Biodegradation and bioconversion of endrin by white rot fungi,Phlebia acanthocystis and Phlebia brevispora

Endrin is persistent organic pollutants that contaminate soil in many parts of the world. In this study, endrin was used as the substrate for a degradation experiment with the white rot fungi of the genus Phlebia. The results of tolerance test showed that the tolerance level of Phlebia acanthocystis and Phlebia brevispora to endrin was higher than that of other fungi, and the tolerance coefficient of both strains to 1.0 mg/L endrin exceeded 0.9 in solid PDA medium. P. acanthocystis and P. brevispora could degrade endrin efficiently in pure culture, especially P. acanthocystis had the highest degradability of more than 80% after 20 d incubation. Compared with low-nitrogen medium, PDB medium is more suitable for the biodegradability of two fungi. Several hydroxylated products such as 8-hydroxyendrin and two monohydroxyendrin were detected, indicating that endrin was initially branched to different monohydroxylated products in fungal degradation. Moreover, a carboxylic acid product was obtained from P. acanthocystis culture, suggesting that the carboxylation reaction occurred in bioconversion of endrin. The fungal cytochrome P450 enzymes play significant role in the in the initial hydroxylation process on endrin degradation. This is the first report that endrin is converted to hydroxylated and carboxylated metabolites by microorganisms.     

Lead,cadmium and nickel removal efficiency of white-rot fungus Phlebia brevispora

Widespread of heavy metals contamination has led to several environmental problems. Some biological methods to remove heavy metals from contaminated wastewater are being widely explored. In the present study, the efficiency of a white-rot fungus, Phlebia brevispora to remove different metals (Pb, Cd and Ni) has been evaluated. Atomic absorption spectroscopy of treated and untreated metal containing water revealed that all the metals were efficiently removed by the fungus. Among all the used metals, cadmium was the most toxic metal for fungal growth. Phlebia brevispora removed maximum Pb (97·5%) from 100 mmol l⁻¹ Pb solution, which was closely followed by Cd (91·6%) and Ni (72·7%). Scanning electron microscopic images revealed that the presence of metal altered the morphology and fine texture of fungal hyphae. However, the attachment of metal on mycelia surface was not observed during energy-dispersive X-ray analysis, which points towards the intracellular compartmentation of metals in vacuoles. Thus, the study demonstrated an application of P. brevispora for efficient removal of Pb, Cd and Ni from the metal contaminated water, which can further be applied for bioremediation of heavy metals present in the industrial effluent.     

Expression of a bacterial 3‐dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency

Lignin confers recalcitrance to plant biomass used as feedstocks in agro‐processing industries or as source of renewable sugars for the production of bioproducts. The metabolic steps for the synthesis of lignin building blocks belong to the shikimate and phenylpropanoid pathways. Genetic engineering efforts to reduce lignin content typically employ gene knockout or gene silencing techniques to constitutively repress one of these metabolic pathways. Recently, new strategies have emerged offering better spatiotemporal control of lignin deposition, including the expression of enzymes that interfere with the normal process for cell wall lignification. In this study, we report that expression of a 3‐dehydroshikimate dehydratase (QsuB from Corynebacterium glutamicum) reduces lignin deposition in Arabidopsis cell walls. QsuB was targeted to the plastids to convert 3‐dehydroshikimate – an intermediate of the shikimate pathway – into protocatechuate. Compared to wild‐type plants, lines expressing QsuB contain higher amounts of protocatechuate, p‐coumarate, p‐coumaraldehyde and p‐coumaryl alcohol, and lower amounts of coniferaldehyde, coniferyl alcohol, sinapaldehyde and sinapyl alcohol. 2D‐NMR spectroscopy and pyrolysis‐gas chromatography/mass spectrometry (pyro‐GC/MS) reveal an increase of p‐hydroxyphenyl units and a reduction of guaiacyl units in the lignin of QsuB lines. Size‐exclusion chromatography indicates a lower degree of lignin polymerization in the transgenic lines. Therefore, our data show that the expression of QsuB primarily affects the lignin biosynthetic pathway. Finally, biomass from these lines exhibits more than a twofold improvement in saccharification efficiency. We conclude that the expression of QsuB in plants, in combination with specific promoters, is a promising gain‐of‐function strategy for spatiotemporal reduction of lignin in plant biomass.     

