Method to determine the decolorization potential of persistent dyes by white rot fungi by colorimetric assays.

Decolorization assays allow to assess the ability of white rot fungi to degrade persistent organic molecules such as textile dyes and can contribute to discover microorganisms that can be used for bioremediation. The decolorization can be overlayed by the absorption from metabolites that are produced by fungi during screening, which interfere with the results. To compensate for this interference a method was developed by using different controls to subtract interfering signals. The method was designed for simple screening in multiwell plates that can be operated with a plate reader. It was applied to four different textile dyes (Reactive Black 5, Reactive Blue 4, Reactive Green 19, and Reactive Orange 16) that were degraded by the white rot fungus Phanerochaete velutina. The four textile dyes showed different results with a different degree of interference. The controls allow to compensate for interfering signals and to calculate kinetic parameters for the decolorization reaction and the enzymatic degradation.•Determine the non-enzymatic degradation of the dyes in experiments without fungi.•Determine the absorbance of metabolites and subtract it from the decolorization data to obtain the degradation of the dye.•Determine kinetic parameters of the degradation to compare the efficiency of the enzymes towards dyes.

Production of Recombinant Laccase From Coprinopsis cinerea and Its Effect in Mediator Promoted Lignin Oxidation at Neutral pH.

Laccases are multi-copper oxidases that use molecular oxygen as the electron acceptor to oxidize phenolic and indirectly also non-phenolic substrates by mechanisms involving radicals. Due to their eco-friendliness and broad substrate specificity, laccases span a wide range of biotechnological applications. We have heterologously expressed a laccase from the coprophilic basidiomycete Coprinopsis cinerea (CcLcc9) in the methylotrophic yeast Pichia pastoris. The recombinant CcLcc9 (rCcLcc9) oxidized 2,6-dimethoxyphenol in the neutral pH range, and showed thermostability up to 70°C. The rCcLcc9 efficiently oxidized veratryl alcohol to veratraldehyde in the presence of low molecular weight mediators syringyl nitrile, methyl syringate and violuric acid, which are syringyl-type plant phenolics that have shown potential as natural co-oxidants for lignocellulosic materials. In addition, rCcLcc9 is able to depolymerize biorefinery hardwood lignin in the presence of methyl syringate and syringyl nitrile as indicated by gel permeation chromatography, and infrared spectral and nucleic magnetic resonance analyses. Furthermore, we showed that several added-value aromatic compounds, such as vanillin, vanillic acid, syringaldehyde, syringic acid and p-hydroxybenzoic acid, were formed during sequential biocatalytic chemical degradation of biorefinery lignin, indicating that rCcLcc9 harbors a great potential for sustainable processes of circular economy and modern biorefineries.

Depolymerization of biorefinery lignin by improved laccases of the white-rot fungus Obba rivulosa.

Fungal laccases are attracting enzymes for sustainable valorization of biorefinery lignins. To improve the lignin oxidation capacity of two previously characterized laccase isoenzymes from the white-rot fungus Obba rivulosa, we mutated their substrate-binding site at T1. As a result, the pH optimum of the recombinantly produced laccase variant rOrLcc2-D206N shifted by three units towards neutral pH. O. rivulosa laccase variants with redox mediators oxidized both the dimeric lignin model compound and biorefinery poplar lignin. Significant structural changes, such as selective benzylic α-oxidation, were detected by nuclear magnetic resonance analysis, although no polymerization of lignin was observed by gel permeation chromatography. This suggests that especially rOrLcc2-D206N is a promising candidate for lignin-related applications.

Investigations on inhibitory effects of nickel and cobalt salts on the decolorization of textile dyes by the white rot fungus Phanerochaete velutina.

Organic aromatic compounds used for dyeing and coloring in the textile industry are persistent and hazardous pollutants that must be treated before they are discharged into rivers and surface waters. Therefore, we investigated the potential of the white rot fungus Phanerochaete velutina to decolorize commonly used reactive dyes. The fungus decolorized in average 55% of Reactive Orange 16 (RO-16) after 14 days at a maximum rate of 0.09 d-1 and a half-life of 8 days. Furthermore, we determined the inhibitory effects of co-present inorganic contaminants Nickel (Ni) and Cobalt (Co) salts on the decolorization potential and determined IC50 values of 5.55 mg l-1 for Co and a weaker inhibition by Ni starting from a concentration of 20 mg l-1. In the decolorization assay for Remazol Brilliant Blue R (RBBR) we observed the interference of a metabolite of P. velutina, which did not allow us to investigate the kinetics of the reaction. The formation of the metabolite, however, could be used to obtain IC50 values of 3.37 mg l-1 for Co and 7.58 mg l-1 for Ni. Our results show that living white rot fungi, such as P. velutina, can be used for remediation of dye polluted wastewater, alternatively to enzyme mixtures, even in the co-presence of heavy metals.

