Bio4Energy would like to acknowledge its researchers who contributed a presentation to the Lignin 2014 conference, held 24-28 August at Umeå, Sweden.

Lignin-2014 Sandra-Winestrand Photo Anna-Strom 265x177Bio4Energy researcher Sandra Winestrand of Umeå University and the Billerud-Korsnäs group.'Smart' packaging designed to prolong the shelf-life of food

Sandra Winestrand and colleagues at Umeå and Karlstad Universities

Edited abstract: Extending the shelf-life of packaged food is a potential way of reducing food waste. One possible way to do this is by using a system that scavenges the oxygen inside a package equipped with an oxygen barrier. A common way to scavenge oxygen inside a package is to insert a small sachet containing iron powder. An alternative to this is to use oxygen-scavenging enzymes that can be incorporated directly in the coating layer of the package.

The phenol-oxidising enzyme laccase uses molecular oxygen as its oxidising substrate, and could therefore be used for the latter type of application. Laccase can use derivatives of lignin as its reducing substrate, which would be interesting from a biorefinery perspective since lignin derivatives are underused co-products in biorefinery based on lignocellulosic feedstock. The aims of the investigation were to understand how the properties of lignin derivatives affected the enzymatic reaction and the quality of the coating layer.

The study involved the use of lignin derivatives and preparations of size-fractionated lignin derivatives from industrial processing of lignocellulose. The molecular properties of the lignin derivatives before and after oxidation by laccase were investigated, as well as the capability of films and coatings to scavenge oxygen. The results indicate that laccase-catalysed cross-linking decreases oxygen levels and improves the water stability of the packaging material.

Ed’s notes: In Chemistry, a scavenger can be a substance added to a mixture to remove or inactivate impurities. A substrate, in Biochemistry, is the substance acted on by an enzyme. A lignin derivate may be seen as a product of lignin.  

The following co-authors are acknowledged: Thomas Gillgren, Kristin Johansson, Lars Järnström and Leif Jönsson.

Henrik-SerkBio4Energy researcher Henrik Serk, Umeå University and the Umeå Plant Science Centre.'Cooperative' lignification of Arabidopsis xylem vessels

Henrik Serk and colleagues at Umeå University (UmU) and Swedish University of Agricultural Sciences (SLU)

Edited abstract: Lignin is the second most abundant natural polymer on earth and is found in the secondary cell wall of specialised cells such as xylem tracheary elements (TEs) which transport water and minerals in vascular land-living plants. This study was performed to improve the understanding of TE lignification at the cellular level. The researchers used xylogenic cell suspension cultures of the model plant Arabidopsis thaliana. Such cell cultures may be created by adding hormones to differentiate in 40-50 per cent of lignified TEs, while the rest of the cells remain parenchymatic (non-TEs).

Bio4Energy researcher This email address is being protected from spambots. You need JavaScript enabled to view it. and colleagues recently showed that TE lignification in xylogenic cell suspensions of Zinnia elegans occurrs after TE programmed cell death (PCD), in a non-cell autonomous manner, during which non-TEs provide lignin monomers to dead TEs. The researchers also found that TE secondary cell wall lignification in Arabidopsis thaliana progressed linearly for several weeks after TE PCD.

To assess the cooperative supply of lignin monomers, phenolic compounds were quantified both in intra- and extracellular spaces along TE formation lines. Extracellular phenolics increased only in the cultures induced to become TEs and appeared to be the limiting factor controlling the extent of TE post-mortem lignification. To demonstrate that non-TEs export lignin precursors, monomer synthesis was initially inhibited, which resulted in unlignified TEs. When the inhibition was removed after all the TEs were dead (only non-TEs remained alive), TEs lignified. This confirms that phenolics exported by non-TEs are lignin precursors. Metabolomic analysis of the extracellular phenolics revealed an increase of lignin intermediates (oligomers), essentially after TE PCD, which confirms the post-mortem progression of TE lignification. Taken together our results confirm that TE lignification in Arabidopsis thaliana occurs post-mortem. They also show that the export of lignin monomers to the apoplast, where TE lignification occurs, is made possible by the activity of parenchymatic cells. 

Ed’s notes: Arabidopsis thaliana and Zinnia elegans are used as a model plants in biomass feedstock research. An apoplast is the free diffusional space outside the plasma membrane of a plant cell. "Cooperative supply" in this context refers to the fact that several types of cells have been shown to contribute to the lignification of xylem vessels in wood.

The following co-authors are acknowledged: Delphine Ménard, Ilka Nicif Abreu, Thomas Moritz and Edouard Pesquet.

Characterisation of lignin-carbohydrate complexes in poplar woodPeter-ImmerzeelBio4Energy researcher Peter Immerzeel of the Swedish University of Agricultural Science and the Umeå Plant Science Centre.

Peter Immerzeel and colleagues at the SLU/Umeå Plant Science Centre (UPSC) and UmU

Edited abstract: Wood is mainly composed of cellulose, various hemicelluloses and lignin. There are structural bonds between these polymers that give rigidity to the cell wall. This is of importance for the living plant. However, the same bonds make processing and purification of cell wall material complicated during pulping, biofuel production or biorefinery involving the production of other "green" chemicals.

We want to study in more detail one group of bonds in the cell wall namely the lignin-carbohydrate complexes (LCCs). Most of the lignin is coupled with cell wall carbohydrates. However, in this large complex only a small number of the primary lignin precursors are covalently linked to carbohydrate polymers. This complicates the analysis of these bonds. It is of scientific interest to have a better understanding of the chemical nature and localisation of LCC formation during wood development and which polysaccharides are involved in these bonds. From an applied perspective more knowledge on LCCs could support the development of more efficient procedures for the breakdown of LCCs in the process of pulp purification or of improved saccharification of wood for the production of biofuels.    

