Project description:The recalcitrance of lignocellulose makes enzymatic hydrolysis of plant biomass for the production of second generation biofuels a major challenge. This work investigates an efficient and economic approach for the enzymatic hydrolysis of sugar beet pulp (SBP), which is a difficult to degrade, hemicellulose-rich by-product of the table sugar industry. Three fungal strains were grown on different substrates and the production of various extracellular hydrolytic and oxidative enzymes involved in pectin, hemicellulose, and cellulose breakdown were monitored. In a second step, the ability of the culture supernatants to hydrolyze thermally pretreated SBP was tested in batch experiments. The supernatant of Sclerotium rolfsii, a soil-borne facultative plant pathogen, was found to have the highest hydrolytic activity on SBP and was selected for further hydrolyzation experiments. A low enzyme load of 0.2 mg g(-1) protein from the culture supernatant was sufficient to hydrolyze a large fraction of the pectin and hemicelluloses present in SBP. The addition of Trichoderma reesei cellulase (1-17.5 mg g(-1) SBP) resulted in almost complete hydrolyzation of cellulose. It was found that the combination of pectinolytic, hemicellulolytic, and cellulolytic activities works synergistically on the complex SBP composite, and a combination of these hydrolytic enzymes is required to achieve a high degree of enzymatic SBP hydrolysis with a low enzyme load.
Project description:An example of the implementation of the principles of the circular economy is the use of sugar beet pulp as animal feed. Here, we investigate the possible use of yeast strains to enrich waste biomass in single-cell protein (SCP). The strains were evaluated for yeast growth (pour plate method), protein increment (Kjeldahl method), assimilation of free amino nitrogen (FAN), and reduction of crude fiber content. All the tested strains were able to grow on hydrolyzed sugar beet pulp-based medium. The greatest increases in protein content were observed for Candida utilis LOCK0021 and Saccharomyces cerevisiae Ethanol Red (ΔN = 2.33%) on fresh sugar beet pulp, and for Scheffersomyces stipitis NCYC1541 (ΔN = 3.04%) on dried sugar beet pulp. All the strains assimilated FAN from the culture medium. The largest reductions in the crude fiber content of the biomass were recorded for Saccharomyces cerevisiae Ethanol Red (Δ = 10.89%) on fresh sugar beet pulp and Candida utilis LOCK0021 (Δ = 15.05%) on dried sugar beet pulp. The results show that sugar beet pulp provides an excellent matrix for SCP and feed production.
Project description:This study was demonstrated with a coculture fermentation system using sugar beet pulp (SBP) as a carbon source combining the cellulose-degrading bacterium Clostridium cellulovorans with microbial flora of methane production (MFMP) for the direct conversion of cellulosic biomass to methane (CH4). The MFMP was taken from a commercial methane fermentation plant and extremely complicated. Therefore, the MFMP was analyzed by a next-generation sequencing system and the microbiome was identified and classified based on several computer programs. As a result, Methanosarcina mazei (1.34% of total counts) and the other methanogens were found in the MFMP. Interestingly, the simultaneous utilization of hydrogen (H2) and carbon dioxide (CO2) for methanogenesis was observed in the coculture with Consortium of C. cellulovorans with the MFMP (CCeM) including M. mazei. Furthermore, the CCeM degraded 87.3% of SBP without any pretreatment and produced 34.0 L of CH4 per 1 kg of dry weight of SBP. Thus, a gas metabolic shift in the fermentation pattern of C. cellulovorans was observed in the CCeM coculture. These results indicated that degradation of agricultural wastes was able to be carried out simultaneously with CH4 production by C. cellulovorans and the MFMP.