Impacts of holmium and lithium to the growth of selected basidiomycetous fungi and their ability to degrade textile dyes.

The impacts of Ho and Li (0, 10, 50, 200 mg/L) were tested towards the growth of four basidiomycetous fungal species, their ability to decolorise synthetic dyes (Reactive Green 19, Reactive Orange 16, Reactive Black 5), and produce oxidative enzymes. All species; Agrocybe dura, Skeletocutis biguttulata, Exidia saccharina and Galerina paludosa; grew with and without supplemented Ho or Li. The growth of S. biguttulata was the most tolerant species towards Ho or Li (200 mg/L), whereas the growth of G. paludosa was the most sensitive of the studied species to both 200 mg Ho or Li/L. All fungi oxidized ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] forming colour zone on plate tests indicating production of lignin modifying laccase enzyme. A. dura and G. paludosa, formed black MnO2 zone in Mn2+ plates, which indicates the production of manganese peroxidase (MnP). A. dura and G. paludosa decolorised Reactive Black 5 indicating the production of versatile peroxide (VP) enzyme. Our study presents two new candidate species able to produce VP. A. dura was capable of decolorising all tested synthetic dyes in the presence of Ho or Li (0-200 mg/L) suggesting that this fungus is a promising species for bioremediation of multi dye-containing wastes.

Newtonian boreal forest ecology: The Scots pine ecosystem as an example.

Isaac Newton's approach to developing theories in his book Principia Mathematica proceeds in four steps. First, he defines various concepts, second, he formulates axioms utilising the concepts, third, he mathematically analyses the behaviour of the system defined by the concepts and axioms obtaining predictions and fourth, he tests the predictions with measurements. In this study, we formulated our theory of boreal forest ecosystems, called NewtonForest, following the four steps introduced by Newton. The forest ecosystem is a complicated entity and hence we needed altogether 27 concepts to describe the material and energy flows in the metabolism of trees, ground vegetation and microbes in the soil, and to describe the regularities in tree structure. Thirtyfour axioms described the most important features in the behaviour of the forest ecosystem. We utilised numerical simulations in the analysis of the behaviour of the system resulting in clear predictions that could be tested with field data. We collected retrospective time series of diameters and heights for test material from 6 stands in southern Finland and five stands in Estonia. The numerical simulations succeeded to predict the measured diameters and heights, providing clear corroboration with our theory.

Mycoremediation of wood and soil from an old sawmill area contaminated for decades.

We investigated the potential of white-rot and litter-decomposing fungi for the treatment of soil and wood from a sawmill area contaminated with aged chlorinated phenols, dibenzo-p-dioxins and furans (PCDD/F). Eight screening assays with emphasis on application of non-sterile conditions were carried out in order to select the strains with capability to withstand indigenous microbes and contamination. Nine fungi were then selected for degrading pentachlorophenol (PCP), and 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP) and mineralizing radiolabelled pentachlorophenol ((14)C-PCP) in non-sterile soil or wood during 15 weeks of incubation. Soil indigenous microbes and fungal inoculated soil (fungal inoculum+indigenous microbes) achieved similar degradation of PCP and 2,3,4,6-TeCP and mineralization of (14)C-PCP. However, the mineralization rate of (14)C-PCP by indigenous microbes was much slower than that boosted by fungal inoculum. The litter-decomposing fungus (LDF) Stropharia rugosoannulata proved to be a suitable fungus for soil treatment. This fungus mineralized 26% of (14)C-PCP and degraded 43% of 2,3,4,6-TeCP and 73% of PCP. Furthermore, S. rugosoannulata attained 13% degradation of PCDD/F (expressed as WHO-Toxic Equivalent). In wood, white-rot fungi grew and degraded chlorophenols better than LDF. No efficient indigenous degraders were present in wood. Interestingly, production of toxic chlorinated organic metabolites (anisoles and veratroles) by LDF in wood was negligible.

Hydrolytic and ligninolytic enzyme activities in the Pb contaminated soil inoculated with litter-decomposing fungi.

