from xylose or lignin polymers. Therefore, a number of new methods and method combinations are necessary in order to arrive at technically usable building blocks for chemical derivatives and thus to provide platform technologies for establishing a lignocellulose biorefinery.
Fraunhofer IGB is working on the eco-efficient use of lignocellulose and the establishment of a biorefinery. Optimal and large-scale digestion methods are adapted to the respective raw material and established. The lignocellulose is fractionated using the suitable digestion method and the enzymatic hydrolysis of the cellulose and hemicellulose fraction is optimized with regard to efficiency and economy. The joint project "Lignocellulose-Biorefinery II" aims to expand the spectrum of available biocatalysts for lignin degradation. For this purpose, white rot fungi and bacterial strains are examined for their suitability for fermentative lignin grading. Suitable enzymes are produced recombinantly and used for cell-free biotechnological processes.
Fraunhofer IGB has established various digestion methods for various renewable lignocellulose-based raw materials. The digestion conditions were optimally adapted for the respective raw material. The results obtained were used, among other things, to design a biorefinery pilot plant, which was subsequently implemented at the Fraunhofer Center for Chemical-Biotechnological Processes CBP in Leuna.
In addition, the saccharification of cellulose in a stirred reactor system was optimized. With this system a hydrolysis with high solids content could be carried out, whereby glucose concentrations of > 100 g/kg were achieved. With a minimum enzyme dosage, a maximum yield of 70 percent could be achieved within 48 hours.
In addition to enzymatic hydrolysis of the cellulose fiber fraction, the fermentative utilization of the sugar-containing (mainly C5 sugar) digestion solution was considered. Furthermore, our research has focused on other complementary detoxification methods to enable selective and complete removal of toxic substances. This means that the sugars obtained can be used microbially as fermentation raw material.
Subsequently, a scale-up of the process steps "digestion" and "hydrolysis" is carried out in cooperation between Fraunhofer IGB in Stuttgart and Fraunhofer CBP in Leuna up to a scale of 1 m3 in order to implement the concept of an integrated process approach from the raw material lignocellulose to product recovery in the sense of a biorefinery.
Research at Fraunhofer IGB focuses on the search for ligninolytic enzymes from fungi and bacteria. In particular, Fraunhofer IGB in Stuttgart is conducting research into the identification, characterization and provision of extracellular ligninolytic enzymes from certain stand fungi, the so-called white rot fungi. Suitable strains and combinations of strains (co-cultures) were characterized and their expression performance optimized by different media composition and inductors in order to produce lignin-splitting enzymes, such as etherases or laccases and peroxidases, in high amounts. In cooperation with the Fraunhofer CBP, the yield of ligninolytic enzymes in submers fungal cultures was further optimized and transferred to a larger scale of up to 10 liters.
In addition to fungi, ligninolytic bacteria are also considered. Both intracellular enzymes of aromatic metabolism and new potential ligninolytic enzymes were identified in the sequenced genomes of selected bacterial strains. A peroxidase of a ligninolytic bacterium has already been actively expressed and purified. In further investigations the catalytic properties of bacterial peroxidases and enzymes of the aromatic metabolism and their potential for lignin modification will be elucidated.
Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB