Novel strategy to improve the colonizing ability of Irpex lacteus in non-sterile wheat straw for enhanced rumen and enzymatic digestibility.

Pretreatment with white rot fungi is a promising method to enhance the digestibility of lignocelluloses; however, sterilization of feedstocks prior to inoculation is one of the costliest steps. To improve the colonizing ability of white rot fungi under non-sterile condition, Irpex lacteus, Pleurotus ostreatus, and Phanerochaete chrysosporium were inoculated in the wheat straw ensiled for 28 days and incubated for 56 days to determine the changes in microbe counts, organic acid content, chemical composition, and rumen and enzymatic digestibility. Results showed that ensiling produced abundant organic acids and suppressed most microbes in wheat straw. Significant growth of I. lacteus was observed after 3 days of incubation, and molds were only detectable at day 7 in the group. At the end of incubation, aerobic bacteria and lactic acid bacteria decreased by 18% and 38% in the wheat straw treated with I. lacteus, but molds, aerobic bacteria, and lactic acid bacteria thrived in those treated with P. ostreatus and P. chrysosporium. Even more, P. ostreatus and P. chrysosporium increased the lignin content of the ensiled wheat straw by 34% and 65%. However, I. lacteus selectively degraded lignin by 28% and improved the rumen and enzymatic digestibility by 18% and 34%. The finding indicates that ensiling prior to fermentation with I. lacteus is an effective method to control spoilage microbes and to enhance the rumen and enzymatic digestibility of wheat straw.

Dynamics of the Phanerochaete carnosa transcriptome during growth on aspen and spruce.

BACKGROUND: The basidiomycete Phanerochaete carnosa is a white-rot species that has been mainly isolated from coniferous softwood. Given the particular recalcitrance of softwoods to bioconversion, we conducted a comparative transcriptomic analysis of P. carnosa following growth on wood powder from one softwood (spruce; Picea glauca) and one hardwood (aspen; Populus tremuloides). P. carnosa was grown on each substrate for over one month, and mycelia were harvested at five time points for total RNA sequencing. Residual wood powder was also analyzed for total sugar and lignin composition. RESULTS: Following a slightly longer lag phase of growth on spruce, radial expansion of the P. carnosa colony was similar on spruce and aspen. Consistent with this observation, the pattern of gene expression by P. carnosa on each substrate converged following the initial adaptation. On both substrates, highest transcript abundances were attributed to genes predicted to encode manganese peroxidases (MnP), along with auxiliary activities from carbohydrate-active enzyme (CAZy) families AA3 and AA5. In addition, a lytic polysaccharide monooxygenase from family AA9 was steadily expressed throughout growth on both substrates. P450 sequences from clans CPY52 and CYP64 accounted for 50% or more of the most highly expressed P450s, which were also the P450 clans that were expanded in the P. carnosa genome relative to other white-rot fungi. CONCLUSIONS: The inclusion of five growth points and two wood substrates was important to revealing differences in the expression profiles of specific sequences within large glycoside hydrolase families (e.g., GH5 and GH16), and permitted co-expression analyses that identified new targets for study, including non-catalytic proteins and proteins with unknown function.

White rot fungi and advanced combined biotechnology with nanomaterials: promising tools for endocrine-disrupting compounds biotransformation.

Endocrine-disrupting compounds (EDCs) can interfere with endocrine systems and bio-accumulate through the food chain and even decrease biodiversity in contaminated areas. This review discusses a critical overview of recent research progress in the biotransformation of EDCs (including polychlorinated biphenyl and nonylphenol, and suspected EDCs such as heavy metals and sulfonamide antibiotics) by white rot fungi (WRF) based on techniques with an emphasis on summarizing and analyzing fungal molecular, metabolic and genetic mechanisms. Not only intracellular metabolism which seems to perform essential roles in the ability of WRF to transform EDCs, but also advanced applications are deeply discussed. This review mainly reveals the removal pathway of heavy metal and antibiotic pollutants because the single pollution almost did not exist in a real environment while the combined pollution has become more serious and close to people's life. The trends in WRF technology and its related advanced applications which use the combined technology, including biocatalysis of WRF and adsorption of nanomaterials, to degrade EDCs have also been introduced. Furthermore, challenges and future research needs EDCs biotransformation by WRF are also discussed. This research, referring to metabolic mechanisms and the combined technology of WRF with nanomaterials, undoubtedly contributes to the applications of biotechnology. This review will be of great benefit to an understanding of the trends in biotechnology for the removal of EDCs.