During the first part of this project a robust procedure will be developed for the efficient separation of mature poplar wood polysaccharides and chemical characterisation of the polysaccharides and attached lignin. LCCs present in these fractions will be characterised with Heteronuclear Single Quantum Coherence (1H-13C HSQC) NMR. This procedure will be used to analyse wood from poplar trees grown in a greenhouse. The motivation for choosing poplar trees as a study object, is to lay bare the process of LCC formation in readily available poplar mutants which have an altered amount of hemicellulose (xylan) or lignin and compare the mutants with those of trees that grow in the wild.

Ed's note: In Chemistry, a covalent bond is the bond formed by the sharing of a pair of electrons by two atoms.

The following co-authors are acknowledged: Junko Takahashi-Schmidt, Mattias Hedenström, Björn Sundberg and Lorenz Gerber.

Carlos-MartínBio4Energy researcher Carlos Martín of Umeå University.Solubilisation of lignin and xylan during sulphuric acid-assisted glycerol pre-treatment of sugarcane bagasse

Carlos Martín of UmU and colleagues from Hamburg University and the Thünen Institute for Wood Research

Edited abstract: The potential of a combined pre-treatment with glycerol and sulphuric acid for increasing the enzymatic convertibility of sugarcane bagasse cellulose has recently been demonstrated (Martín et al., 2013). Lignin and xylan are partially solubilised during this pre-treatment. In the current work, a central composite rotatable experimental design (CCRED) was applied to evaluate the main and interactive effects of glycerol concentration (55.4 – 79.6%), sulphuric acid concentration (0.0 – 1.1%), temperature (150 – 199oC), and pre-treatment time (0.7 – 2.3 h) on the solubilisation of lignin and xylan. Xylan solubilisation, ranging between six and 94 per cent, rose significantly with the increase of temperature, time and H2SO4 concentration, but dropped with the increase of glycerol amount. Glycerol restricted the solubilisation and full hydrolysis of xylans and the degradation of xylose. Lignin solubilisation (20.6 – 49.4%) rose with the increase of all the experimental factors. A model based on the experimental results predicted maximal lignin solubilisation at 187.7oC, 2.3 h, 79.6 per cent glycerol, 0.64 per cent H2SO4. These rates were confirmed in control experiments.

Ed’s note: Hydrolysis may be defined briefly as a chemical reaction in which the interaction of a compound with water results in the decomposition of that compound. It can also be thought of as a process by which a compound is split into fragments with the addition of water, such as is used to break polymers down to smaller units. Xylan is a type of hemicellulose.

The following co-authors are acknowledged: Jürgen Puls, Andreas Schreiber and Bodo Saake.

Sacha-EscamezBio4Energy researcher Sacha Escamez of Umeå University and the Umeå Plant Science Centre.PERSEPHONE-interacting-bHLH transcription factor controls expression of lignin biosynthetic genes

Sacha Escamez and colleagues at UmU/the UPSC

One the one hand, wood represents an abundant and renewable source of biomass because most of its cells deposit thick secondary cell walls (SCWs) rich in carbohydrates. On the other, the SCWs also contain large amounts of lignin; its recalcitrance must be overcome for the production of pulp or biofuels. Therefore our strategy has been to identify and engineer biological regulators of xylem lignification. We have already identified the protein PERSEPHONE and the PERSEPHONE-interacting-bHLH transcription factor (PIB) as putative regulators of lignification.

Indeed, we have seen that lignin composition is affected in plants lacking PERSEPHONE or PIB. Plants with no or low PIB tend to have a reduced lignin content compared with plants in which PIB levels are normal. Consequently, in our study the absence of PIB protein resulted in a decrease in expression of several genes involved in lignin monomer biosynthesis. Hence our preliminary results suggest that PIB, possibly in a complex with PERSEPHONE, is a regulator of lignification acting at the level of gene expression. Despite having less lignin, the plants lacking PIB seemed to grow normally. In the context of this study, this renders PIB an attractive target for genetic engineering of lignin.

The following co-authors are acknowledged: Bo Zhang and Hannele Tuominen.

Thomas-GillgrenBio4Energy researcher Thomas Gillgren of Umeå University.Studying lignin reactions in real-time

Thomas Gillgren and colleagues at UmU

Despite a lot of effort and great successes in the field of lignin research, there are still aspects of chemical reactions involving lignin that are unknown. This is true both for lignification in plants and for industrial processes used to degrade lignin. A deeper understanding of these reactions would give us even greater insight.

Fourier Transform Infrared (FTIR) Spectroscopy is a tool for the characterisation of lignin. It has been used for decades in the field of lignin analysis. Notably, it may be employed to obtain information about functional groups. The samples studied with FTIR have generally been powders extracted from lignin derivatives or milled wood. By contrast, we have developed a method which allows us to watch reactions take place in real-time.

For data processing we use Multivariate Curve Resolution – Alternating Least Squares (MCR-ALS). This kind of data analysis identifies the components that contribute to each spectrum, as well as their concentration changes over time. This allows us to monitor, simultaneously, changes in concentration, chemical composition and structure of all the reaction components, including intermediates and final products.

Ed's note: In Chemistry, degrade can mean "to break down" or "to decompose".

The following co-authors are acknowledged: Andras Gorzsas and Leif Jönsson.

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