Browsing by Subject "Lignocellulose"

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  • Biswal, Ajaya K.; Soeno, Kazuo; Gandla, Madhavi Latha; Immerzeel, Peter; Pattathil, Sivakumar; Lucenius, Jessica; Serimaa, Ritva; Hahn, Michael G.; Moritz, Thomas; Jonsson, Leif J.; Israelsson-Nordstrom, Maria; Mellerowicz, Ewa J. (2014)
  • Pakarinen, Annukka; Haven, Mai Ostergaard; Djajadi, Demi Tristan; Varnai, Aniko; Puranen, Terhi; Viikari, Liisa (2014)
  • Ellilä, Simo; Bromann, Paul; Nyyssönen, Mari; Itävaara, Merja; Koivula, Anu; Paulin, Lars; Kruus, Kristiina (2019)
    Xylanases are in important class of industrial enzymes that are essential for the complete hydrolysis of lignocellulosic biomass into fermentable sugars. In the present study, we report the cloning of novel xylanases with interesting properties from compost metagenomics libraries. Controlled composting of lignocellulosic materials was used to enrich the microbial population in lignocellulolytic organisms. DNA extracted from the compost samples was used to construct metagenomics libraries, which were screened for xylanase activity. In total, 40 clones exhibiting xylanase activity were identified and the thermostability of the discovered xylanases was assayed directly from the library clones. Five genes, including one belonging to the more rare family GH8, were selected for subcloning and the enzymes were expressed in recombinant form in E. coli. Preliminary characterization of the metagenome-derived xylanases revealed interesting properties of the novel enzymes, such as high thermostability and specific activity, and differences in hydrolysis profiles. One enzyme was found to perform better than a standard Trichoderma reesei xylanase in the hydrolysis of lignocellulose at elevated temperatures.
  • Ellilä, Simo; Bromann, Paul; Nyyssönen, Mari; Itävaara, Merja; Koivula, Anu; Paulin, Lars; Kruus, Kristiina (Springer Berlin Heidelberg, 2019)
    Abstract Xylanases are in important class of industrial enzymes that are essential for the complete hydrolysis of lignocellulosic biomass into fermentable sugars. In the present study, we report the cloning of novel xylanases with interesting properties from compost metagenomics libraries. Controlled composting of lignocellulosic materials was used to enrich the microbial population in lignocellulolytic organisms. DNA extracted from the compost samples was used to construct metagenomics libraries, which were screened for xylanase activity. In total, 40 clones exhibiting xylanase activity were identified and the thermostability of the discovered xylanases was assayed directly from the library clones. Five genes, including one belonging to the more rare family GH8, were selected for subcloning and the enzymes were expressed in recombinant form in E. coli. Preliminary characterization of the metagenome-derived xylanases revealed interesting properties of the novel enzymes, such as high thermostability and specific activity, and differences in hydrolysis profiles. One enzyme was found to perform better than a standard Trichoderma reesei xylanase in the hydrolysis of lignocellulose at elevated temperatures.
  • Oghenekaro, Abbot O.; Raffaello, Tommaso; Kovalchuk, Andriy; Asiegbu, Fred O. (2016)
    Background: The basidiomycete Rigidoporus microporus is a fungus that causes the white rot disease of the tropical rubber tree, Hevea brasiliensis, the major source of commercial natural rubber. Besides its lifestyle as a pathogen, the fungus is known to switch to saprotrophic growth on wood with the ability to degrade both lignin and cellulose. There is almost no genomic or transcriptomic information on the saprotrophic abilities of this fungus. In this study, we present the fungal transcriptomic profiles during saprotrophic growth on rubber wood. Results: A total of 266.6 million RNA-Seq reads were generated from six libraries of the fungus growing either on rubber wood or without wood. De novo assembly produced 34, 518 unigenes with an average length of 217(bp. Annotation of unigenes using public databases; GenBank, Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG), Cluster of Orthologous Groups (COG) and Gene Ontology (GO) produced 25, 880 annotated unigenes. Transcriptomic profiling analysis revealed that the fungus expressed over 300 genes encoding lignocellulolytic enzymes. Among these, 175 genes were up-regulated in rubber wood. These include three members of the glycoside hydrolase family 43, as well as various glycosyl transferases, carbohydrate esterases and polysaccharide lyases. A large number of oxidoreductases which includes nine manganese peroxidases were also significantly up-regulated in rubber wood. Several genes involved in fatty acid metabolism and degradation as well as natural rubber degradation were expressed in the transcriptome. Four genes (acyl-CoA synthetase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA acetyltransferase) potentially involved in rubber latex degradation pathway were also induced. A number of ATP binding cassette (ABC) transporters and hydrophobin genes were significantly expressed in the transcriptome during saprotrophic growth. Some genes related to energy metabolism were also induced. Conclusions: The analysed data gives an insight into the activation of lignocellulose breakdown machinery of R. microporus. This study also revealed genes with relevance in antibiotic metabolism (e.g. cephalosporin esterase) as well as those with potential applications in fatty acid degradation. This is the first study on the transcriptomic analysis of R. microporus on rubber wood and should serve as a pioneering resource for future studies of the fungus at the genomic or transcriptomic level.