The impact of Pb contamination was tested to five hydrolytic (beta-glucosidase, beta-xylosidase, beta-cellobiosidase, alpha-glucosidase and sulphatase) and two ligninolytic (manganese peroxidase, MnP and laccase) enzyme activities in the humus layer in the forest soil. The ability of eight selected litter-degrading fungi to grow and produce extracellular enzymes in the heavily Pb (40 g Pb of kg ww soil(-1)) contaminated and non-contaminated soil in the non-sterile conditions was also studied. The Pb content in the test soil was close to that of the shooting range at Hälvälä (37 g Pb of kg ww soil(-1)) in Southern Finland. The fungi were Agaricus bisporus, Agrocybe praecox, Gymnopus peronatus, Gymnopilus sapineus, Mycena galericulata, Gymnopilus luteofolius, Stropharia aeruginosa and Stropharia rugosoannulata. The Pb contamination (40 g Pb of kg ww soil(-1)) was deleterious to all five studied hydrolytic enzyme activities after five weeks of incubation. All five hydrolytic enzyme activities were significantly higher in the soil than in the extract of the soil indicating that a considerable part of enzymes were particle bound in the soils. Hydrolytic enzyme activities were higher in the non-contaminated soil than in the Pb contaminated soil. Fungal inocula increased the hydrolytic enzyme activities beta-cellobiosidase and beta-glucosidase in non-contaminated soils. All five hydrolytic enzyme activities were similar with fungi and without fungi in the Pb contaminated soil. This was in line that Pb contamination (40 g Pb of kg ww soil(-1)) depressed the growth of all fungi compared to those grown without Pb in the soil. Laccase and MnP activities were low in both Pb contaminated and non-contaminated soil cultures. MnP activities were higher in soil cultures containing Pb than without Pb. Our results showed that Pb in the shooting ranges decreased fungal growth and microbial functioning in the soil.

Microbial activities in soils of a former sawmill area.

To find out microbial metabolic functioning and toxicity in a former sawmill area, carbon dioxide evolution, methane oxidation potential, 10 hydrolytic enzyme activities, Vibrio fischeri test, fluorescein diacetate hydrolysis activity (FDA), soil pH, carbon, nitrogen and pentachlorophenol (PCP) content were measured at four sites. The area is contaminated with aged chlorophenols. Chlorophenol content of soil was analyzed with a novel HPLC-MS technique, which allowed to measure chlorophenols without derivatization. The sites had a pollution gradient from 0.5 to 15 microg PCP g dw of soil(-1). Endogenous carbon dioxide evolution, methane oxidation potential, butyrate-esterase, acetate-esterase, sulphatase and aminopeptidase activities were lower at the site 2 than 3, although the site 2 and 3 had similar content of carbon and nitrogen. The soil was toxic in V. fischeri test at the site 2, which had high content of PCP (3.93+/-1.00 microg PCP g dw of soil(-1)). The results indicated that endogenous carbon dioxide evolution, methane oxidation potential, butyrate-esterase, acetate-esterase, sulphatase and aminopeptidase activities were sensitive to PCP in the soil. The results indicated that alpha-glucosidase, beta-glucosidase, beta-xylosidase, beta-cellobiosidase, phosphomonoesterase, N-acetyl-glucosaminidase activity and FDA hydrolysis activity were not sensitive to PCP in the soil. Soil processes involved in the cycling of carbon, nitrogen, sulphur and phosphorus were only slightly vulnerable in the former sawmill area and most sensitive microbial species were probably replaced with more tolerant ones to maintain and recover functioning of the former sawmill soils.

Vertical distribution of sediment enzyme activities involved in the cycling of carbon, nitrogen, phosphorus and sulphur in three boreal rural lakes.

Vertical distribution of eleven hydrolytic enzyme activities were investigated with fluorogenic model substrates in boreal rural lake sediments, Lake Uurainen (13 km(2)), Lake Jämijärvi (9 km(2)) and Lake Pyylampi (0.068 km(2)), in Finland. The eleven hydrolytic enzyme activities were high into deep sediment layers indicating potential for turnover of organic matter in the permanently anoxic zones. The activities of beta-glucosidase, sulphatase and N-acetyl-glucosaminidase were similar in the three lakes independent on the ecological status of the lake. Acetate- and butyrate-esterase were more active than the other enzymes in the three lakes. These unspecific esterases had sediment activities in forest Lake Pyylampi close to those reported for boreal coniferous forest soils in Finland. Similar beta-cellobiosidase activities throughout the sediment depths indicated constant depolymerisation potential for cellulose.