Biological pretreatment of corn stover with Phlebia brevispora NRRL‐13108 for enhanced enzymatic hydrolysis and efficient ethanol production

Biological pretreatment of lignocellulosic biomass by white‐rot fungus can represent a low‐cost and eco‐friendly alternative to harsh physical, chemical, or physico‐chemical pretreatment methods to facilitate enzymatic hydrolysis. In this work, solid‐state cultivation of corn stover with Phlebia brevispora NRRL‐13018 was optimized with respect to duration, moisture content and inoculum size. Changes in composition of pretreated corn stover and its susceptibility to enzymatic hydrolysis were analyzed. About 84% moisture and 42 days incubation at 28°C were found to be optimal for pretreatment with respect to enzymatic saccharification. Inoculum size had little effect compared to moisture level. Ergosterol data shows continued growth of the fungus studied up to 57 days. No furfural and hydroxymethyl furfural were produced. The total sugar yield was 442 ± 5 mg/g of pretreated corn stover. About 36 ± 0.6 g ethanol was produced from 150 g pretreated stover per L by fed‐batch simultaneous saccharification and fermentation (SSF) using mixed sugar utilizing ethanologenic recombinant Eschericia coli FBR5 strain. The ethanol yields were 32.0 ± 0.2 and 38.0 ± 0.2 g from 200 g pretreated corn stover per L by fed‐batch SSF using Saccharomyces cerevisiae D5A and xylose utilizing recombinant S. cerevisiae YRH400 strain, respectively. This research demonstrates that P. brevispora NRRL‐13018 has potential to be used for biological pretreatment of lignocellulosic biomass. This is the first report on the production of ethanol from P. brevispora pretreated corn stover. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:365–374, 2017     

p-Coumaroylation of poplar lignins impacts lignin structure and improves wood saccharification

The enzymatic hydrolysis of cellulose into glucose, referred to as saccharification, is severely hampered by lignins. Here, we analyzed transgenic poplars (Populus tremula × Populus alba) expressing the Brachypodium (Brachypodium distachyon) p-coumaroyl-Coenzyme A monolignol transferase 1 (BdPMT1) gene driven by the Arabidopsis (Arabidopsis thaliana) Cinnamate 4-Hydroxylase (AtC4H) promoter in the wild-type (WT) line and in a line overexpressing the Arabidopsis Ferulate 5-Hydroxylase (AtF5H). BdPMT1 encodes a transferase which catalyzes the acylation of monolignols by p-coumaric acid (pCA). Several BdPMT1-OE/WT and BdPMT1-OE/AtF5H-OE lines were grown in the greenhouse, and BdPMT1 expression in xylem was confirmed by RT-PCR. Analyses of poplar stem cell walls (CWs) and of the corresponding purified dioxan lignins (DLs) revealed that BdPMT1-OE lignins were as p-coumaroylated as lignins from C3 grass straws. For some transformants, pCA levels reached 11 mg·g⁻¹ CW and 66 mg·g⁻¹ DL, exceeding levels in Brachypodium or wheat (Triticum aestivum) samples. This unprecedentedly high lignin p-coumaroylation affected neither poplar growth nor stem lignin content. Interestingly, p-coumaroylation of poplar lignins was not favored in BdPMT1-OE/AtF5H-OE transgenic lines despite their high frequency of syringyl units. However, lignins of all BdPMT1-OE lines were structurally modified, with an increase of terminal unit with free phenolic groups. Relative to controls, this increase argues for a reduced polymerization degree of BdPMT1-OE lignins and makes them more soluble in cold NaOH solution. The p-coumaroylation of poplar samples improved the saccharification yield of alkali-pretreated CW, demonstrating that the genetically driven p-coumaroylation of lignins is a promising strategy to make wood lignins more susceptible to alkaline treatments used during the industrial processing of lignocellulosics.

The expression of a grass p-coumaroyl-CoA:monolignol transferase induces high p-coumaroylation of poplar lignins and better saccharification of alkali-pretreated poplar wood without growth penalty.     

Degradation of endocrine disrupting chemicals by genetic transformants in Irpex lacteus with an inducible laccase gene of Phlebia tremellosa

Irpex lacteus was genetically transformed using an laccase expression vector to get increased laccase producing strains. Stable integration of the vector was confirmed by PCR using the vector-specific primers, and the transformants showed increased laccase activities. When the transformants were grown with several endocrine disrupting chemicals, laccase activity of each transformant was induced up to six times higher than that of the wild type. They showed increased degrading activities against EDCs as well as increased removal rates of estrogenic activities generated by the EDCs than the wild type, and the laccase expression was increased during the degradations of the EDCs.     

