Chrysene bioconversion by the white rot fungus <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> D1

The effect of cultivation conditions on chrysene bioconversion by the fungus Pleurotus ostreatus D1 was shown. Under the laccase production conditions, transformation of this polycyclic aromatic hydrocarbon occurs with accumulation of the quinone metabolite. Under both the laccase and versatile peroxidase production conditions, chrysene degradation occurs, with the stages leading to phthalic acid formation and its further utilization. The formation of phthalic acid as a metabolite of chrysene degradation by white rot fungi was revealed for the first time. The data obtained suggest that the laccase revealed on the mycelial surface and the extracellular laccase are probably involved at the initial stages of chrysene metabolism, whereas versatile peroxidase seems to be required for oxidizing the metabolites formed.     

Influence of cultivation conditions on pyrene degradation by the fungus Pleurotus Ostreatus D1

For the first time the dependence of completeness of pyrene degradation by the white-rot fungus Pleurotus ostreatus D1 on cultivation conditions was found. In Kirk’s medium about 65.6 ± 0.9% of the initial pyrene was metabolized after 3 weeks, with pyrene-4,5-dihydrodiol accumulating. This process was accompanied by laccase production only. In basidiomycetes rich medium, P. ostreatus D1 metabolized up to 89.8 ± 2.3% of pyrene within 3 weeks without pyrene-4,5-dihydrodiol accumulation throughout the time of cultivation. Phenanthrene and phthalic acid were identified as the metabolites produced from pyrene degradation under these conditions. Accumulation of phenanthrene with its subsequent disappearance was observed. One more metabolite probably was the product of phenanthrene degradation. Pyrene metabolism in basidiomycetes rich medium was accompanied first by laccase and tyrosinase production and later by versatile peroxidase production. The cell-associated activities of laccase, tyrosinase, and versatile peroxidase were found. The data obtained indicate that both enzymes (laccase and versatile peroxidase) are necessary for complete degradation of pyrene. Furthermore, both cell-associated and extracellular laccases can catalyse the first stages of pyrene degradation, and versatile peroxidase can be necessary for oxidation of the resulting metabolites.     

Effect of polycyclic aromatic hydrocarbons on laccase production by white rot fungus <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> D1

The effect of polycyclic aromatic hydrocarbons (PAHs) on the time course of laccase production by the fungus Pleurotus ostreatus D1 under conditions of submerged cultivation on Kirk’s medium has been studied. It has been shown that phenanthrene, fluoranthene, pyrene, and chrysene actively induce this enzyme, whereas fluorene and anthracene had a smaller effect. Addition of Mn²⁺ ions to cultivation medium elevates the laccase activity twofold and more in the presence of all the studied PAHs. Electrophoresis under nondenaturing conditions demonstrates induction of additional laccase forms by xenobiotics. Ligninolytic peroxidase activities are undetectable under the conditions used.     

Emulsifying Agent Production During PAHs Degradation by the White Rot Fungus <Emphasis Type="Italic">Pleurotus Ostreatus</Emphasis> D1

For the first time the production of an emulsifying agent during phthalic, 2,2′-diphenic and α-hydroxy-β-naphthoic acids, phenanthrene, anthracene, fluorene, pyrene, fluoranthene, and chrysene degradation by white rot fungus Pleurotus ostreatus was found. The emulsifying activity of the cultivation medium after degradation of these compounds was assessed. Maximal activities were found in the presence of chrysene (48.4%) and α-hydroxy-β-naphthoic acid (52.2%). Emulsifying activity inversely dependent on the water solubility of the compounds used. Versatile peroxidase was produced concurrently with the emulsifying agent.     

