Isolation of functional ligninolytic Bacillus aryabhattai from paper mill sludge and its lignin degradation potential.
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ABSTRACT:
SUBMITTER: Singh A
PROVIDER: S-EPMC9307452 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:This study reports the degradation and decolourization capability of a manganese peroxidase enzyme producing bacterium isolated from pulp and paper mill wastewater. The isolate was identified as Bacillus aryabhattai based on biochemical analysis and 16S rRNA gene sequencing. The strain was designated MG966493. This bacterium was able to reduce 67% and 54% colour and lignin, respectively, from the pulp and paper mill wastewater after 144 h of treatment at 32 °C, pH 7.6 and 120 rpm. Further, FT-IR analysis showed that during the lignin degradation process a number of metabolites were produced comprising different functional groups such as carbonyl (C=C), carboxyl (-COOH), alkene (C=C), amines (-NH2), sulphonic (-SO3) and nitro (-NO2). In addition, the SEM analysis showed that the bacterial cells exposed to pulp and paper mill wastewater have rough surfaces with reduced size as compared to the unexposed cells with smooth surfaces. This study concluded that the isolated bacterium B. aryabhattai has significant potential for the bioremediation of pulp and paper mill wastewater and thus, can be applied for their treatment at an industrial scale.
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Project description:Among others, isolate PSK1 was selected and identified by 16 S rDNA sequencing as Bacillus aryabhattai. Growth optimization of PSK1 and physicochemical parameters affected bioflocculant production was carried out by Plackett-Burman design and resulted in increasing in the activity by 4.5%. Bioflocculant production by entrapped and adsorbed immobilized microbial cells was performed using different techniques and revealed enhancement in the activity in particular with pumice adsorption. HPLC analysis of sugars and amino acids composition, FTIR and the effect of different factors on the purified PSK1 biopolymer such as presence of cations, thermal stability, pH range and clay concentration was carried out. Scanning electron microscopy (SEM) of free, immobilized cells, PSK1 bioflocculant and formed flocs were performed. The results revealed that bioflocculant PSK1 is mainly glycoprotein consists of glucose and rhamnose with a large number of amino acids in which arginine and phenylalanine were the major. SEM analysis demonstrated that PSK1 have a clear crystalline rod shaped structure. FTIR spectrum reported the presence of hydroxyl and amino groups which are preferred in flocculation process. PSK1 was soluble in water and insoluble in all other tested organic solvents, while it was thermally stable from 40 to 80 °C. Among examined cations, CaCl2 was the best coagulant. The maximum flocculation activity of the PSK1 recorded at 50 °C (92.8%), pH 2.0 (94.56%) with clay concentration range 5-9 g/l. To obtain a large amount of PSK1 bioflocculant with high flocculating activity, batch fermentation was employed. The results recorded ?6 g/l yield after 24 h of fermentation.
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Project description:The valorization of a South African paper mill waste sludge into an activated biocarbon electrode material for energy storage application is reported. The valorization method is a two-step synthesis that comprises hydrothermal carbonization and NaOH activation of paper mill waste at 700 °C to produce activated biocarbon. The development of high porosity carbon material with a surface area of 1139 m2/g was observed. The synthesized biocarbon electrode exhibited good specific capacitance (C sp) values of 206 and 157 Fg-1, from a three-electrode cell in neutral (1 M Na2SO4) and alkali (3 M KOH) electrolytes, respectively. The electrolyte concentration purportedly has a considerable effect on specific capacitance. In both electrolytes, symmetric triangular curves in galvanostatic charge-discharge point to a quick charge-discharge process. Synthesized material testing with a two-electrode cell in 3 M KOH and 1 M Na2SO4 electrolytes, respectively, delivered specific capacitances of 125 and 152 Fg-1, with the corresponding energy densities of 17.4 and 21.1 Wh kg-1. The material had capacity retention efficiencies of 83 and 92% after 5000 cycles in 3 M KOH and 1 M Na2SO4 electrolytes, respectively. The electrode material performance of the activated biocarbon from paper sludge clearly shows its potential for electrochemical energy storage. The reported results present an exciting potential contribution of the pulp and paper industry toward the transition to green energy.