Effect of steam explosion on solid-state fermentation of maize stalk by Penicillium decumbens and Phanerochaete chrysosporium for animal feed production.

Pre-treated maize stalk could be used for animal feed due to the improved digestibility. Steam explosion (SE) and solid-state fermentation (SSF) are two methods in maize stalk pre-treatment. To evaluate the effect of combination of SE and SSF on the maize stalk pre-treatment, the maize stalk was treated with steam explosion and microbial fermentation with Penicillium decumbens and Phanerochate chrysporium in this study. The steam explosion severity (logR0 ) affected component of the corn stover and then affected the SSF. The highest weight loss of substrate (23.20%), protein (12.71%) and cellulase filter paper activity (FPA) (7.42IU/g) could be obtained at logR0  = 3.64 and the IVDMD of SE-SSF-treated straw could reach 75.8%. The SE-SSF pre-treatment method could improve the utilization of straw as animal feed and overcome the shortage of feed source in animal husbandry.

Effect of selenite on the morphology and respiratory activity of Phanerochaete chrysosporium biofilms.

The temporal and spatial effects of selenite (SeO3(2-)) on the physical properties and respiratory activity of Phanerochaete chrysosporium biofilms, grown in flow-cell reactors, were investigated using oxygen microsensors and confocal laser scanning microscopy (CLSM) imaging. Exposure of the biofilm to a SeO3(2-) load of 1.67mgSeL(-1)h(-1) (10mgSeL(-1) influent concentration), for 24h, resulted in a 20% reduction of the O2 flux, followed by a ∼10% decrease in the glucose consumption rate. Long-term exposure (4days) to SeO3(2-) influenced the architecture of the biofilm by creating a more compact and dense hyphal arrangement resulting in a decrease of biofilm thickness compared to fungal biofilms grown without SeO3(2-). To the best of our knowledge, this is the first time that the effect of SeO3(2-) on the aerobic respiratory activity on fungal biofilms is described.

Impact of Phanerochaete chrysosporium on the Functional Diversity of Bacterial Communities Associated with Decaying Wood.

Bacteria and fungi naturally coexist in various environments including forest ecosystems. While the role of saprotrophic basidiomycetes in wood decomposition is well established, the influence of these fungi on the functional diversity of the wood-associated bacterial communities has received much less attention. Based on a microcosm experiment, we tested the hypothesis that both the presence of the white-rot fungus Phanerochaete chrysosporium and the wood, as a growth substrate, impacted the functional diversity of these bacterial communities. Microcosms containing sterile sawdust were inoculated with a microbial inoculum extracted from a forest soil, in presence or in absence of P. chrysosporium and subsequently, three enrichment steps were performed. First, bacterial strains were isolated from different microcosms previously analyzed by 16S rRNA gene-based pyrosequencing. Strains isolated from P. chrysosporium mycosphere showed less antagonism against this fungus compared to the strains isolated from the initial forest soil inoculum, suggesting a selection by the fungus of less inhibitory bacterial communities. Moreover, the presence of the fungus in wood resulted in a selection of cellulolytic and xylanolytic bacterial strains, highlighting the role of mycospheric bacteria in wood decomposition. Additionally, the proportion of siderophore-producing bacteria increased along the enrichment steps, suggesting an important role of bacteria in iron mobilization in decaying-wood. Finally, taxonomic identification of 311 bacterial isolates revealed, at the family level, strong similarities with the high-throughput sequencing data as well as with other studies in terms of taxonomic composition of the wood-associated bacterial community, highlighting that the isolated strains are representative of the wood-associated bacterial communities.

The GSTome Reflects the Chemical Environment of White-Rot Fungi.