  • Veloz Villavicencio, Eliana Estefanía (Helsingin yliopisto, 2019)
    Fungal wood-decayers play an important role in the recycling of biomass and circulation of nutrients in nature. Fungi are capable to convert cellulose, hemicellulose, pectin and lignin, by the action of carbohydrate-acting enzymes (CAZymes) secreted and also by non-enzymatic reactions, depending on the ecology and decay strategy of the fungus. In the present study, four Basidiomycota fungi with different decay strategies were studied to compare their enzyme activity profiles. The white rot fungus Phlebia radiata, brown rot fungus Fomitopsis pinicola and “grey rot” fungus Schizophyllum commune were cultivated on birch (Betula pendula) wood pieces for twelve weeks, whereas the litter-decomposing fungus Coprinopsis cinerea was cultivated on cut barley (Hordeum vulgare) straw for six weeks. All fungi were also cultivated on liquid medium (malt extract 2%) for four weeks. Laccase, manganese peroxidase (MnP), β-glucosidase, xylanase and endoglucanase activities were followed weekly by measuring the absorbances on 96-well plates. The pH and the production of organic acids at each time point were also followed. The results showed that P. radiata produced high laccase and MnP activities. Additionally, high amounts of succinic acid in the aqueous phase of the solid-state cultivations were detected. F. pinicola had a notable production of xylanase activity on birch, in contrast to the moderate β-glucosidase and endoglucanase activities observed on the same substrate. S. commune was a strong producer of β-glucosidase, but especially xylanase activity on solid substrate. Lastly, the litter-decomposer C. cinerea seemed to have a poor performance in enzymatically decomposing the lignin portion from barley straw, whereas a preference on hemicellulose decomposition was observed. Overall, the results indicated the ability of the studied fungi in decomposing the components of the plant cell wall to different extents according to their decay strategy, which is key in the understanding of the ecophysiology of wood-decay and litter-decomposing fungi, and the potential of fungal enzymes for biotechnological applications.
  • Ma, Yibo; Stubb, Jonas; Kontro, Inkeri; Nieminen, Kaarlo; Hummel, Michael; Sixta, Herbert (2018)
    Man-made lignocellulosic fibres were successfully prepared from unbleached birch kraft pulps by using the Ioncell-F technology. Pulps with different lignin content were produced by tailored kraft pulping with varying intensity. The degree of polymerization of the pulps was adjusted by acid-catalyzed hydrolysis and electron beam treatment. All substrates were completely soluble in 1,5-diazabicyclo[4.3.0]non-5-enium acetate ([DBNH] OAc) and the respective solutions were spinnable to yield fibres with good to excellent mechanical properties despite the use of only mildly refined wood pulp. The tensile properties decreased gradually as the lignin concentration in the fibres increased. Changes in the chemical composition also affected the structure and morphology of the fibres. Both the molecular orientation and the crystallinity decreased while the presence of lignin enhanced the water accessibility. The effects of the crystallite size and lignin content on monolayer water adsorption are discussed.
  • Mattila, Hans K; Mäkinen, Mari; Lundell, Taina (BioMed Central, 2020)
    Abstract Background Fungal decomposition of wood is considered as a strictly aerobic process. However, recent findings on wood-decaying fungi to produce ethanol from various lignocelluloses under oxygen-depleted conditions lead us to question this. We designed gene expression study of the white rot fungus Phlebia radiata (isolate FBCC0043) by adopting comparative transcriptomics and functional genomics on solid lignocellulose substrates under varying cultivation atmospheric conditions. Results Switch to fermentative conditions was a major regulator for intracellular metabolism and extracellular enzymatic degradation of wood polysaccharides. Changes in the expression profiles of CAZy (carbohydrate-active enzyme) encoding genes upon oxygen depletion, lead into an alternative wood decomposition strategy. Surprisingly, we noticed higher cellulolytic activity under fermentative conditions in comparison to aerobic cultivation. In addition, our results manifest how oxygen depletion affects over 200 genes of fungal primary metabolism including several transcription factors. We present new functions for acetate generating phosphoketolase pathway and its potential regulator, Adr1 transcription factor, in carbon catabolism under oxygen depletion. Conclusions Physiologically resilient wood-decomposing Basidiomycota species P. radiata is capable of thriving under respirative and fermentative conditions utilizing only untreated lignocellulose as carbon source. Hypoxia-response mechanism in the fungus is, however, divergent from the regulation described for Ascomycota fermenting yeasts or animal-pathogenic species of Basidiomycota.
