Biological pretreatment of wheat straw by Phanerochaete chrysosporium supplemented with inorganic salts

Inorganic salts and tween 80 are known to induce the lignin degrading peroxidase expression of Phanerochaete chrysosporium in submerged culture. In this study, the wheat straw pretreatment supplemented with inorganic salts (salts group), tween 80 (plus) and no supplementation to the biomass (minus) were examined. Among the solid state fermentation groups, salts group resulted in a substantial degradation of wheat straw within one week, along with the highest lignin loss (25%) and ～250% higher efficiency for the total sugar release through enzymatic hydrolysis. The results were correlated with pyrolysis GC-MS (Py-GC-MS), thermogravimetric (TG)/differential thermogravimetric (DTG) and X-ray diffraction (XRD). The results suggested that the supplementation of inorganic salts in the solid state fermentation of wheat straw significantly enhances the degradation rate of the biomass by P. chrysosporium which can be exploited as an alternative means to existing pretreatment technologies.     

Ultrastructural aspects of wheat straw degradation by Phanerochaete chrysosporium and Trametes versicolor

The ultrastructural patterns characterizing wheat straw degradation by the ligninolytic fungi Phanerochaete chrysosporium and Trametes versicolor were studied. During fungal attack, the less lignified tissues were degraded first, whereas the xylematic and sclerenchymatic fibers underwent a delayed attack. In straw samples degraded by T. versicolor, partial delignification, defibrillation and swelling of cell walls, often causing separation between primary and secondary walls, were observed. By contrast, the formation of erosions and fissures, with minor lignin removal, characterized the attack to the cell wall by P. chrysosporium. At an advanced stage of decay, KMnO₄ staining demonstrated abundant electron-dense material around hyphae and in the proximity of the cell-wall surface. In the case of P. chrysosporium, spherical black bodies were found in the erosions and fissures produced during fungal attack.     

Solid-state production of biopulp by Phanerochaete chrysosporium using steam-exploded wheat straw as substrate.

A novel material for biopulp-making, steam-exploded wheat straw (SEWS), was studied. During the steam explosion process, the hemicellulose was partly degraded and became water-soluble sugar as the carbon resource of the chosen microbe growth, and compared with non-SEWS, the degradation amount of cellulose decreased and the degradation amount of lignin increased for the fermented steam-exploded wheat straw (FSEWS) cultured with Phanerochaete chrysosporium ME-446. Under the optimum conditions of solid-state ferrmentation (SSF), the degradation amount of lignin reached 60% on the 5th day and the fermented straw residue could be used directly as the material for pulp making.     

黄孢原毛平革菌产絮凝剂营养条件优化

微生物絮凝剂与传统化学絮凝剂相比,安全无毒、无二次污染,具有开发潜力.黄孢原毛平革菌(Phanerochaete chrysosporium)能产生微生物絮凝剂,但目前缺少对其产絮凝剂营养条件的优化.使用高岭土并利用单因素法研究碳源、氮源、碳氮比、接种量对Phanerochaete chrysosporium产絮凝剂的...     

Ligninase production in agitated conditions by Phanerochaete chrysosporium

Abstract Extracellular H₂O₂-dependent ligninase activity of Phanerochaete chrysosporium was produced in agitated culture conditions when veratryl alcohol or veratraldehyde were added to the cultures. The enzyme production was suppressed by cycloheximide indicating that true protein synthesis occurred. The activated cultures were also able to degrade synthetic lignin. Reduction of veratraldehyde to corresponding alcohol during secondary metabolism was a good indicator of the effect of agitation on cell metabolism. Too high agitation speed led to complete inhibition of both the reduction reaction and the ligninolytic activity.     

A scalable membrane gradostat reactor for enzyme production using Phanerochaete chrysosporium

This is the first demonstration of process scale-up of a membrane gradostat reactor for continuous enzyme production using Phanerochaete chrysosporium ME446. The fungus was immobilised by reverse filtration on to externally unskinned, ultrafiltration capillary membranes and then nutrient gradients were induced across the biofilm. A 10-fold scale-up from a single capillary bioreactor to a 2.4 l multi-capillary unit resulted in a 7-fold increase in enzyme productivity with a peak at 209 U l^–1 d^–1. Subsequent scale effects on the spore distribution, continuous manganese peroxidase production profile and biofilm development are discussed.     