White-rot fungi possess the unique ability to degrade and mineralize all the different components of wood. In other respects, wood durability, among other factors, is due to the presence of extractives that are potential antimicrobial molecules. To cope with these molecules, wood decay fungi have developed a complex detoxification network including glutathione transferases (GST). The interactions between GSTs from two white-rot fungi, Trametes versicolor and Phanerochaete chrysosporium, and an environmental library of wood extracts have been studied. The results demonstrate that the specificity of these interactions is closely related to the chemical composition of the extracts in accordance with the tree species and their localization inside the wood (sapwood vs heartwood vs knotwood). These data suggest that the fungal GSTome could reflect the chemical environment encountered by these fungi during wood degradation and could be a way to study their adaptation to their way of life.

Fungal pretreatment by Phanerochaete chrysosporium for enhancement of biogas production from corn stover silage.

Corn stover silage (CSS) was pretreated by Phanerochaete chrysosporium in solid-state fermentation (SSF), to enhance methane production via subsequent anaerobic digestion (AD). Effects of washing of corn stover silage (WCSS) on the lignocellulosic biodegradability in the fungal pretreatment step and on methane production in the AD step were investigated with comparison to the CSS. It was found that P. chrysosporium had the degradation of cellulose, hemicellulose, and lignin of CSS up to 19.9, 32.4, and 22.6 %, respectively. Consequently, CSS pretreated by 25 days achieved the highest methane yield of 265.1 mL/g volatile solid (VS), which was 23.0 % higher than the untreated CSS. However, the degradation of cellulose, hemicellulose, and lignin in WCSS after 30 days of SSF increased to 45.9, 48.4, and 39.0 %, respectively. Surface morphology and Fourier-transform infrared spectroscopy analyses also demonstrated that the WCSS improved degradation of cell wall components during SSF. Correspondingly, the pretreatment of WCSS improved methane production by 19.6 to 32.6 %, as compared with untreated CSS. Hence, washing and reducing organic acids (such as lactic acid, acetic acid, propionic acid, and butyric acid) present in CSS has been proven to further improve biodegradability in SSF and methane production in the AD step.

Hydroxylation of bisphenol A by hyper lignin-degrading fungus Phanerochaete sordida YK-624 under non-ligninolytic condition.

Bisphenol A (BPA) is one of the representative compounds of the endocrine disrupting compounds group and the highest volume chemicals produced worldwide. As a result, BPA is often detected in many soil and water environments. In this study, we demonstrated the transformation of BPA from liquid cultures inoculated with hyper lignin-degrading fungus Phanerochaete sordida YK-624. Under non-ligninolytic condition, approximately 80% of BPA was eliminated after 7d of incubation. High-resolution electrospray ionization mass spectra and nuclear magnetic resonance analyses of a metabolite isolated from the culture supernatant suggested that BPA was metabolized to hydroxy-BPA, 4-(2-(4-hydroxyphenyl)propan-2-yl)benzene-1,2-diol, which has a much lower estrogenic activity than BPA. In addition, we investigated the effect of the cytochrome P450 inhibitor piperonyl butoxide (PB) on the hydroxylation of BPA, markedly lower transformation activity of BPA was observed in cultures containing PB. These results suggest that cytochrome P450 plays an important role in the hydroxylation of BPA by P. sordida YK-624 under non-ligninolytic condition.

Novel application of fungal Phanerochaete sp. and xylanase for reduction in pollution load of paper mill effluent.

Four different strategies of pulping and bleaching were carried out to develop alternative mechanistic ecoenvironmental friendly approaches and generated effluent was characterised. Strategy-I included Phanerochaete sp. fungal pretreatment followed by conventional bleaching, whereas in strategy-II, fungal pretreatment was followed by enzyme xylanase aided bleaching. Strategy-III also included xylanase supplement but without prior fungal pretreatment. Chemically driven pulping and bleaching was the IV strategy. Conventional C(D)E(OP)D1D2 sequence of bleaching was used for strategy-I and IV whereas XC(D)E(OP)D1D2 sequence was applied to strategy-I and III. Strategy-II was responsible for 27.5% reduction in Kappa no. whereas the maximum (27.5%) reduction in refining energy was observed with strategy-II. Biobleaching strategies-II and III were helpful in saving 37.3 and 20.3% of elemental chlorine (Cl2) and 30.8 and 23.1% of chlorine dioxide (ClO2) respectively. In comparison to control (strategy-IV), strategy II resulted in maximum pollution load reduction of chemical oxygen demand (COD), biological oxygen demand (BOD), color and adsorbable organic halides (AOX) up to 57, 60, 30 and 43.6%, respectively.