Influence of PAHs on ligninolytic enzymes of the fungus <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> D1

Polycyclic aromatic hydrocarbons (PAHs), their derivatives, and their degradation products were assayed for the ability to enhance activities of ligninolytic enzymes (laccase and versatile peroxidase) of the fungus Pleurotus ostreatus D1. The activities of both laccase and versatile peroxidase were induced by the PAHs, their derivatives, and their degradation products. Laccase was produced mostly in the first 7–10 days, whereas the production of versatile peroxidase began after 5–7 days of cultivation. Non-denaturing PAGE showed the presence of additional forms of laccase and versatile peroxidase in the presence of the xenobiotics in the cultivation medium. The difference in the production time for these enzymes may reflect that laccases are involved in the first stages of PAHs degradation and that versatile peroxidase can be necessary for oxidation of some degradation products. This is the first report on versatile peroxidase induction by PAHs and their derivatives.     

Promising cellulolytic fungi isolates for rice straw degradation

The objective of this study was to evaluate the potential of eight fungal isolates obtained from soils in rice crops for straw degradation in situ. From the initial eight isolates, Pleurotus ostreatus T1.1 and Penicillium sp. HC1 were selected for further characterization based on qualitative cellulolytic enzyme production and capacity to use rice straw as a sole carbon source. Subsequently, cellulolytic, xylanolytic, and lignolytic (Pleurotus ostreatus) activity on carboxymethyl cellulose, oat xylan, and rice straw with different nitrogen sources was evaluated. From the results obtained it was concluded both isolates are capable to produce enzymes necessary for rice straw degradation. However, their production is dependent upon carbon and nitrogen source. Last, it was established that Pleurotus ostreatus T1.1 and Penicillium sp. HC1 capability to colonize and mineralize rice straw, in mono-and co-culture, without affecting nitrogen soil content.     

Establishment of an efficient transformation system for <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

Pleurotus ostreatus is widely cultivated worldwide, but the lack of an efficient transformation system regarding its use restricts its genetic research. The present study developed an improved and efficient Agrobacterium tumefaciens-mediated transformation method in P. ostreatus. Four parameters were optimized to obtain the most efficient transformation method. The strain LBA4404 was the most suitable for the transformation of P. ostreatus. A bacteria-to-protoplast ratio of 100:1, an acetosyringone (AS) concentration of 0.1 mM, and 18 h of co-culture showed the best transformation efficiency. The hygromycin B phosphotransferase gene (HPH) was used as the selective marker, and EGFP was used as the reporter gene in this study. Southern blot analysis combined with EGFP fluorescence assay showed positive results, and mitotic stability assay showed that more than 75% transformants were stable after five generations. These results showed that our transformation method is effective and stable and may facilitate future genetic studies in P. ostreatus.     

Effect of polycyclic aromatic hydrocarbons on laccase production by white rot fungus Pleurotus ostreatus D1

The effect of polycyclic aromatic hydrocarbons (PAHs) on the dynamics of laccase production by the fungus Pleurotus ostreatus D1 under conditions of submerged cultivation on Kirk's medium has been studied. It has been shown that phenanthrene, fluoranthene, pyrene, and chrysene actively induce this enzyme, whereas fluorene and anthrecene had a smaller effect. Addition of Mn2+ ions to cultivation medium elevates the laccase activity twofold and more in the presence of all the studied PAHs. Electrophoresis under nondenaturing conditions demonstrates induction of additional laccase species by xenobiotics. Ligninolytic peroxidase activities are undetectable under the conditions used.     

The degradation of three-ringed polycyclic aromatic hydrocarbons by wood-inhabiting fungus Pleurotus ostreatus and soil-inhabiting fungus Agaricus bisporus

The degradation of two isomeric three-ringed polycyclic aromatic hydrocarbons by the white rot fungus Pleurotus ostreatus D1 and the litter-decomposing fungus Agaricus bisporus F-8 was studied. Despite some differences, the degradation of phenanthrene and anthracene followed the same scheme, forming quinone metabolites at the first stage. The further fate of these metabolites was determined by the composition of the ligninolytic enzyme complexes of the fungi. The quinone metabolites of phenanthrene and anthracene produced in the presence of only laccase were observed to accumulate, whereas those formed in presence of laccase and versatile peroxidase were metabolized further to form products that were further included in basal metabolism (e.g. phthalic acid). Laccase can catalyze the initial attack on the PAH molecule, which leads to the formation of quinones, and that peroxidase ensures their further oxidation, which eventually leads to PAH mineralization.A. bisporus, which produced only laccase, metabolized phenanthrene and anthracene to give the corresponding quinones as the dominant metabolites. No products of further utilization of these compounds were detected. Thus, the fungi's affiliation with different ecophysiological groups and their cultivation conditions affect the composition and dynamics of production of the ligninolytic enzyme complex and the completeness of PAH utilization.     