Project description:Valorization of the lignocellulosic side and waste streams is key to making industrial processes more efficient from both an economic and ecological perspective. Currently, the production of sugars from beets results in pulps in large quantities. However, there is a lack of promising opportunities for upcycling these materials despite their promising properties. Here, we investigate beet pulps from two different stages of the sugar manufacturing process as raw materials for supercapacitor electrodes. We demonstrate that these materials can be efficiently converted to activated, highly porous carbons. The carbons exhibit pore dimensions approaching the size of the desolvated K+ and SO42- ions with surface areas up to 2600 m2 g-1. These carbons were subsequently manufactured into electrodes, assembled in supercapacitors, and tested with environmentally friendly aqueous electrolytes (6 M KOH and 1 M H2SO4). Further analysis demonstrated the presence of capacitance-enhancing functionalities, and up to 193 and 177 F g-1 in H2SO4 and KOH, respectively, were achieved, which outperformed supercapacitors prepared from commercial YP80 F. Overall, our study suggests that side streams from sugar manufacturing offer a hidden potential for use in high-performance energy storage devices.
Project description:Pectic oligosaccharides (POS) have been indicated as novel candidate prebiotics. Traditionally, POS are produced from pectin-rich by-products using a two-step process involving extraction of the pectin, followed by its hydrolysis into POS. A one-step approach, in which the POS is directly produced from the raw material, might provide a more efficient alternative. Thus, the main aim of this paper was to investigate a one-step enzymatic hydrolysis approach to directly produce POS from sugar beet pulp (SBP). The POS yield was investigated as a function of the process parameters, as well as raw material characteristics. A statistically-based response surface methodology, using a central composite design was applied, to investigate the individual as well as the combined influences of the diverse parameters. The model was confirmed by a validation experiment, carried out at 135 g/l substrate concentration, 0.75 FPU/g SBP enzyme concentration, 0.8 mm particle size and 3 h hydrolysis time. Under these conditions, a POS-rich hydrolysate was obtained, containing rhamnose, arabinose, galactose, xylose and galacturonic acid, at 0.9, 15.2, 5.1, 1.4, and 13.2 g/l, respectively, enzymes were added each at 20 FPU/g dry matter (DM).
Project description:BACKGROUND:Enzymatic degradation of plant biomass requires a complex mixture of many different enzymes. Like most fungi, thermophilic Myceliophthora species therefore have a large set of enzymes targeting different linkages in plant polysaccharides. The majority of these enzymes have not been functionally characterized, and their role in plant biomass degradation is unknown. The biotechnological challenge is to select the right set of enzymes to efficiently degrade a particular biomass. This study describes a strategy using sexual crossing and screening with the thermophilic fungus Myceliophthora heterothallica to identify specific enzymes associated with improved sugar beet pulp saccharification. RESULTS:Two genetically diverse M. heterothallica strains CBS 203.75 and CBS 663.74 were used to generate progenies with improved growth on sugar beet pulp. One progeny, named SBP.F1.2.11, had a different genetic pattern from the parental strains and had improved saccharification activity after the growth on 3 % sugar beet pulp. The improved SBP saccharification was not explained by altered activities of the major (hemi-)cellulases. Exo-proteome analysis of progeny and parental strains after 7-day growth on sugar beet pulp showed that only 17 of the 133 secreted CAZy enzymes were more abundant in progeny SBP.F1.2.11. Particularly one enzyme belonging to the carbohydrate esterase family 5 (CE5) was more abundant in SBP.F1.2.11. This CE5-CBM1 enzyme, named as Axe1, was phylogenetically related to acetyl xylan esterases. Biochemical characterization of Axe1 confirmed de-acetylation activity with optimal activities at 75-85 °C and pH 5.5-6.0. Supplementing Axe1 to CBS 203.75 enzyme set improved release of xylose and glucose from sugar beet pulp. CONCLUSIONS:This study identified beneficial enzymes for sugar beet pulp saccharification by selecting progeny with improved growth on this particular substrate. Saccharification of sugar beet pulp was improved by supplementing enzyme mixtures with a previously uncharacterized CE5-CBM1 acetyl xylan esterase. This shows that sexual crossing and selection of M. heterothallica are the successful strategy to improve the composition of enzyme mixtures for efficient plant biomass degradation.