Responses of Phanerochaete chrysosporium to toxic pollutants: physiological flux, oxidative stress, and detoxification.

The white-rot fungus Phanerochaete chrysosporium has been widely used for the treatment of waste streams containing heavy metals and toxic organic pollutants. The development of fungal-based treatment technologies requires detailed knowledge of the relationship between bulk water quality and the physiological responses of fungi. A noninvasive microtest technique was used to quantify real-time changes in proton, oxygen, and cadmium ion fluxes following the exposure of P. chrysosporium to environmental toxic (2,4-dichlorophenol and cadmium). Significant changes in H(+) and O(2) flux occurred after exposure to 10 mg/L 2,4-dichlorophenol and 0.1 mM cadmium. Cd(2+) flux decreased with time. Reactive oxygen species formation and antioxidant levels increased after cadmium treatment. Superoxide dismutase activity correlated well with malondialdehyde levels (r(2) = 0.964) at low cadmium concentrations. However, this correlation diminished and malondialdehyde levels significantly increased at the highest cadmium concentration tested. Real-time microscale signatures of H(+), O(2), and Cd(2+) fluxes coupled with oxidative stress analysis can improve our understanding of the physiological responses of P. chrysosporium to toxic pollutants and provide useful information for the development of fungal-based technologies to improve the treatment of wastes cocontaminated with heavy metals and organic pollutants.

Elimination of carbamazepine in a non-sterile fungal bioreactor.

A properly configured bioreactor is in need to transfer the fungal biodegradation of recalcitrant pollutants into real applications. In this study, a novel plate bioreactor was designed to eliminate carbamazepine (CBZ), a widely concerned pharmaceutical, with the white rot fungus Phanerochaete chrysosporium grown on polyether foam under non-sterile conditions. The bioreactor was operated in both sequence batch and continuous modes. It was found that the sufficient supply with nutrients is crucial for an effective elimination of CBZ. Given the conditions, a high elimination of CBZ (60-80%) was achieved. The effective elimination was stable in a continuous operation for a long term (around 100 days). The high elimination of CBZ could also be achieved under real conditions with the effluent from a municipal wastewater treatment plant.

Growth-induced mass flows in fungal networks.

Cord-forming fungi form extensive networks that continuously adapt to maintain an efficient transport system. As osmotically driven water uptake is often distal from the tips, and aqueous fluids are incompressible, we propose that growth induces mass flows across the mycelium, whether or not there are intrahyphal concentration gradients. We imaged the temporal evolution of networks formed by Phanerochaete velutina, and at each stage calculated the unique set of currents that account for the observed changes in cord volume, while minimizing the work required to overcome viscous drag. Predicted speeds were in reasonable agreement with experimental data, and the pressure gradients needed to produce these flows are small. Furthermore, cords that were predicted to carry fast-moving or large currents were significantly more likely to increase in size than cords with slow-moving or small currents. The incompressibility of the fluids within fungi means there is a rapid global response to local fluid movements. Hence velocity of fluid flow is a local signal that conveys quasi-global information about the role of a cord within the mycelium. We suggest that fluid incompressibility and the coupling of growth and mass flow are critical physical features that enable the development of efficient, adaptive biological transport networks.

Fungal network responses to grazing.

Mycelial networks operate on scales from microscopic to many m(2) and naturally persist for extended periods. As fungi exhibit highly adaptive development, it is important to test behavioural responses on natural substrata with realistic nutrient levels across a range of spatial scales and extended time periods. Here we quantified network responses over 7.5 months in large (57 x 57cm) microcosms to test whether grazing shifts the network to a more resilient architecture. Resource limitation constrained any ability to respond at all, with both grazed and ungrazed networks gradually thinning out over time. Added resources sustained further exploratory growth, but only transiently increased cross-connectivity and network resilience, when tested by simulated damage in silico. Grazed networks were initially weaker and emergence of new exploratory growth was curtailed. However, increased interstitial proliferation led to new cross-links, consolidating the existing mycelial network and increasing the resilience of the network to further attack.