  • Sun, Mengyi (Helsingfors universitet, 2016)
    As the sustainable energy is becoming increasingly important, utilization of lignocellulosic biomass for biofuel production is the central part of this area. Fungal enzymes play an important role in lignocellulose degradation. Glucuronoyl esterase (GE) is a less studied fungal enzyme which degrades the ester linkage between lignin alcohol and hemicellulose side chain 4-O-methyl D-glucuronic acids. Genes encoding GE have been identified from various fungal species and they have been expressed in different production systems to be able to study their biochemical properties in detail. The gene encoding GE from the basidiomycete litter-decomposing fungus Stropharia coronilla was cloned and heterologous expressed in Pichia pastoris yeast. The expression and secretion of GE was induced by growing S. coronilla on lignocellulose supplemented cultivations. ScGE activity can be detected after the fifth day cultivation and it peaked on the 14th day. The heterologous expression of ScGE in P. pastoris showed that ScGE was produced as an enzymatically active protein. The commercial K-URONIC kit supplemented with a GE specific substrate benzyl-D-glucuronate was used to determine GE activity.
  • Faruq, Mohammad Omar (Helsingin yliopisto, 2020)
    In this research, lignocellulose decomposition and bioethanol production potentiality of the white rot fungus Phlebia radiata 79 was studied at different atmospheric conditions on several solid substrate mixtures containing spent brewery barley mash (SBBM), barley straw, spruce wood sawdust, and birch wood sawdust. The fungus was capable of growing on all substrate mixtures, subsequently converting them into fermentable sugars like glucose, producing various primary metabolites involving ethanol, acetate, and glycerol. Ethanol accumulation was always dominant under nitrogen flushed anaerobic conditions as well as semi-aerobic conditions. The highest concentration of ethanol accumulated on the second week of cultivation on all solid substrate mixtures. Under anaerobic conditions, the detected amount of ethanol was 88 mmol/l, 87 mmol/l, and 108 mmol/l, after two weeks of cultivation of the fungus on lignocellulose substrates containing SBBM mixed with barley straw, spruce wood sawdust, or birch wood sawdust, respectively. Under semi-aerobic conditions, corresponding concentrations of ethanol - 90 mmol/l, 61 mmol/l, and 105 mmol/l - accumulated in the cultures after two weeks of cultivation on the same substrate mixtures, respectively. Under aerobic conditions, only small amounts of ethanol were detected during the first two weeks of cultivation. Another part of the study was to establish an enzyme assay method for pectin degradation and conversion, in order to measure the activities of specific carbohydrate-active enzymes (CAZymes) involving pectinase, as well as cellulolytic β-glucosidase and cellobiohydrolase (CBH) activities. The highest β-glucosidase activity (5.2 nkat/ml) was observed under aerobic conditions in cultures on the substrate mixture of SBBM and barley straw. CBH activity was also prominent under aerobic conditions, and the maximal activity (0.7 nkat/ml) was detected on the substrate mixture of SBBM and spruce wood sawdust, while elevated pectinase activity (83 nkat/ml) was recorded under aerobic conditions on substrate mixtures containing SBBM and barley straw. Thus, the conclusions are that composition of the solid waste lignocellulose substrate mixture affected enzyme production by the fungus, whereas production of ethanol was mainly controlled by the cultivation atmosphere. Interestingly, both anaerobic and semi-aerobic atmospheric conditions supported similar bioconversion efficiency resulting with similar high levels of bioethanol production in the fungal cultures within two weeks.
  • Marinovic, Mila; Aguilar-Pontes, Maria Victoria; Zhou, Miaomiao; Miettinen, Otto Kullervo; de Vries, Ronald; Mäkelä, Miia Riitta; Hilden, Sari Kristiina (2018)
    The basidiomycete white-rot fungus Obba rivulosa, a close relative of Gelatoporia (Ceriporiopsis) subvermispora, is an efficient degrader of softwood. The dikaryotic O. rivulosa strain T241i (FBCC949) has been shown to selectively remove lignin from spruce wood prior to depolymerization of plant cell wall polysaccharides, thus possessing potential in biotechnological applications such as pretreatment of wood in pulp and paper industry. In this work, we studied the time-course of the conversion of spruce by the genome-sequenced monokaryotic O. rivulosa strain 3A-2, which is derived from the dikaryon T241i, to get insight into transcriptome level changes during prolonged solid state cultivation. During 8-week cultivation, O. rivulosa expressed a constitutive set of genes encoding putative plant cell wall degrading enzymes. High level of expression of the genes targeted towards all plant cell wall polymers was detected at 2-week time point, after which majority of the genes showed reduced expression. This implicated non-selective degradation of lignin by the O. rivulosa monokaryon and suggests high variation between mono- and dikaryotic strains of the white-rot fungi with respect to their abilities to convert plant cell wall polymers.