Project description:Degradation of plant biomass to fermentable sugars is of critical importance for the use of plant materials for biofuels. Filamentous fungi are ubiquitous organisms and major plant biomass degraders. Single colonies of some fungal species can colonize massive areas as large as five soccer stadia. During growth, the mycelium encounters heterogeneous carbon sources. Here we assessed whether substrate heterogeneity is a major determinant of spatial gene expression in colonies of Aspergillus niger. We analyzed whole-genome gene expression in five concentric zones of 5-day-old colonies utilizing sugar beet pulp as a complex carbon source. Growth, protein production and secretion occurred throughout the colony. Genes involved in carbon catabolism were expressed uniformly from the centre to the periphery whereas genes encoding plant biomass degrading enzymes and nitrate utilization were expressed differentially across the colony. A combined adaptive response of carbon-catabolism and enzyme production to locally available monosaccharides was observed. Finally, our results demonstrate that A. niger employs different enzymatic tools to adapt its metabolism as it colonizes complex environments.
Project description:In nature, filamentous fungi are exposed to diverse nutritional sources and changes in substrate availability. Conversely, in submerged cultures, mycelia are continuously exposed to the existing substrates, which are depleted over time. Submerged cultures are the preferred choice for experimental setups in laboratory and industry and are often used for understanding the physiology of fungi. However, to what extent the cultivation method affects fungal physiology, with respect to utilization of natural substrates, has not been addressed in detail. Here, we compared the transcriptomic responses of Aspergillus niger grown in submerged culture and solid culture, both containing sugar beet pulp (SBP) as a carbon source. The results showed that expression of CAZy (Carbohydrate Active enZyme)-encoding and sugar catabolic genes in liquid SBP was time dependent. Moreover, additional components of SBP delayed the A. niger response to the degradation of pectin present in SBP. In addition, we demonstrated that liquid cultures induced wider transcriptome variability than solid cultures. Although there was a correlation regarding sugar metabolic gene expression patterns between liquid and solid cultures, it decreased in the case of CAZyme-encoding genes. In conclusion, the transcriptomic response of A. niger to SBP is influenced by the culturing method, limiting the value of liquid cultures for understanding the behavior of fungi in natural habitats. IMPORTANCE Understanding the interaction between filamentous fungi and their natural and biotechnological environments has been of great interest for the scientific community. Submerged cultures are preferred over solid cultures at a laboratory scale to study the natural response of fungi to different stimuli found in nature (e.g., carbon/nitrogen sources, pH). However, whether and to what extent submerged cultures introduce variation in the physiology of fungi during growth on plant biomass have not been studied in detail. In this study, we compared the transcriptomic responses of Aspergillus niger to growth on liquid and solid cultures containing sugar beet pulp (a by-product of the sugar industry) as a carbon source. We demonstrate that the transcriptomic response of A. niger was highly affected by the culture condition, since the transcriptomic response obtained in a liquid environment could not fully explain the behavior of the fungus in a solid environment. This could partially explain the differences often observed between the phenotypes on plates compared to liquid cultures.
Project description:Penicillium purpurogenum is a filamentous fungus, which grows on a variety of natural carbon sources and secretes a large number of enzymes involved in cellulose, hemicelluloses and pectin biodegradation. The purpose of this work has been to identify potential lignocellulolytic enzymes and to compare the secreted enzymes produced when the fungus is grown on sugar beet pulp (rich in cellulose and pectin) and corn cob (rich in cellulose and xylan). Culture supernatants were subjected to two-dimensional nano-liquid chromatography/tandem mass spectrometry. Using MASCOT and a genome-derived protein database, the proteins present in the supernatant were identified. The putative function in the degradation of the polysaccharides was determined using dbCAN software. The results show that there is a good correlation between the polysaccharide composition of the carbon sources and the function of the secreted enzymes: both cultures are rich in cellulases, while sugar beet pulp induces pectinases and corncob, xylanases. The eventual biochemical characterisation of these enzymes will be of value for a better understanding of the biodegradation process performed by the fungus and increase the availability of enzymes for biotechnological methods associated with this process.