• Energy Research

Plant cell walls have protective and structural functions conferring resistance to degradation. The lignin and hemicellulose network surrounding the cellulose microfibrils is insoluble unless subjected to harsh treatments. As lignin, pectin and xylan are effective barriers to cellulose extraction and hydrolysis, reducing their presence in cell walls improves saccharification. Microorganisms that can depolymerise lignin are of extreme interest to the biofuel industry. White rot fungi can be effective in pretreatment of lignocellulosic biomass prior to saccharification. Here, we show the cumulative effects of pretreating biomass with two white rot fungi, Phanerochaete chrysosporium and Trametes cingulata, on tobacco lines with reduced lignin or xylan, caused by suppression of the CINNAMOYL-CoA REDUCTASE, CINNAMATE-4-HYDROXYLASE, TOBACCO PEROXIDASE 60 or UDP-GLUCURONATE DECARBOXYLASE and on Arabidopsis thaliana with reduced de-esterified homogalacturonan content, obtained by overexpressing a pectin methyl esterase inhibitor or constitutively expressing the Aspergillus niger POLYGALACTURONASE II gene. Tests were extended to fresh material from an Arabidopsis mutant for a cell wall peroxidase. We demonstrate that fungal pretreatment is a reliable method of improving cellulose accessibility in biofuel feedstocks, fresh material and cell wall residues from different plants. These results contribute to the understanding of the consequences of primary and secondary cell wall perturbations on lignocellulosic biomass accessibility to white rot fungi and on saccharification yield. A comparison of the effects of P. chrysosporium and T. cingulata on tobacco saccharification also highlights the limitation of current knowledge in this research field and the necessity to systematically test culture conditions to avoid generalisations.

One of the main limitations of commercial production of algae is the cost of cultivation, largely attributed to the cost of the nutrients; hence, finding cheap alternative substrates has been a significant line of research in this field. The dairy industry produces large amounts of wastewater that might be used as a cost-effective nutrient alternative, containing lactose as a main carbon source, as well as other essential nutrients like nitrogen and phosphate. Nevertheless, just a few algal species of commercial value can grow on any organic carbon sources, and even less are able to utilize lactose. In this work we have identified a Parachlorella kessleri strain capable of utilizing lactose for growth, and have characterized its ability to accumulate metabolites of commercial interest under heterotrophic growth conditions. P. kessleri was capable to utilize lactose from dairy wastewater, and to accumulate several amino acids, dicarboxylic acids, such as tartaric acid and succinic acid, and triacylglycerols in heterotrophic conditions. These metabolites have applications in the food, feed, and pharmaceutical industries, and in the green chemical industry for the production of bio-based green polymers and biofuels. The significance of these findings for future product development is discussed.

Lignocellulosic biomass from agriculture wastes is a potential source of biofuel, but its use is currently limited by the recalcitrance of the plant cell wall to enzymatic digestion. Modification of the wall structural components can be a viable strategy to overcome this bottleneck. We have previously shown that the expression of a fungal polygalacturonase (pga2 from Aspergillus niger) in Arabidopsis and tobacco plants reduces the levels of de-esterified homogalacturonan in the cell wall and significantly increases saccharification efficiency. However, plants expressing pga2 show stunted growth and reduced biomass production, likely as a consequence of an extensive loss of pectin integrity during the whole plant life cycle. We report here that the expression in Arabidopsis of another pectic enzyme, the pectate lyase 1 (PL1) of Pectobacterium carotovorum, under the control of a chemically inducible promoter, results, after induction of the transgene, in a saccharification efficiency similar to that of plants expressing pga2. However, lines with high levels of transgene induction show reduced growth even in the absence of the inducer. To overcome the problem of plant fitness, we have generated Arabidopsis plants that express pga2 under the control of the promoter of SAG12, a gene expressed only during senescence. These plants expressed pga2 only at late stages of development, and their growth was comparable to that of WT plants. Notably, leaves and stems of transgenic plants were more easily digested by cellulase, compared to WT plants, only during senescence. Expression of cell wall-degrading enzymes at the end of the plant life cycle may be therefore a useful strategy to engineer crops unimpaired in biomass yield but improved for bioconversion.

Plant cell walls represent an abundant, renewable source of biofuel and other useful products. The major bottleneck for the industrial scale-up of their conversion to simple sugars (saccharification), to be subsequently converted by microorganisms into ethanol or other products, is their recalcitrance to enzymatic saccharification. We investigated whether the structure of pectin that embeds the cellulose-hemicellulose network affects the exposure of cellulose to enzymes and consequently the process of saccharification. Reduction of de-methyl-esterified homogalacturonan (HGA) in Arabidopsis plants through the expression of a fungal polygalacturonase (PG) or an inhibitor of pectin methylesterase (PMEI) increased the efficiency of enzymatic saccharification. The improved enzymatic saccharification efficiency observed in transformed plants could also reduce the need for acid pretreatment. Similar results were obtained in PG-expressing tobacco plants and in PMEI-expressing wheat plants, indicating that reduction of de-methyl-esterified HGA may be used in crop species to facilitate the process of biomass saccharification.