1. COST ACTION FP1105 Janis Gravitis Is the biosynthesis of lignins controlled by genes or physico- chemical factors? Lignins self-assembled compact or fractal cluster stuctures. Overview. Riga, Latvia, LSIWC, e-mail: jgravit@edi.lv COST MEETING Donostia-San Sebastian, May 25-27, 2015

2. From genes to cells. The case of lignin?

3. In lignin biosynthesis interpretation recently we can recognize two contradictory and sometimes aggressive to each other viewpoints: The first one is based on idea that lignin biosynthesis is the same as proteins, polysaccharides, DNA/RNA formed by condensations, thermodynamically driven by coupled dephosphorylations of nucleotide triphosphates and catalyzed by proteins giving strictly gene controlled chemical structure. That interpretation emphasize role in regio- and stereoselective monolignol radical-radical coupling is catalyzed by the dirigent proteins (DiP) discovered some years ago. (Norman G. Levis, Laurence B. Davin. 59 th Appita Annual Conference and Exhibition incorporating the 13 th ISWFPC, Vol.2, 2005).

4. The second one interprets that at least lignin and suberin are formed by oxidative coupling, where the monomers are oxidized into resonance stabilized radicals, which couple by uncatalized radical- radical reactions. Such coupling are followed by nucleophilic reactions on quinone methide intermediates. The DiPs are probably not involved in the lignin biosynthesis, since they only produce optically active products. Lignins are completely racemic polymers. In a  -ether 110-mer, there are actually only 218 optical centers, and therefore 2 117 physically distinct isomers – an astronomically large number. This is lignin random assembly combinatorial biochemistry approach. Natural lignin to be produced by slow end-wise type polymerization. (John Ralf. 59th Appita Annual Conference and Exhibition incorporating the 13th ISWFPC, Vol.2, 2005. Anders Holmgren et al. 59th Appita Annual Conference and Exhibition incorporating the 13th ISWFPC, Vol.3, 2005. ).

5. Freeze fracture replicas of rosettes terminal complexes associated with cellulose microfibril biogenesis. C. Haigler, unpublished data, adopted from D. Delmer

6. Hypothetical structure of the terminal proteins nano-machine for cellulose synthesis according to D. Delmer

9. Computer simulation of DL Agreagation of P-Cl (Vitten-Sander model)

10. It was concluded: the Witten-Sander diffusion-limited particle-cluster aggregation (DLA P-Cl) model approximated growing lignin fragments. DLA P-Cl is the universal model for the systems formed under the following limiting conditions: the lignin C 9 (~ 0.7 nm) monomer makes the Brownian motion to approach a growing lignin fragment. The growing cluster is much larger than the unit, and the probability of joining after the particle C 9 and the growing cluster contact is sufficiently high to add the particle to the exterior part of the cluster without entering its interior part. Existing carbohydrates environment creates diffusion barrier for monomer movement. The phenylpropane free radical generated from C 9 random recombination in vivo and in vitro satisfies such DLA P-CL limiting conditions. The model allows the cycles inside the branches, and growing proceeds in non-equilibrium conditions. The DLA P-Cl model describes adequately the lignin structure in wall layer S 2 for the degree of a polymerization range of  20 – 100 (~14-70 nm) with the scaling indexes: fractal dimension  2.5, spectral dimension, which characterizes the relaxation process in the system, is  1.2 - 1.4 and minimal dimension which characterizes the connection of the C 9 monomers is one.

11. Lignin in wood S 2 layer is a network of weakly connected polydisperse DLA fractals. For a lignin clusters with a scale less as DP 20, a compact structure with D f = 3 seems to be more possible. This model concerns the inter-cell wall layer ML (SAXS, USAXS).

12. Recently (Ulla Vainio, Ritva Serimaa et al.), lignin fractal dimension is measured using small- (SAXS) and ultra small- (USAXS) X-ray scattering. SAXS is performed at the X-ray laboratory of the University of Helsinki and USAX by using synchrotron facilities in Hamburg and Grenoble. The length scale for SAXS is 0.4 – 20nm.

13. X-RAY DIFFRACTOMETER at Department of Physical Sciences, University of Helsinki Basic structure: crystalline, amorphous

15. Grenoble 844 m diameter

16. Measurements show that the SAXS intensities obey a power law at small values of q, where q is the length of the scattering vector, at room temperature. The SAXS intensities were corrected for absorption and air scattering, and the wide-angle x-ray scattering (WAXS) background was subtracted. A mass fractal with mass fractal dimension D­m can be shown to give a scattering law of the form I  q - , where  is Dm (mass) and a surface fractal of surface fractal dimension Ds obeys also a power law with  = 6 - Ds. For a mass fractal Ds = Dm and for a surface fractal Dm is always 3. For compact particle with a sharp phase boundary Dm = 3, Ds = 2 and  = 4. Porous solids with continuous charge density transition have  > 4. However, measurements concern solid sedimented lignins. So, there is second aggregation of primary nano-particles. For instance, usually we can recognize secondary lignin aggregation spherical particles on the cell wall after steam explosion autohydrolysis (SEA) treatment.

18. SAXS curves of different lignins (Ulla Vainio et al. 2004)

19. Kerr and Goring

21. (J. Gravitis, A. Kokorevics & V. Ozols-Kalnins)

22. Terachima honey-comb arrangement model with beads-like lignin modules, 2005

23. Free and associated CEF (~4-10 nm) after SE treatment. TEM. Urve Kallavus & Janis Gravitis

24. Secondary aggregates of the primary lignin nano-clusters on wood cell wall after SE treatment. TEM. Urve Kallavus & Janis Gravitis

26. Conclusions The hierarchical cell wall polymers bio-composite structure recently is studied from the viewpoint of nano-materials and nano-technologies. In current study the steam explosion autohydrolysis (SEA) have been used as a method for exposing main polymer components biodegradable nano- particles. TEM, SEM, solid-state 13 C CP/MAS NMR proton spin-lattice relaxation time T 1H measurements, SAXS, USAXS gave strong evidence that the cell walls during SEA treatment segregates in the nano-domains, where primary nano- particles are cellulose nano-fibrils and lignin compact or fractal type nano- clusters. The latter studies are disturbed by secondary aggregation of primary structures. Studies of natural nano-cell walls structure and dynamics open diverse opportunities for learning natural nano-composites and natural nano- technologies. The nano-machine – cellulose rosettes type enzyme complex role in cellulose nano-fibrils synthesis is futuristic challange for development.

27. Applied aspects of wood industry nanotechnology The multidisciplinary nature of nanotechnology makes the exploitation of new technologies and ideas from other clusters especially important. The nanotechnologies reduced consumption of materials, emissions and facilitate decrease of impact to environment. Nano-approach increase the quality of life. Opportunities for nanotechnologies in cellulose industry and composite materials containing cellulose and/or lignin. Opportunities for nanotechnologies in paper industry and other industries closely related to papermaking – such as printing and packaging. Opportunities for nanotechnologies in wood protection. Commercial development is still hindered by a “chicken-and-egg”scenarios where are no applications of the new nano-materials and as a consequence is lack of production capacity to allow applications development.