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Project description:This study utilized pulp and paper mill sludge as a carbon source to produce activated biochar adsorbents. The response surface methodology (RSM) application for predicting and optimizing the activated biochar preparation conditions was investigated. Biochars were prepared based on a Box-Behnken design (BBD) approach with three independent factors (i.e., pyrolysis temperature, holding time, and KOH:biomass ratio), and the responses evaluated were specific surface area (SSA), micropore area (S micro), and mesopore area (S meso). According to the RSM and BBD analysis, a pyrolysis temperature of 800 °C for 3 h of holding and an impregnation ratio of 1:1 (biomass:KOH) are the optimum conditions for obtaining the highest SSA (885 m2 g-1). Maximized S micro was reached at 800 °C, 1 h and the ratio of 1:1, and for maximizing S meso (569.16 m2 g-1), 800 °C, 2 h and ratio 1:1.5 (445-473 m2 g-1) were employed. The biochars presented different micro- and mesoporosity characteristics depending on pyrolysis conditions. Elemental analysis showed that biochars exhibited high carbon and oxygen content. Raman analysis indicated that all biochars had disordered carbon structures with structural defects, which can boost their properties, e.g., by improving their adsorption performances. The hydrophobicity-hydrophilicity experiments showed very hydrophobic biochar surfaces. The biochars were used as adsorbents for diclofenac and amoxicillin. They presented very high adsorption performances, which could be explained by the pore filling, hydrophobic surface, and π-π electron-donor-acceptor interactions between aromatic rings of both adsorbent and adsorbate. The biochar with the highest surface area (and highest uptake performance) was subjected to regeneration tests, showing that it can be reused multiple times.
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Project description:Lignin is a recalcitrant macromolecule formed by three alcohols (monolignols) predominantly connected by ?-aryl ether linkages and is one of the most abundant organic macromolecules in the biosphere. However, the role played by environmental bacteria in lignin degradation is still not entirely understood. In this study, we identified an environmental Klebsiella strain isolated from sediment collected from an altitudinal region in a unique Brazilian biome called Caatinga. This organism can also grow in the presence of kraft lignin as a sole source of carbon and aromatic compounds. We performed whole-genome sequencing and conducted an extensive genome-based metabolic reconstruction to reveal the potential mechanisms used by the bacterium Klebsiella variicola P1CD1 for lignin utilization as a carbon source. We identified 262 genes associated with lignin-modifying enzymes (LMEs) and lignin-degrading auxiliary enzymes (LDAs) required for lignin and aromatic compound degradation. The presence of one DyP (Dye-decolorizing Peroxidase) gene suggests the ability of P1CD1 strain to access phenolic and nonphenolic structures of lignin molecules, resulting in the production of catechol and protocatechuate (via vanillin or syringate) along the peripheral pathways of lignin degradation. K. variicola P1CD1 uses aldehyde-alcohol dehydrogenase to perform direct conversion of vanillin to protocatechol. The upper funneling pathways are linked to the central pathways of the protocatechuate/catechol catabolic branches via ?-ketoadipate pathways, connecting the more abundant catabolized aromatic compounds with essential cellular functions, such as energy cellular and biomass production (i.e., via acetyl-CoA formation). The combination of phenotypic and genomic approaches revealed the potential dissimilatory and assimilatory ability of K. variicola P1CD1 to perform base-catalyzed lignin degradation, acting on high- and low-molecular-weight lignin fragments. These findings will be relevant for developing metabolic models to predict the ligninolytic mechanism used by environmental bacteria and shedding light on the flux of carbon in the soil.