Progress in biopulping of non-woody materials: Chemical, enzymatic and ultrastructural aspects of wheat straw delignification with ligninolytic fungi from the genus Pleurotus

Abstract: During screening of basidiomycetes for wheat straw delignification, considerable lignin degradation with a limited attack to cellulose was attained with Pleurotus eryngii . Straw solid-state fermentation (SSF) was optimized, and the enzymatic mechanisms for lignin degradation were investigated. No lignin peroxidase was detected under liquid or SSF conditions, but high laccase and aryl-alcohol oxidase levels were found. The latter enzyme has been fully characterized in PI. eryngii and it seems to be involved in a cyclic redox system for H₂0₂ generation from aromatic compounds. Results obtained using homoveratric acid suggest that Pleurotus laccase could be involved in degradation of phenolic and non-phenolic lignin moieties. Histological and ultrastructural studies provided some general morphological characteristics of the fungal attack on wheat straw. Whereas a simultaneous degradation pattern was observed in straw treated with Phanerochaete chrysosporium , PI. eryngii caused partial degradation of middle lamella and separation of individual sclerenchymatic fibers. When these straw samples were subjected to refining tests, energy saving after biological treatment was the highest in the case of straw treated with PI. eryngii , which also produced the lowest substrate loss. From these results, a correlation between preferential removal of lignin, separation of sclerenchymatic fibers and pulping properties was provided during fungal treatment of wheat straw.     

Combination of steam explosion and laccase-mediator treatments prior to Eucalyptus globulus kraft pulping

The effect of a pretreatment consisting of steam explosion (SE) followed by a laccase mediator system (LMS) stage on Eucalyptus globulus kraft pulping has been evaluated and compared with fungal pretreatments. Pretreatment with SE and LMS was more efficient than pretreatments using Pycnoporus sanguineus and Trametes sp. I-62. Steam explosion not only improved the enzyme penetration into the wood chips and shortened the pulping process by 60%, but also extracted around 50% of the hemicelluloses which could be converted into value-added products. The optimal conditions for the LMS treatment were 3 h, 3 UA/g and 40 °C. Compared to SE, the SE/LMS treatment yielded an increase in delignification of 13.9% without affecting pulp properties, provided a similar screened kraft yield, and reduced consumption of chemical reagents Na₂S and NaOH by 11.5% and 6.3%, respectively. Therefore, SE/LMS is a promising pretreatment for converting the pulp mill into a forest bio-refinery.     

Enhanced delignification of mixed wood pulp by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> laccase mediator system

An environmentally sound biobleaching to get high quality paper pulp from mixed wood pulp was attempted employing laccase from Aspergillus fumigatus VkJ2.4.5 for lignin removal. Laccase treatment was performed in the presence of a mediator N-hydroxybenzotriazole (HBT, 1.5% w/w), resulting into notably higher level of delignification of the pulp. Enzyme at 10 Ug⁻¹ of pulp at 50°C, pH 6.0, for 2 h with a pulp consistency of 10% was found suitable for enabling maximum decrease in the kappa number. The kappa number and yellowness decreased by 14 and 4% whereas ISO brightness improved by 7%. The presence of a characteristic peak at 280 nm indicated the presence of lignin in the effluent during biobleaching. Analysis of FTIR spectra of residual lignin revealed characteristic modifications following enzymatic bleaching by laccase mediator system (LMS). Variations in morphology and crystallinity of pulp were evaluated by scanning electron microscopy and X-ray diffraction analysis.     

Thermogravimetry study of xylanase- and laccase/mediator-treated eucalyptus pulp fibres

Thermogravimetric analyses (TGA) was applied to study the effects of enzymatic bleaching of eucalyptus pulp with xylanase and a laccase-mediator system. The thermal degradation profile of the pulps was sensitive to the enzymatic treatments. Xylanase treatment produced an ordered and clean microfibril, whereas laccase oxidized surface cellulose chains and increased the amorphous (paracrystalline) cellulose content. In this case, pulp viscosity decreased from 972 to 859 mL/g and apparent pulp crystallinity calculated from TGA data decreased almost 50%. Alkaline extraction was necessary to recover pulp crystallinity and to remove oxidized lignin in the laccase-treated samples. TGA data allowed differentiating and quantifying crystalline and amorphous cellulose. This thermogravimetric approach is a simple method in order to monitor superficial changes in cellulosic microfibrils.     

Performance of a mobile mechanical screen to improve the commercial quality of wood chips for energy

The study analyzed the performance of a mobile screening device for upgrading coarse wood chips to residential user standards, by removing oversize particles and fines. The machine was designed for transportation to forest landings, logistic terminals and plant chip yards. Average productivity was 1.9 oven-dry tons (odt) h⁻¹, corresponding to a screening cost of 28.5 € odt⁻¹. This figure was lower than the price increase obtained by upgrading industrial chips to residential user standards. Hence, screening offered a profit of 4.7 € odt⁻¹, or 16% of the original screening cost. The screening process was capable of upgrading chips from industrial to residential specifications, by reducing the incidence of oversize particles below the 1% critical threshold. Screening also allowed a substantial reduction in the content of fines. A similar effect was not verified for crushed wood, which failed to meet the specifications for residential fuel.     