Oil degradation by basidiomycetes in soil and peat at low temperatures

A total of 17 basidiomycete strains causing white rot and growing on oil-contaminated substrates have been screened. Three strains with high (Steccherinum murashkinskyi), average (Trametes maxima), and low (Pleurotus ostreatus) capacities for the colonization of oil-contaminated substrates have been selected. The potential for degrading crude oil hydrocarbons has been assessed with the use of fungi grown on nonsterile soil and peat at low temperatures. Candida sp. and Rhodococcus sp. commercial strains have been used as reference organisms with oil-degrading ability. All microorganisms introduced in oil-contaminated soil have proved to be ineffective, whereas the inoculation of peat with basidiomycetes and oil-degrading microorganisms accelerated the destruction of oil hydrocarbons. The greatest degradation potential of oil-aliphatic hydrocarbons has been found in S. murashlinskyi. T. maxima turned out to be the most successful in degrading aromatic hydrocarbons. It has been suggested that aboriginal microflora contributes importantly to the effectiveness of oil-destructing microorganisms. T. maxima and S. murashkinskyi strains are promising for further study as oil-oxidizing agents during bioremediation of oil-contaminated peat soil under conditions of low temperatures.     

Laccase Production and Humic Acids Decomposition by Microscopic Soil Fungi

Laccase (para-diphenol:oxygen oxidoreductase, EC 1.10.3.2) is a phenol oxidase widespread in fungi and bacteria. In basidiomycetes, this enzyme is involved in the transformation of lignin and humic substances (HS) in soil. The role of laccases of soil ascomycetes and deuteromycetes in HS degradation is not established, and conditions of the enzyme production have been poorly studied. In the present work soil micromycetes, potential laccase producers, were isolated from typical soils of the forest, steppe, and foreststeppe zones of European Russia by plating on agar media with ABTS (2,2'-azino-bis(3-ethylbenzothiazoline- 6-sulphonic acid, sodium salt)) as the substrate. Their abundance, species composition, conditions of laccase production, and its relation to humic acids (HA) degradation in liquid and solid media were studied. Out of 68 strains isolated, 20 exhibited ABTS oxidation at initial plating on agar media. In pure cultures on agar media, oxidation was less pronounced, but in the presence of HA laccase production by some strains was higher than without HA. Significant and weak extracellular laccase production in liquid medium was observed for Acremonium murorum (Corda) W. Gams Z1710 and Botritis cinerea Pers. ex Fries Z1711, respectively. The level of laccase production by A. murorum was the same without inducers and in the presence of HA, while B. cinerea produced laccase only without inducers. No direct correlation was found between the presence of laccase and/or its activity and ability of the fungi to decolorize (degrade) HA. In liquid media active laccase producer A. murorum caused lower HA decolorization (43%) than B. cinerea (62%) and the fungi lacking extracellular laccase (54–81%). The role of micromycete oxidative systems in HA degradation requires further investigation.     

The composition of volatile components of cepe (<Emphasis Type="Italic">Boletus edulis</Emphasis>) and oyster mushrooms (<Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>)

The composition of aroma compounds in cooked and canned cepe (Boletus edulis) and in cooked oyster mushrooms (Pleurotus ostreatus) is studied using capillary gas chromatography and chromatographymass spectrometry. It is found that unsaturated alcohols and ketones containing eight atoms of carbon determine the aroma of raw mushrooms and take part in the formation of the aroma of cooked mushrooms as well. The content of these compounds was the highest in canned cepes. In oyster mushrooms, the concentration of these alcohols and ketones was lower in comparison with cepes. The content of aliphatic and aromatic aldehydes was much higher in oyster mushrooms. Volatile aliphatic and heterocyclic Maillard reaction products and isomeric octenols and octenones formed the aroma of cooked and canned mushrooms.     