Transcriptome and secretome analyses of Phanerochaete chrysosporium reveal complex patterns of gene expression.

The wood decay basidiomycete Phanerochaete chrysosporium was grown under standard ligninolytic or cellulolytic conditions and subjected to whole-genome expression microarray analysis and liquid chromatography-tandem mass spectrometry of extracellular proteins. A total of 545 genes were flagged on the basis of significant changes in transcript accumulation and/or peptide sequences of the secreted proteins. Under nitrogen or carbon limitation, lignin and manganese peroxidase expression increased relative to nutrient replete medium. Various extracellular oxidases were also secreted in these media, supporting a physiological connection based on peroxide generation. Numerous genes presumed to be involved in mobilizing and recycling nitrogen were expressed under nitrogen limitation, and among these were several secreted glutamic acid proteases not previously observed. In medium containing microcrystalline cellulose as the sole carbon source, numerous genes encoding carbohydrate-active enzymes were upregulated. Among these were six members of the glycoside hydrolase family 61, as well as several polysaccharide lyases and carbohydrate esterases. Presenting a daunting challenge for future research, more than 190 upregulated genes are predicted to encode proteins of unknown function. Of these hypothetical proteins, approximately one-third featured predicted secretion signals, and 54 encoded proteins detected in extracellular filtrates. Our results affirm the importance of certain oxidative enzymes and, underscoring the complexity of lignocellulose degradation, also support an important role for many new proteins of unknown function.

Limitations of a membrane gradostat bioreactor designed for enzyme production from biofilms of Phanerochaete chrysosporium.

Growing interest has been shown in the continuous production of high-value products such as extracellular secondary metabolites used in the biotechnology, bioremediation and pharmaceutical industries. These high-value extracellular secondary metabolites are mostly produced in submerged fermentations. However, the use of continuous membrane bioreactors was determined to be highly productive. A novel membrane bioreactor, classified as a membrane gradostat reactor (MGR) was developed to immobilize biofilms to produce extracellular secondary metabolites continuously using an externally unskinned and internally skinned membrane. Anaerobic zones were identified in the MGR system when air was used for aeration. To improve the MGR system, limitations related to the performance of the bioreactor were determined using P. chrysosporium. A DO penetration depth of +/-450 microm was identified after 264 h, with the anaerobic zone thickness reaching approximately 1,943 microm in the immobilised biofilms. The penetration ratio, decreased from 0.42 after 72 h to 0.14 after 264 h. This led to the production of ethanol in the range of 10 to 56 mg/L in the MCMGR and 7 to 54 mg/L in SCMGR systems. This was attributed to an increase in beta-glucan within immobilised biofilms when an oxygen enriched aeration source was used. Increasing lipid peroxidation and trace element accumulation was observed with the use of an oxygen enriched aeration source.

Quantifying dynamic resource allocation illuminates foraging strategy in Phanerochaete velutina.

Saprotrophic woodland fungi forage for mineral nutrients and woody resources by extension of a mycelial network across the forest floor. Different species explore at different rates and establish networks with qualitatively differing architecture. However, detailed understanding of fungal foraging behaviour has been hampered by the absence of tools to quantify resource allocation and growth accurately and non-invasively. To solve this problem, we have used photon-counting scintillation imaging (PCSI) to map and quantify nutrient allocation and localised growth simultaneously in heterogeneous resource environments. We show that colonies spontaneously shift to an asymmetric growth pattern, even in the absence of added resources, often with a distinct transition between the two growth phases. However, the extent of polarisation was much more pronounced and focussed in the presence of an additional cellulose resource. In this case, there was highly localised growth, often at the expense of growth elsewhere in the colony, and marked accumulation of (14)C-AIB in the sector of the colony with the added resource. The magnitude of the response was greatest when resource was added around the time of the endogenous developmental transition. The focussed response required a metabolisable resource, as only limited changes were seen with glass fibre discs used to mimic the osmotic and thigmotropic stimuli upon resource addition. Overall the behaviour is consistent with an adaptive foraging strategy, both to exploit new resources and also to redirect subsequent foraging effort to this region, presumably with an expectation that the probability of finding additional resources is increased.