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Project description:Lignin is a complex natural organic polymer and is one of the primary components of lignocellulose. The efficient utilization of lignocellulose is limited because it is difficult to degrade lignin. In this study, we screened a lacz1 gene fragment encoding laccase from the macrotranscriptome data of a microbial consortium WSC-6, which can efficiently degrade lignocellulose. The reverse transcription-quantitative PCR (RT-qPCR) results demonstrated that the expression level of the lacz1 gene during the peak period of lignocellulose degradation by WSC-6 increased by 30.63 times compared to the initial degradation period. Phylogenetic tree analysis demonstrated that the complete lacz1 gene is derived from a Bacillus sp. and encoded laccase. The corresponding protein, LacZ1, was expressed and purified by Ni-chelating affinity chromatography. The optimum temperature was 75°C, the optimum pH was 4.5, and the highest enzyme activity reached 16.39 U/mg. We found that Cu2+ was an important cofactor needed for LacZ1 to have enzyme activity. The molecular weight distribution of lignin was determined by gel permeation chromatography (GPC), and changes in the lignin structure were determined by 1H nuclear magnetic resonance (1H NMR) spectra. The degradation products of lignin by LacZ1 were determined by gas chromatography and mass spectrometry (GC-MS), and three lignin degradation pathways (the gentian acid pathway, benzoic acid pathway, and protocatechuic acid pathway) were proposed. This study provides insight into the degradation of lignin and new insights into high-temperature bacterial laccase. IMPORTANCE Lignin is a natural aromatic polymer that is not easily degraded, hindering the efficient use of lignocellulose-rich biomass resources, such as straw. Biodegradation is a method of decomposing lignin that has recently received increasing attention. In this study, we screened a gene encoding laccase from the lignocellulose-degrading microbial consortium WSC-6, purified the corresponding protein LacZ1, characterized the enzymatic properties of laccase LacZ1, and speculated that the degradation pathway of LacZ1 degrades lignin. This study identified a new, high-temperature bacterial laccase that can degrade lignin, providing insight into lignin degradation by this laccase.
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Project description:Ligninolytic bacteria degrading lignin were isolates and identified, and their biodegradation mechanism of alkaline-lignin was investigated. Four strains with lignin degradation capability were screened and identified from the soil, straw, and silage based on their decolorizing capacity of aniline blue and colony size on alkaline-lignin medium. The degradation ratio of Bacillus aryabhattai BY5, Acinetobacter johnsonii LN2, Acinetobacter lwoffii LN4, and Micrococcus yunnanensis CL32 have been assayed using alkaline-lignin as the unique carbon source. Further, the Lip (lignin peroxidase) and Mnp (manganese peroxidase) activities of strains were investigated. Lip activity of A. lwoffii LN4 was highest after 72 h of incubation and reached 7151.7 U · l-1. Mnp activity of M. yunnanensis CL32 was highest after 48 h and reached 12533 U · l-1. The analysis of alkaline-lignin degradation products by GC-MS revealed that the strains screened could utilize aromatic esters compounds such as dibutyl phthalate (DBP), and decomposite monocyclic aromatic compounds through the DBP aerobic metabolic pathway. The results indicate that B. aryabhattai BY5, A. johnsonii LN2, A. lwoffii LN4, and M. yunnanensis CL32 have high potential to degrade alkaline-lignin, and might utilize aromatic compounds by DBP aerobic metabolic pathway in the process of lignin degradation.
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Project description:A key challenge in chemicals biomanufacturing is the maintenance of stable, highly productive microbial strains to enable cost-effective fermentation at scale. A "cookie-cutter" approach to microbial engineering is often used to optimize host stability and productivity. This can involve identifying potential limitations in strain characteristics followed by attempts to systematically optimize production strains by targeted engineering. Such targeted approaches however do not always lead to the desired traits. Here, we demonstrate both 'hit and miss' outcomes of targeted approaches in attempts to generate a stable Escherichia coli strain for the bioproduction of the monoterpenoid linalool, a fragrance molecule of industrial interest. First, we stabilized linalool production strains by eliminating repetitive sequences responsible for excision of pathway components in plasmid constructs that encode the pathway for linalool production. These optimized pathway constructs were then integrated within the genome of E. coli in three parts to eliminate a need for antibiotics to maintain linalool production. Additional strategies were also employed including: reduction in cytotoxicity of linalool by adaptive laboratory evolution and modification or homologous gene replacement of key bottleneck enzymes GPPS/LinS. Our study highlights that a major factor influencing linalool titres in E. coli is the stability of the genetic construct against excision or similar recombination events. Other factors, such as decreasing linalool cytotoxicity and changing pathway genes, did not lead to improvements in the stability or titres obtained. With the objective of reducing fermentation costs at scale, the use of minimal base medium containing paper mill wastewater secondary paper fiber as sole carbon source was also investigated. This involved simultaneous saccharification and fermentation using either supplemental cellulase blends or by co-expressing secretable cellulases in E. coli containing the stabilized linalool production pathway. Combined, this study has demonstrated a stable method for linalool production using an abundant and low-cost feedstock and improved production strains, providing an important proof-of-concept for chemicals production from paper mill waste streams. For scaled production, optimization will be required, using more holistic approaches that involve further rounds of microbial engineering and fermentation process development.
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