Anticomplementary Activity of Antitumour Polysaccharides

THE polysaccharides obtained from some basidiomycetes, Pleurotus ostreatus (Jacq. ex Fr.) Quel.¹, Flammulina velutipes (Curt, ex Fr.) Sing.², Lentinus edodes (Berk.) Sing.^2–4, Polia cocos, Wolf (Bukuryo)⁵ and Schizophyllum commune Fr.⁷, strongly inhibit the growth of sarcoma 180 and their anti-tumour activity is host-mediated^8,9.     

Strains of <Emphasis Type="Italic">Bacillus</Emphasis> ssp. regulate wheat resistance to <Emphasis Type="Italic">Septoria nodorum</Emphasis> Berk.

The ability of Bacillus subtilis Cohn and Bacillus thuringiensis Berliner to induce systemic resistance in wheat plants to the casual agent of Septoria nodorum Berk., blotch has been studied. It has been shown that strains of Bacillus ssp. that possess the capacity for endophytic survival have antagonistic activity against this pathogen in vitro. A reduction of the degree of Septoria nodorum blotch development on wheat leaves under the influence of Bacillus spp. was accompanied by the suppression of catalase activity, an increase in peroxidase activity and H₂O₂ content, and expression of defence related genes such us PR-1, PR-6, and PR-9. It has been shown that B. subtilis 26 D induces expression levels of wheat pathogenesis-related (PR) genes which marks a SA-dependent pathway of sustainable development and that B. thuringiensis V-5689 and V-6066 induces a JA/ET-dependent pathway. These results suggest that these strain Bacillus spp. promotes the formation of wheat plant resistance to S. nodorum through systemic activation of the plant defense system. The designed bacterial consortium formed a complex biological response in wheat plants infected phytopathogen.     

Identification and genomic analysis of antifungal property of a tomato root endophyte <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. p21

Pseudomonas sp., which occupy a variety of ecological niches, have been widely studied for their versatile metabolic capacity to promote plant growth, suppress microbial pathogens, and induce systemic resistance in plants. In this study, a Pseudomonas sp. strain p21, which was isolated from tomato root endophytes, was identified as having antagonism against Aspergillus niger. Further analysis showed that this strain had the ability to biosynthesise siderophores and was less effective in inhibiting the growth of A. niger with the supplementation of Fe³⁺ in the agar medium. Genomic sequencing and the secondary metabolite cluster analysis demonstrated that Pseudomonas sp. p21 harboured 2 pyoverdine biosynthetic gene clusters, which encode compounds with predicted core structures and two variable tetra-peptide or eleven-peptide chains. The results indicated that siderophore-mediated competition for iron might be an important mechanism in Pseudomonas suppression of the fungal pathogen A. niger and in microbe-pathogen-plant interactions.     

Dearomatization of diesel oil using <Emphasis Type="Italic">Pseudomonas</Emphasis> sp.

Objectives

To improve the quality of diesel fuel via removal of aromatic compounds using Pseudomonas sp.

Results

In the present study Pseudomonas sp. was able to remove 94% of fluorene, 59% of phenanthrene, 49% of anthracene, 52% of fluoranthene, 45% of pyrene and 75% carbazole present in diesel oil. Additionally, it also does not affect the aliphatic content of fuel thus maintaining the carbon backbone of the fuel.

Conclusions

Pseudomonas sp. is a potential biocatalyst that can be used in the refining industry.

     

Use of the yeast-like cells of <Emphasis Type="Italic">Tremella fuciformis</Emphasis> as a cell factory to produce a <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> hydrophobin

Objectives

To obtain hydrophobin, a Class I hydrophobin gene, Po.hyd from Pleurotus ostreatus, was transformed into the yeast-like cells of Tremella fuciformis using Agrobacterium tumefaciens.

