Uncovering the lignin-degrading potential of Serratia quinivorans AORB19: insights from genomic analyses and alkaline lignin degradation.

BACKGROUND: Lignin is an intricate phenolic polymer found in plant cell walls that has tremendous potential for being converted into value-added products with the possibility of significantly increasing the economics of bio-refineries. Although lignin in nature is bio-degradable, its biocatalytic conversion is challenging due to its stable complex structure and recalcitrance. In this context, an understanding of strain's genomics, enzymes, and degradation pathways can provide a solution for breaking down lignin to unlock the full potential of lignin as a dominant valuable bioresource. A gammaproteobacterial strain AORB19 has been isolated previously from decomposed wood based on its high laccase production. This work then focused on the detailed genomic and functional characterization of this strain based on whole genome sequencing, the identification of lignin degradation products, and the strain's laccase production capabilities on various agro-industrial residues. RESULTS: Lignin degrading bacterial strain AORB19 was identified as Serratia quinivorans based on whole genome sequencing and core genome phylogeny. The strain comprised a total of 123 annotated CAZyme genes, including ten cellulases, four hemicellulases, five predicted carbohydrate esterase genes, and eight lignin-degrading enzyme genes. Strain AORB19 was also found to possess genes associated with metabolic pathways such as the ß-ketoadipate, gentisate, anthranilate, homogentisic, and phenylacetate CoA pathways. LC-UV analysis demonstrated the presence of p-hydroxybenzaldehyde and vanillin in the culture media which constitutes potent biosignatures indicating the strain's capability to degrade lignin. Finally, the study evaluated the laccase production of Serratia AORB19 grown with various industrial raw materials, with the highest activity detected on flax seed meal (257.71 U/L), followed by pea hull (230.11 U/L), canola meal (209.56 U/L), okara (187.67 U/L), and barley malt sprouts (169.27 U/L). CONCLUSIONS: The whole genome analysis of Serratia quinivorans AORB19, elucidated a repertoire of genes, pathways and enzymes vital for lignin degradation that widens the understanding of ligninolytic metabolism among bacterial lignin degraders. The LC-UV analysis of the lignin degradation products coupled with the ability of S. quinivorans AORB19 to produce laccase on diverse agro-industrial residues underscores its versatility and its potential to contribute to the economic viability of bio-refineries.

Lignocellulosic biomass pretreatment: Industrial oriented high-solid twin-screw extrusion method to improve biogas production from forestry biomass resources.

Forestry lignocellulosic waste is an important, largely untapped source of biomass for producing clean energy. In this study, a high-solids twin-screw extrusion approach is developed as a novel pretreatment method to effectively increase the biogas production rate to better fit commercial requirements. Multiple screw designs are progressively introduced with increasingly intensified mechanical shear. The experiments also looked at the impact of feed solids content and several cost-effective processing aids along with these screw designs. Various characterization methods were used to relate the physical state of the biomass based on its specific surface area and volatile fraction, to the rate of biomethane generation possible from a 14- and 31-day biomethane potential test. An increase in biomethane production over this period by up to 190% was possible with the optimal screw design compared to a benchmark sample. This is a promising finding for the industrialization of biomethane production from forestry lignocellulosic biomass.

Assay Development for Metal-Dependent Enzymes-Influence of Reaction Buffers on Activities and Kinetic Characteristics.

Buffers are often thought of as innocuous components of a reaction, with the sole task of maintaining the pH of a system. However, studies had shown that this is not always the case. Common buffers used in biochemical research, such as Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl), can chelate metal ions and may thus affect the activity of metalloenzymes, which are enzymes that require metal ions for enhanced catalysis. To determine whether enzyme activity is influenced by buffer identity, the activity of three enzymes (BLC23O, Ro1,2-CTD, and trypsin) was comparatively characterized in N-2- hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), Tris-HCl, and sodium phosphate buffer. The pH and temperature optima of BLC23O, a Mn2+-dependent dioxygenase, were first identified, and then the metal ion dissociation constant (Kd) was determined in the three buffer systems. It was observed that BLC23O exhibited different Kd values depending on the buffer, with the lowest (1.49 ± 0.05 µM) recorded in HEPES under the optimal set of conditions (pH 7.6 and 32.5 °C). Likewise, the kinetic parameters obtained varied depending on the buffer, with HEPES (pH 7.6) yielding overall the greatest catalytic efficiency and turnover number (kcat = 0.45 ± 0.01 s-1; kcat/Km = 0.84 ± 0.02 mM-1 s-1). To corroborate findings, the characterization of Fe3+-dependent Ro1,2-CTD was performed, resulting in different kinetic constants depending on the buffer (Km (HEPES, Tris-HCl, and Na-phosphate) = 1.80, 6.93, and 3.64 µM; kcat(HEPES, Tris-HCl, and Na-phosphate) = 0.64, 1.14, and 1.01 s-1; kcat/Km(HEPES, Tris-HCl, and Na-phosphate)= 0.36, 0.17, and 0.28 µM-1 s-1). In order to determine whether buffer identity influenced the enzymatic activity of nonmetalloenzymes alike, the characterization of trypsin was also carried out. Contrary to the previous results, trypsin yielded comparable kinetic parameters independent of the buffer (Km (HEPES, Tris-HCl, and Na-Phosphate) = 3.14, 3.07, and 2.91 mM; kcat(HEPES, Tris-HCl, and Na-phosphate) = 1.51, 1.47, and 1.53 s-1; kcat/Km (HEPES, Tris-HCl, and Na-phosphate) = 0.48, 0.48, and 0.52 mM-1 s-1). These results showed that the activity of tested metalloenzymes was impacted by different buffers. While selected buffers did not influence the tested nonmetalloenzyme activity, other research had shown impacts of buffers on other enzyme activities. As a result, we suggest that buffer selection be optimized for any new enzymes such that the results from one lab to another can be accurately compared.

