Dissertation committee:
Δημήτρης Χατζηνικολάου, Αναπληρωτής Καθηγητής, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Γεώργιος Διαλλινάς, Καθηγητής, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Αμαλία Καραγκούνη - Κύρτσου, Ομότιμη Καθηγήτρια, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Κωνσταντίνος Βοργιάς, Καθηγητής, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Ζαχαρούλα Γκόνου - Ζάγκου, Επίκουρη Καθηγήτρια, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Ιορδάνης Χατζηπαυλίδης, Αναπληρωτής Καθηγητής, Τμήμα Φυτικής Παραγωγής, Γεωπονικό Πανεπιστήμιο Αθηνών
Διομή Μαμμά, Επίκουρη Καθηγήτρια, Τμήμα Χημικών Μηχανικών, Εθνικό Μετσόβιο Πολυτεχνείο
Summary:
Lignocellulose biorefineries can harness the immense amounts of reduced carbon in the structural components of plant cell walls, via the conversion of residual biomass into biofuels and bio-based chemicals. The use of bacterial enzymes for lignocellulose valorization has recently gained growing attention and has been proposed as an alternative approach to the current use of fungal hydrolytic enzymes, due to the lower production cost of bacterial enzymes and their higher stability against the harsh conditions usually employed in biorefineries. Moreover, bacteria naturally endowed or engineered with the ability to funnel diverse lignin-derived molecules into specific high added-value products can replace chemical processes for lignin valorization. The aims of this thesis were to discover novel lignin and plant polysaccharide degrading bacteria, to detect efficient hydrolytic and oxidative enzymes and to elucidate pathways involved in bacterial degradation of lignin. The outcomes of this thesis are expected to aid the efforts towards designing efficient enzymatic and microbial biocatalysts for the complete valorization of lignocellulosic biomass.
To detect aerobic, mesophilic bacterial lignocellulose degraders, five uniformly distributed surface soil samples were collected from Keri Lake, at the island of Zakynthos, in western Greece. This area represents a unique environment, dominated by a marsh mainly composed of reeds, with increased biomass degradation, where, in parallel, for more than 2.500 years asphalt springs release crude oil, rich in aromatic hydrocarbons. For the isolation of bacteria, an enrichment strategy based on organosolv lignin, xylan from birchwood and carboxymethyl amorphous cellulose (CMC), as sole carbon and energy sources was used. A total of 63 colonies were isolated from enrichment cultures of all five soil samples. Characterization of the 16S rRNA gene of all the isolates generated 24 different genera. A wide diversity of Pseudomonas species was enriched in organosolv lignin cultures. Complex bacterial consortia were enriched in cultures with xylan or CMC, belonging to Actinobacteria, Proteobacteria, Bacilli, Sphingobacteriia and Flavobacteria. Several individual isolates could target amorphous and crystalline cellulose, or xylan, by expressing the corresponding hydrolytic activities, providing evidence of endoglycolytic, exoglycolytic and xylanolytic enzyme activity. A series of screening tests have featured the ability of strain Pseudomonas kilonensis ZKA7 to grow on organosolv lignin, on lignin hydrolysates generated from agricultural residues of corn stover and wheat straw, by implementing a mild alkali pretreatment method, and on lignin-derived aromatic monomers such as ferulic, caffeic and vanillic acid. NMR analysis revealed that this strain was also able to generate structural modifications in corn stover lignin hydrolysate, arising from degradation of lignin aromatic moieties.
Three novel oxidoreductases from strain ZKA7 were selected for investigation of their ligninolytic and oxidative potential, a multicopper oxidase (Pk-CopA), a dye decolorizing peroxidase (Pk-DypB) and a catalase-peroxidase (Pk-katG). The recombinant proteins were expressed as N-terminal 6xHis-fusion proteins in E. coli expression system and were purified by nickel affinity chromatography and gel filtration chromatography. Pk-CopA was able to oxidize lignin prepared from alkali pretreated corn stover, in the presence of ABTS as a mediator, generating a new product, as indicated by HPLC analysis. It was also able to oxidize the lignin-associated aromatic monomers ferulic acid, caffeic acid, syringic acid and catechol. Pk-CopA exhibited activity at a higher pH range than most bacterial laccases, retaining over 80% of its optimal activity between pH 4.5 - 6.5. It also exhibited high pH and thermal stability, maintaining 80% of its initial activity after 2 h of preincubation at pH 11and almost 70% of its initial activity within 6 hours of incubation at 60°C.
Pk-DypB was active towards ABTS, while it was able to act at a less acidic pH (6.0) than other characterized dyp-type peroxidases. However, its thermal stability and pH stability under alkaline and acidic conditions were lower than other enzymes. Under the conditions tested, no oxidative activity was detected for Pk-DypB towards lignin hydrolysate from alkali pretreated corn stover. Pk-KatG exhibited both catalase and peroxidase activity though it exhibited higher affinity and catalytic efficiency towards peroxidase substrates than the natural catalase substrate, H2O2. The biochemical characterization of Pk-KatG also provides the first report of the oxidation of syringaldazine, a typical substrate of ligninolytic enzymes, by a catalase-peroxidase, as well as the first report of the oxidation of the aromatic monomers catechol and pyrogallol, mediated by the presence of ABTS, and the oxidation of the synthetic dye Remazol Brilliant Blue R, a substrate used to detect ligninolytic activity. These findings may suggest a potential lignin oxidative activity that remains to be investigated.
For the identification of pathways involved in lignin degradation a proteomic analysis was conducted on Pseudomonas kilonensis ZKA7 cells, grown on lignin hydrolysate from corn stover, ferulic, caffeic, and vanillic acid, and acetate as a reference substrate. Protein samples were collected from cells grown on each substrate, separated into intracellular and extracellular fractions and subjected to proteomic analysis, using an LC-MS/MS system. Statistical analysis revealed the upregulation of genes involved in upper and central pathways of lignin-derived aromatic monomers degradation, as well as genes encoding transport proteins, transcriptional regulators, stress response proteins, oxidative enzymes and proteins of unknown function.