Results

The hydrophobin Po.HYD from P. ostreatus was heterogeneously expressed by the yeast-like cells of T. fuciformis. Plasmids harboring the Po.hyd gene driven by endogenous glyceraldehyde-3-phosphate dehydrogenase promoter were transformed by A. tumefaciens. The integration and expression of the rPo.HYD in the T. fuciformis cells were confirmed by PCR, Southern blot, fluorescence microscopy and quantitative real-time PCR. SDS-PAGE demonstrated that the rPo.HYD was extracted with the expected MW of 14 kDa. The yield of purified rPo.HYD was 0.58 mg/g dry wt. The protein, with its ability to stabilize oil droplets, exhibited a better emulsifying activity than the typical food emulsifiers Tween 20 and sodium caseinate.

Conclusion

Tremella fuciformis can be used as a cell factory to produce hydrophobin on a large scale for the food industry.

     

Extending our tools and resources in the non-conventional industrial yeast <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis> through the application of metabolite profiling methodologies

Introduction

Xanthophyllomyces dendrorhous is a non-conventional industrial yeast. It has the unique ability among yeasts to produce geranylgeranyl pyrophosphate derived terpenoids such as carotenoids and in particular the high value pigment astaxanthin.

Objective

In order to fully exploit the industrial potential of Xanthophyllomyces using modern industrial biotechnology approaches the further development of “omic” resources in this organism are required to build on the now sequenced and annotated genome. To contribute to this goal, the present study has developed and implemented an efficient metabolite profiling system comprised of, quenching, extraction and associated GC–MS and UPLC analysis.

Method

Four quenching methods and five extraction methods compatible with GC–MS and UPLC profiling were tested and validated by analysing steady state metabolite changes of Xanthophyllomyces cultivated at laboratory scale in liquid shake culture at lag, exponential and early and late stationary phases.

Results

A customised Automated Mass Spectral Deconvolution and Identification System (AMDIS) library has been created for Xanthophyllomyces, over 400 compounds are present in the library of which 78 are detected and quantified routinely in polar and non-polar derived extracts. A preliminary biochemical network has been constructed. Over a standardised laboratory growth cycle, changes in metabolite levels have been determined to create reference point for future strain improvement approaches and the initial biochemical network construction. Correlation analysis has illustrated that astaxanthin formation correlates positively with different sectors of intermediary metabolism (e.g. the TCA cycle intermediates and amino acid formation), “short” saturated fatty acids and β-carotene, while other metabolites are reduced in response to astaxanthin production. These sectors of intermediary metabolism offer potential future targets for the manipulation resulting in the generation of strains with improved titres of given terpenoids.

Discussion

In summary a robust metabolite profiling system for Xanthophyllomyces is in place to further our understanding and potential exploitation of this underutilised industrial yeast.

     

Degradation of fluorene,anthracene, phenanthrene,fluoranthene, and pyrene lacks connection to the production of extracellular enzymes by Pleurotus ostreatus and Bjerkandera adusta

The degradation of polycyclic aromatic hydrocarbons (PAH) was studied in liquid cultures of Bjerkandera adusta and Pleurotus ostreatus during 7 weeks of cultivation. During only 3 days of incubation, B. adusta removed 56% and 38% of fluorene and anthracene, while P. ostreatus degraded 43% and 60% of these compounds; other PAH were degraded to a lower extent. Except for anthracene in cultures of P. ostreatus, all PAH were removed uniformly during the cultivation time but fluorene and anthracene were degraded faster than other PAH. Supplementation of liquid cultures with milled wood decreased the concentration of PAH in the solution and diminished the degradation of PAH. The fungi produced valuable activity of manganese-dependent peroxidase; laccase was secreted only by P. ostreatus and was strongly induced by the addition of milled wood. The production of the oxidative enzymes did not correlate directly to the metabolisation of PAH.