A high throughput screening process and quick isolation of novel lignin-degrading microbes from large number of natural biomasses.

High throughput screening approaches can significantly speed up the identification of novel enzymes from natural microbial consortiums. A two-step high throughput screening process was proposed and explored to screen lignin-degrading microorganisms. By employing this modified culture enrichment method and screening based on enzyme activity, a total of 82 bacterial and 46 fungal strains were isolated from fifty decayed wood samples (100 liquid cultures) collected from the banks of the Ottawa River in Canada. Among them, ten bacterial and five fungal strains were selected and identified based on their high laccase activities by 16S rDNA and ITS gene sequencing, respectively. The study identified bacterial strains from various genera including Serratia, Enterobacter, Raoultella, and Bacillus, along with fungal counterparts including Mucor, Trametes, Conifera and Aspergillus. Moreover, Aspergillus sydowii (AORF21), Mucor sp. (AORF43), Trametes versicolor (AORF3) and Enterobacter sp. (AORB55) exhibited xylanase and ß- glucanase activities in addition to laccase production. The proposed approach allowed for the quick identification of promising consortia and enhanced the chance of isolating desired strains based on desired enzyme activities. This method is not limited to lignocellulose and lignin-degrading microorganisms but can be applied to identify novel microbial strains and enzymes from different natural samples.

Identification and Characterization of a New Serratia proteamaculans Strain That Naturally Produces Significant Amount of Extracellular Laccase.

Natural biodegradation processes hold promises for the conversion of agro-industrial lignocellulosic biomaterials into biofuels and fine chemicals through lignin-degrading enzymes. The high cost and low stability of these enzymes remain a significant challenge to economic lignocellulosic biomass conversion. Wood-degrading microorganisms are a great source for novel enzyme discoveries. In this study, the decomposed wood samples were screened, and a promising γ-proteobacterial strain that naturally secreted a significant amount of laccase enzyme was isolated and identified as Serratia proteamaculans AORB19 based on its phenotypic and genotypic characteristics. The laccase activities in culture medium of strain AORB19 were confirmed both qualitatively and quantitatively. Significant cultural parameters for laccase production under submerged conditions were identified following a one-factor-at-a-time (OFAT) methodology: temperature 30°C, pH 9, yeast extract (2 g/l), Li+, Cu2+, Ca2+, and Mn2+ (0.5 mM), and acetone (5%). Under the selected conditions, a 6-fold increase (73.3 U/L) in laccase production was achieved when compared with the initial culturing conditions (12.18 U/L). Furthermore, laccase production was enhanced under alkaline and mesophilic growth conditions in the presence of metal ions and organic solvents. The results of the study suggest the promising potential of the identified strain and its enzymes in the valorization of lignocellulosic wastes. Further optimization of culturing conditions to enhance the AORB19 strain laccase secretion, identification and characterization of the purified enzyme, and heterologous expression of the specific enzyme may lead to practical industrial and environmental applications.

A novel Bacillus ligniniphilus catechol 2,3-dioxygenase shows unique substrate preference and metal requirement.

Identification of novel enzymes from lignin degrading microorganisms will help to develop biotechnologies for biomass valorization and aromatic hydrocarbons degradation. Bacillus ligniniphilus L1 grows with alkaline lignin as the single carbon source and is a great candidate for ligninolytic enzyme identification. The first dioxygenase from strain L1 was heterologously expressed, purified, and characterized with an optimal temperature and pH of 32.5 °C and 7.4, respectively. It showed the highest activity with 3-ethylcatechol and significant activities with other substrates in the decreasing order of 3-ethylcatechol > 3-methylcatechol > 3-isopropyl catechol > 2, 3-dihydroxybiphenyl > 4-methylcatechol > catechol. It did not show activities against other tested substrates with similar structures. Most reported catechol 2,3-dioxygenases (C23Os) are Fe2+-dependent whereas Bacillus ligniniphilus catechol 2,3-dioxygenase (BLC23O) is more Mn2+- dependent. At 1 mM, Mn2+ led to 230-fold activity increase and Fe2+ led to 22-fold increase. Sequence comparison and phylogenetic analyses suggested that BL23O is different from other Mn-dependent enzymes and uniquely grouped with an uncharacterized vicinal oxygen chelate (VOC) family protein from Paenibacillus apiaries. Gel filtration analysis showed that BLC23O is a monomer under native condition. This is the first report of a C23O from Bacillus ligniniphilus L1 with unique substrate preference, metal-dependency, and monomeric structure.