Fungal pretreatment of lignocellulose by Phanerochaete chrysosporium to produce ethanol from rice straw

Phanerochaete chrysosporium is a wood‐rot fungus that is capable of degrading lignin via its lignolytic system. In this study, an environmentally friendly fungal pretreatment process that produces less inhibitory substances than conventional methods was developed using P. chrysosporium and then evaluated by various analytical methods. To maximize the production of manganese peroxidase, which is the primary lignin‐degrading enzyme, culture medium was optimized using response surface methodologies including the Plackett–Burman design and the Box–Behnken design. Fermentation of 100 g of rice straw feedstock containing 35.7 g of glucan (mainly in the form of cellulose) by cultivation with P. chrysosporium for 15 days in the media optimized by response surface methodology was resulted in a yield of 29.0 g of glucan that had an enzymatic digestibility of 64.9% of the theoretical maximum glucose yield. In addition, scanning electronic microscopy, confocal laser scanning microscopy, and X‐ray diffractometry revealed significant microstructural changes, fungal growth, and a reduction of the crystallinity index in the pretreated rice straw, respectively. When the fungal‐pretreated rice straw was used as a substrate for ethanol production in simultaneous saccharification and fermentation (SSF) for 24 h, the ethanol concentration, production yield and the productivity were 9.49 g/L, 58.2% of the theoretical maximum, and 0.40 g/L/h, respectively. Based on these experimental data, if 100 g of rice straw are subjected to fungal pretreatment and SSF, 9.9 g of ethanol can be produced after 96 h, which is 62.7% of the theoretical maximum ethanol yield. Biotechnol. Bioeng. 2009; 104: 471–482 © 2009 Wiley Periodicals, Inc.     

Ligninolytic enzyme production by Phanerochaete chrysosporium under conditions of nitrogen sufficiency

Abstract A lignin-degrading enzyme has been detected in culture supernatants of Phanerochaete chrysosporium strain INA-12 grown under non-limiting nitrogen conditions. Highest levels of enzyme activity were observed when glycerol served as carbon source. Veratryl alcohol, a known secondary metabolite of P. chrysosporium , was also produced in high nitrogen/glycerol cultures of strain INA-12 and closely followed the development of the 'ligninase' activity. Evolution of ¹⁴CO₂ from ¹⁴C-ring-DHP was readily observed when a hydrogen peroxide-generating system was added to 5-day-old high nitrogen/glycerol cultures which contained high amounts of enzyme.     

Metabolism of cyanide by Phanerochaete chrysosporium

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 (LiP H2) from the white rot fungus Phanerochaete chrysosporium was strongly inhibited by sodium cyanide. The I50 was estimated to be about 2-3 microM. In contrast, sodium cyanide binds to the native enzyme with an apparent sodium cyanide dissociation constant Kd of about 10 microM. Inhibition of the veratryl alcohol oxidase activity of LiP H2 by cyanide was reversible. Ligninolytic cultures of P. chrysosporium mineralized cyanide at a rate that was proportional to the concentration of cyanide to 2 mM. The N-tert-butyl-alpha-phenylnitrone-cyanyl radical adduct was observed by ESR spin trapping upon incubation of LiP H2 with H2O2 and sodium cyanide. The identity of the spin adduct was confirmed using 13C-labeled cyanide. Six-day-old cultures of the fungus were more tolerant to sodium cyanide toxicity than spores. Toxicity measurements were based on the effect of sodium cyanide on respiration of the fungus as determined by the metabolism of [14C]glucose to [14C]CO2. We propose that this tolerance of the mature fungus was due to its ability to mineralize cyanide and that this fungus might be effective in treating environmental pollution sites contaminated with cyanide.     

Removal of surfactant solubilized polycyclic aromatic hydrocarbons by Phanerochaete chrysosporium in a rotating biological contactor reactor

White rot fungi can oxidize surfactant solubilized polycyclic aromatic hydrocarbons (PAH). The objective of this study was to evaluate the performance of immobilized white rot fungus, Phanerochaete chrysosporium, to remove surfactant Tween 80 solubilized PAH i.e. phenanthrene, pyrene and benzo(alpha)pyrene in a rotating biological contactor (RBC) reactor. Results indicated that the immobilized P. chrysosporium in the RBC reactor system in continuous operation could effectively remove the three tested PAH at specific hydraulic loading rates and concentrations tested for each individual PAH. Batch operation of RBC reactor showed that the immobilized P. chrysosporium was stable and effective for the eight successive batch treatments of PAH in solution medium i.e. PAH removal was greater than 90% after 60 h, although only low levels of ligninolytic enzyme activity were detected. The removal of phenanthrene and pyrene in solution medium has been found to be a first order reaction in batch operation. A mass balance calculation indicated that biological oxidation was the main factor for removal of benzo(alpha)pyrene i.e. 95.7% in the RBC reactor. However, for phenanthrene and pyrene, both biological oxidation (i.e. 49 and 56%, respectively) and RBC disc foam adsorption (i.e. 44 and 34%, respectively) made a significant contribution to the removal of PAH.     

