Sourcing and bioprocessing of brown seaweed for maximizing glucose release

The research undertaken for this PhD thesis has been part of a larger research program “The MacroAlgaeBiorefinery – sustainable production of third generation (3G) bioenergy carriers and high value aquatic fish feed from macroalgae (MAB3)”. The research has been based on the overall hypothesis that brown seaweeds represent a huge unexploited bioresource of the sea which can be upgraded to energy carriers via degradation to fermentable sugars. The research in the PhD thesis has aimed at optimizing pretreatment and enzymatic saccharification of Saccharina latissima and Laminaria digitata to release maximum levels of glucose. The first requirement was to develop a robust methodology, including acid hydrolysis and analytical composition analysis, to quantitatively estimate the carbohydrate composition of the brown seaweeds. The monosaccharide composition of four different samples of brown seaweeds Laminaria digitata and Saccharina latissima were compared by different high performance anion exchange chromatography (HPAEC) methods after 3 different acid hydrolysis treatments or a cellulase treatment. HPAEC analysis with pulsed amperometric detection (PAD) preceded 2-step pretreatment with 72 % sulfuric acid (H2SO4) for 1 h at 30 °C and followed by 4 % H2SO4 at 120 °C for 40 min allowed quantitative determination of the carbohydrate composition of brown seaweed. The use of guluronic, glucuronic and galacturonic acid standards enabled quantification of the uronic acids. The variation in the biochemical composition of four populations of Saccharina latissima and Laminaria digitata from three different locations from Danish waters was documented. The chemical composition of brown seaweed varied mainly in regard to the season but differed also with respect to species, location, between the years and even within the population. Concentrations of ash and protein levels varied inversely to the carbohydrate levels, and total carbohydrate concentration varied seasonally, in particular through the storage of carbohydrates glucose and mannitol. Generally, alginate was the most abundant carbohydrate at all sites from December to summer with up to 36 % w/wDM by weight before glucose levels were at least at the same magnitude. Total alginate concentration was relatively independent of seasonal changes but mannuronic (M) and guluronic acid (G) differed strongly throughout the year. M/G ratios varied regarding season, species or location from 1.3 to 3.6 but without a general pattern. The highest concentrations of glucan were found in August for wild growing L. digitata from the North Sea, with the glucose potential lying >50 % w/wDM for three sequential years (2012-2014) accompanied by mannitol levels of about 10 % w/wDM and low ash levels of 10-11 % w/wDM. Generally spoken, glucose levels of L. digitata appeared to be superior to those of S. latissima. Cultivation of S. latissima in the Limfjorden, Denmark to obtain high glucan levels was not possible due to the incidence of biofouling in the summer. The average N-to-protein conversion factor was 3.7 but ranged from 2.1 to 5.9. Hence, application of a common factor cannot be recommended since total nitrogen content was more variable than the protein content. Post washing L. digitata harvested from the Danish North Sea in August 2012 had a total organic matter of 84 % mostly accounted for glucose (51 % w/wDM), including a smaller contribution of mannitol (8 % w/wDM), making this material an ideal feedstock for biocatalytical processing to achieve maximum glucose release. The influence of milling as pretreatment to enhance enzymatic degradation was studied on the glucan rich L. digitata (North Sea, August 2012). Wet refiner milling, using rotating disc distances of 0.1-2 mm, generated differently sized particle populations with particles having decreasing average surface area (100-0.1 mm2) with increased milling severity. Milling with disc distances below the thickness of the algae (≤1 mm) increased the particle volume of the milled seaweed slurries and higher milling severity (lower rotating disc distance) also induced higher carbohydrate solubilization from the material, particularly for glucan and mannitol. However, particle size diminution did not improve the enzymatic glucose release. Milling was thus not required for enzymatic saccharification because all available glucose was released even from unmilled material during the combined treatment of alginate lyase and the cellulase preparation Cellic®CTec2. Apparently, the alginate lyase (Sigma Aldrich) activity catalyzed the cleavage of alginate on the substrate, which both decreases the viscosity of the substrate alginate and catalytically solubilizes the alginate to provide access to the glucan in the brown seaweed cell wall matrix. The impact of alginate lyase in addition to cellulase on the brown seaweed degradation was studied further for L. digitata degradation. Therefore, two bacterial alginate endo-lyases (EC 4.2.2.-) from Sphingomonas sp. (SALy) and Flavobacterium sp. (FALy) were selected for heterologous, monocomponent expression in Escherichia coli. The optimal pH range for SALy was pH 5.5-7.0 with optimum at pH 6. The optimum for FALy and the commercially available alginate lyase from Sigma Aldrich (SigmALy) was pH 7.5. The investigated reaction temperatures of 30-50 °C had no influence on the activity. The thermal stability was reduced above 50 °C, for SigmALy above 40 °C. The FALy preferred poly-mannuronic acid as substrate, but also exhibited activity on poly-guluronic acid, whereas SALy had higher activity on poly-guluronic acid and SigmALy was only active on poly-guluronic acid. Subsequently, the alginate lyases were applied together with the commercial, fungally derived cellulase preparation Cellic®CTec2 at pH 6 and 40 °C on the glucan rich L. digitata. A decrease in viscosity decrease ensued in the initial minutes while alginate degradation occurred primarily within the first 1-2 hours of reaction. The level of released mannuronic acid blocks was inversely proportional to the glucose release indicating that the degradation of mannuronic acid blocks inhibited the cellulase catalyzed glucose release from L. digitata. Only the selective activity of SigmALy on guluronic acid enabled a 90 % glucose release within 8 hours by the cellulase preparation Cellic®CTec2. Nevertheless, combined alginate lyase and cellulase treatment for 24 hours released all potential glucose regardless of the applied lyase. Treatment with a mixture of 1 % w/wDM SigmALy and 10 % v/wDM Cellic®CTec2 at pH 5 and 40 °C released the available glucose during 8 hours. Two-thirds of the glucose was released with lower enzyme loading. Simple application of only the cellulase preparation enabled the release of only half of the present glucose after 8 h. Analysis after the enzymatic treatment indicated a potential extraction of proteins from the solid residue and the sulfated polysaccharide fucoidan solubilized in the saccharified liquid. The results of this PhD study demonstrated that brown seaweed can be completely degraded enzymatically by combined cellulase and alginate lyase treatment after milling. The work also showed, that biorefining of brown seaweed with current state of art technology is highly dependent on the cultivation, in particular growth site and season, of a suitable feedstock for achieving maximal glucan content and in turn allowing maximum glucose release.