Oxidation of milled wood lignin with laccase, tyrosinase and horseradish peroxidase

In this paper the oxidation of milled wood lignin (MWL), catalysed by three enzymes, i.e. laccase, tyrosinase and horseradish peroxidase (HRP) was studied. The oxidation was followed by measuring the consumption of O₂ during laccase and tyrosinase treatment and of H₂O₂ during HRP treatment. Both laccase and HRP were found to oxidise lignin effectively, whereas the effect of tyrosinase was negligible. The changes in MWL molecular-weight distributions caused in the reactions were analysed by gel permeation chromatography. Both laccase and HRP treatments were found to polymerise MWL. Peroxidase treatment was found to decrease the amount of phenolic hydroxyls in MWL, whereas no such effect could be detected in the laccase-treated sample. Both laccase and HRP treatments were, however, found to increase the amount of conjugated structures in MWL. The formation of phenoxy radicals during the treatments was studied by electron paramagnetic resonance spectroscopy. Phenoxy radicals were detected in both laccase and HRP-treated samples. The amount of the formed phenoxy radicals was found to be essentially constant during the detected time (i.e. 20–120 min after the addition of enzyme).     

Effect of different supplements on bioprocessing of wheat straw by Phlebia brevispora: changes in its chemical composition, in vitro digestibility and nutritional properties

Bioprocessing of wheat straw was carried out by Phlebia brevispora under solid state conditions. Effect of different supplements on lignocellulolytic enzymes production, degradation of straw cell wall fibers and its resultant effect on nutritional quality of wheat straw were studied. Ammonium chloride and malt extract were more effective in terms of ligninolysis and enhanced in vitro digestibility. The concentration of the selected supplements and the moisture content was worked out using response surface methodology in order to minimize the loss in total organic matter so as to selectively degrade lignin. The experiment was scaled up to batches of 200 g under optimized conditions and the degraded substrate was analyzed for its biochemical properties. P. brevispora degraded 290 g/kg of lignin and enhanced the in vitro digestibility from 150 to 268 g/kg (78%). Crude protein, amino acids, total phenolic contents and antioxidant properties were significantly higher in degraded straw.     

Laccase catalysed modification of lignin subunits and coupling to p-aminobenzoic acid

Laccase catalysed oxidation of syringyl and guaiacyl subunits of lignin and their modification with an aromatic amine, p-aminobenzoic acid (PABA) were investigated. Laccase from Galerina sp. HC1 isolated earlier by us was used as the main catalyst, and Trametes versicolor laccase was used for comparison. Among the syringyl compounds, syringic acid and syringaldehyde were oxidised to 2,6-dimethoxy-1,4-benzoquinone, and in the presence of PABA yielded a cross-coupling imine product. The reaction with methyl syringol resulted in several products whose structures were determined. The possible oxidative coupling pathways were proposed for the formation of the identified products. Oxidation of syringol and the guaiacyl compounds resulted mainly in homooligomers by free radical mechanism, with a negligible tendency of reaction with the nucleophilic group of PABA. Similar treatment of Eucalyptus Kraft lignin, which is rich in syringyl moieties, showed the presence of identical products obtained with syringic acid and syringaldehyde.     

Towards industrially-feasible delignification and pitch removal by treating paper pulp with Myceliophthora thermophila laccase and a phenolic mediator

The ability of two natural phenols to act as mediators of the recombinant Myceliophthora thermophila laccase (MtL) in eucalypt-pulp delignification was investigated. After alkaline peroxide extraction, the properties of the enzymatically-treated pulps improved with respect to the control. The pulp brightness increased (3.1 points) after the enzymatic treatment with MtL alone, but the highest improvements were obtained after the MtL treatment using syringaldehyde (4.7 points) and especially methyl syringate (8.3 points) as mediators. Likewise, a decrease in kappa number up to 2.7 points was obtained after the MtL-methyl syringate treatment, followed by decreases of 1.4 and 0.9 points after the treatments with MtL-syringaldehyde and MtL alone, respectively. On the other hand, removal of the main lipophilic extractives present in eucalypt pulp was observed after the above laccase-mediator treatments. Finally, the doses of both MtL and methyl syringate were reduced, and results compatible with industrial implementation were obtained.