Biocatalytic decolourisation of molasses by Phanerochaete chrysosporium

Bioremediation potential of Phanerochaete chrysosporium strains NCIM 1073, NCIM 1106 and NCIM 1197 to decolourise molasses in solid and liquid molasses media was studied. Strains varied in the pattern of molasses decolourisation on solid medium by Giant colony method. Under submerged cultivation conditions, strain NCIM 1073 did not decolourise molasses while, strains NCIM 1106 and NCIM 1197 could decolourise molasses up to 82% and 76%, respectively. Under stationary cultivation conditions, none of the strains could decolourise molasses. This was overcome by increasing the surface area of the culture in flat bottom glass bottles under stationary cultivation conditions. Under submerged cultivation conditions, growth was more or less same in all strains. However, the lignin peroxidase and manganese peroxidase activities were significantly less in the strain NCIM 1073. Under stationary cultivation conditions, none of the strains could produce enzymes lignin peroxidase, manganese peroxidase and laccase. However, all of them could produce lignin peroxidase and manganese peroxidase when cultivated in flat bottom glass bottles under stationary cultivation conditions.     

Metabolism of phenanthrene by Phanerochaete chrysosporium.

The white rot fungus Phanerochaete chrysosporium metabolized phenanthrene when it was grown for 7 days at 37 degrees C in a medium containing malt extract, D-glucose, D-maltose, yeast extract, and Tween 80. After cultures were grown with [9-14C]phenanthrene, radioactive metabolites were extracted from the medium with ethyl acetate, separated by high-performance liquid chromatography, and detected by liquid scintillation counting. Metabolites from cultures grown with unlabeled phenanthrene were identified as phenanthrene trans-9,10-dihydrodiol, phenanthrene trans-3,4-dihydrodiol, 9-phenanthrol, 3-phenanthrol, 4-phenanthrol, and the novel conjugate 9-phenanthryl beta-D-glucopyranoside. Identification of the compounds was based on their UV absorption, mass, and nuclear magnetic resonance spectra. Since lignin peroxidase was not detected in the culture medium, these results suggest the involvement of monooxygenase and epoxide hydrolase activity in the initial oxidation and hydration of phenanthrene by P. chrysosporium.     

Effect of oxygenation conditions on submerged cultures of Phanerochaete chrysosporium

Summary The effect of the oxygen supply pattern on the onset and development of the lignolytic enzyme system of Phanerochaete chrysosporium was studied in submerged culture employing the serum bottle approach. Periodic or continuous flushing through the head phase, and continuous bubbling through the liquid phase with either oxygen (O₂) or air were applied. The nature of the O₂ supply had a crucial regulatory effect not only on the formation of lignin-degrading peroxidases but also on their decay and on the production of extracellular protease activity and polysaccharides. Continuous oxygenation or aeration increased the glucose consumption rate, extracellular protease activity and polysaccharides. Gassing with air, whether continuous or periodic, sustained Mn-peroxidase activity while ligninase was undetectable. Continuous O₂ supply speeded up ligninase decay, displaying a sharper maximum, while a broader maximum and slower decay of ligninase activity were observed when supplying periodic O₂. Cultures initially grown with free exposure to air displayed a higher but sharper ligninase activity maximum when shifted to continuous rather than periodic O₂ supply. In general, the higher levels of either polysaccharides or protease activity corresponded to the lower levels and faster decay of ligninase and Mn-peroxidase activities. Offprint requests to: H. E. Grethlein     

Decolourization of synthetic wastewater containing azo dyes by immobilized Phanerochaete chrysosporium in a continuously operated RBC reactor

A laboratory-scale rotating biological contactor (RBC) reactor with immobilized fungal biomass of Phanerochaete chrysosporium was investigated for its performance in decolourizing synthetic wastewater containing single or mixture of azo dyes, Direct Red-80 (DR-80) and Mordant Blue-9 (MB-9). Decolourization efficiency in the continuously operated bioreactor was studied by varying dye inlet concentration and disc rotation speed at two different wastewater hydraulic retention times (HRTs), i.e. 24and 48 h. Results from the single dye-containing experiments showed that the system could completely decolourize the wastewater for a maximum inlet dye concentration within the range 25–200 mg L⁻¹ and 48 h HRT in the reactor; for an inlet dye concentration above 200 mg L⁻¹, the decolourization efficiency slightly reduced up to 85% for the same HRT. However, wastewater containing DR-80 was found to be decolourized more efficiently compared to that containing MB-9. Further, the effect of increase in the disc rotation speed from 2 to 6 rpm in the study revealed no large differences in the decolourization efficiencies of the wastewaters. Similar results were obtained with wastewater containing the dyes together at various concentration combinations as per the two-level factorial design of experiments. Enzyme activities of lignin peroxidase and manganese peroxidase by the fungus were also analysed in the study, and the results indicated that while DR-80 showed a large negative effect on both the enzymes, MB-9 affected mainly the MnP activity by the fungus.     