Reorganization of mycelial networks of Phanerochaete velutina in response to new woody resources and collembola (Folsomia candida) grazing.

Mycelial development of Phanerochaete velutina extending from wood inocula in 57 x 57 cm trays of non-sterile soil was characterized after adding: (1) collembola; (2) new wood resources; (3) both new wood resources and collembola; and (4) no new resources and no collembola. After 99 d, all systems had produced distinct mycelial cords, much of the diffuse mycelium and thinner cords that were produced early on having regressed. Systems to which new resources (but no collembola) had been added developed thick cords interconnecting inocula with new resources, and much of the non-connected mycelium regressed. Nonetheless, these systems had significantly greater hyphal coverage and mass fractal dimension than the other treatments, resulting from outgrowth from the new resources. Unexpectedly, morphology of grazed systems with no added resources was very similar to that of ungrazed systems with no added resources, apparently because the collembola grazed on senescing hyphae that would ultimately have regressed. Where new resources and collembola were added, there was proliferation of fine mycelium along connective cords and elsewhere, but this was not as extensive as in the new resource/no collembola systems, the fine mycelium apparently being grazed in patches. Fungus gnat (family Sciaridae) larvae contaminated eight (out of 14) trays with no added collembola, but none of the systems to which collembola had been added. They burrowed around the wood and caused cords to be severed.

[Production of ligninolytic enzymes in bioreactor].

Production of ligninolytic enzymes under nitrogen limited conditions(C/N = 56/2.2) was studied in a 5-L stirred tank bioreactor with a working volume of 2 L for obtaining higher production of ligninolytic enzymes by white rot fungus Phanerochaete chrysosporium BKM-F-1767 and its control strategy. Results show that the manganese peroxidase (MnP) and laccase (Lac) reached peak at the sixth day and the seventh day, respectively, and the variation of them with time in a batch cultivation are similar to the results by agitated Erlenmeyer flasks; however higher enzyme activity was not achieved by applying a fed-batch strategy, in which nitrogen limited medium was fed to the reactor. In addition, variation of pH during cultivation was related to the growth of P. chrysosporium and enzymes production during both batch and fed-batch cultivation. The pH value of liquid medium began to decline when the enzyme activity occurred in the system, and the decline became more and more slow along with the decrease of enzyme activity at the end of fermentation. So, pH would be as a control parameter to find out the growth of P. chrysosporium and enzymes production during incubating P. chrysosporium. However, fed-batch strategy still need further study.

Ligninolytic enzyme production by Phanerochaete chrysosporium in plastic composite support biofilm stirred tank bioreactors.

Phanerochaete chrysosporium (ATCC 24725) produced lignin peroxidase (LiP) and manganese peroxidase (MnP) in defined medium in plastic composite support (PCS) biofilm stirred tank reactors. Laccase was not detected. The formation of the Ph. chrysosporium biofilm on the PCS was essential for the production of MnP and LiP. The bioreactor was operated as a repeat batch, and no reinoculation was required between batches. Peroxidase production was influenced by 5 min purging of the bioreactor with pure oxygen or continuous aerating with a mixture of air and oxygen at a flow rate of 0.005 vvm. Continuous aeration and 300 rpm agitation with 3 mM veratryl alcohol addition on days 0 and 3 demonstrated the highest lignin peroxidase production on day 6 with means of 50.0 and 47.0 U/L. Addition of veratryl alcohol and MnSO(4) on day 0 with 300 rpm agitation and continuous aeration at 0.005 vvm (air flow rate in L/min divided by the reactor working volume in liters) hastens the production of MnP with final yield of 63.0 U/L after 3 days. Fourteen repeated batches fermentation were performed without contamination due to low pH (4.5) and aseptic techniques employed.