Production of Phanerochaete chrysosporium lignin peroxidase under various culture conditions

Lignin peroxidase production by the white-rot fungus Phanerochaete chrysosporium is markedly influenced by the buffer system employed. In immobilized P. chrysosporium cultures with carbon-limited glucose medium, the use of acetate buffer resulted in higher lignin peroxidase activities than tartrate. With acetate as the buffer in shake-flask cultures a 20% to over 100% improvement in lignin peroxidase production was obtained as compared to tartrate-buffered systems. Of trace elements, Cu²⁺, Mn²⁺ and Zn²⁺ seemed to have the greatest influence on lignin peroxidase production. Furthermore, an increase in the Cu²⁺ and Zn²⁺ concentrations resulted in considerably higher ligninase activities. Although it has been shown previously that high manganese levels repress ligninase production, for maximum ligninase production the presence of some Mn²⁺ appeared to be necessary. The concentration of phosphorus had surprisingly little effect on ligninase production. Highest lignin peroxidase activities were obtained with lower phosphorus concentrations, but reasonably high activities were obtained within the whole studied phosphorus range of 0.12–4.60 g l^–1. Diammonium tartrate alone was a better nitrogen source than a mixture of diammonium tartrate, proteose peptone and yeast extract. The addition of solid manganese (IV) oxide to 3-day-old immobilized biocatalyst cultures increased the maximum ligninase activity obtained by about one-third. Correspondence to: S. Linko     

Kinetics of endosulfan degradation by Phanerochaete chrysosporium

The chlorinated pesticide, endosulfan, could be degraded by Phanerochaete chrysosporium under non-ligninolytic conditions, and this did not require direct contact with mycelium. The major metabolites formed were endosulfan sulfate and endosulfan diol. The rate of degradation depended on the initial concentration. With 2.5 mg endosulfan l^–1, degradation was at 0.23 mg l^–1 day^–1. The degradation could be described using a nonlinear rate expression that was similar to the Michaelis–Menten equation.     

Biodegradation of polycyclic hydrocarbons by Phanerochaete chrysosporium

The ability of the white rot fungus Phanerochaete chrysosporium to degrade polycyclic aromatic hydrocarbons (PAHs) that are present in anthracene oil (a distillation product obtained from coal tar) was demonstrated. Analysis by capillary gas chromatography and high-performance liquid chromatography showed that at least 22 PAHs, including all of the most abundant PAH components present in anthracene oil, underwent 70 to 100% disappearance during 27 days of incubation with nutrient nitrogen-limited cultures of this fungus. Because phenanthrene is the most abundant PAH present in anthracene oil, this PAH was selected for further study. In experiments in which [14C]phenanthrene was incubated with cultures of P. chrysosporium containing anthracene oil for 27 days, it was shown that 7.7% of the recovered radiolabeled carbon originally present in [14C]phenanthrene was metabolized to 14CO2 and 25.2% was recovered from the aqueous fraction, while 56.1 and 11.0% were recovered from the methylene chloride and particulate fractions, respectively. High-performance liquid chromatography of the 14C-labeled material present in the methylene chloride fraction revealed that most (91.9%) of this material was composed of polar metabolites of [14C]phenanthrene. These results suggest that this microorganism may be useful for the decontamination of sites in the environment contaminated with PAHs.     

Biodegradation of Rose Bengal by Phanerochaete chrysosporium

Rose Bengal (tetrachloro-tetraiodo-fluorescein) was not able to limit the spreading growth of the ligninolytic fungus, Phanerochaete chrysosporium in the presence of Tween 80, and when added to the 5 d old liquid cultures of this organism it was almost completely degraded in 5 h. Thin layer chromatography analysis showed the formation of a single degradation product.     

Novel bioconversion of wheat straw to bio-organic fertilizer in a solid-state bioreactor

In order to increase the eco-efficiency and overall availability of naturally renewable resource, the novel bioconversion of steam-exploded wheat straw to bio-organic fertilizer containing N₂-fixer, P and K solubilizers was investigated. The conversion was performed in solid-state fermentation (SSF) with periodic air-forced pressure oscillation (PAPO). The results showed that SSF-PAPO was competitive with the conventional solid-state fermentation (cSSF) in biomass accumulation and wheat straw digestion. With solid–liquid ratio 1:3, microbial biomass production at 72 h was high up to 2 × 10¹¹ cfu g⁻¹, nearly twice as that in cSSF. The degradation rate of cellulose, hemicellulose and lignin after fermentation in SSF-PAPO reached 48.57 ± 10.66, 84.77 ± 2.75 and 2.15 ± 10.11, respectively, which was greater than that of 29.30 ± 10.28%, 33.47 ± 4.85% and 0.53 ± 9.07% in cSSF, correspondingly. The SSF-PAPO system displayed unique advantage, by a novel gas phase control strategy on gas concentration and heat gradient, on the bioconversion of wheat straw to the bio-organic fertilizer.