A time course analysis of the extracellular proteome of Aspergillus nidulans growing on sorghum stover

Background

Fungi are important players in the turnover of plant biomass because they produce a broad range of degradative enzymes. Aspergillus nidulans, a well-studied saprophyte and close homologue to industrially important species such as A. niger and A. oryzae, was selected for this study.

Results

A. nidulans was grown on sorghum stover under solid-state culture conditions for 1, 2, 3, 5, 7 and 14?days. Based on analysis of chitin content, A. nidulans grew to be 4-5% of the total biomass in the culture after 2?days and then maintained a steady state of 4% of the total biomass for the next 12?days. A hyphal mat developed on the surface of the sorghum by day one and as seen by scanning electron microscopy the hyphae enmeshed the sorghum particles by day 5. After 14?days hyphae had penetrated the entire sorghum slurry. Analysis (1-D PAGE LC-MS/MS) of the secretome of A. nidulans, and analysis of the breakdown products from the sorghum stover showed a wide range of enzymes secreted. A total of 294 extracellular proteins were identified with hemicellulases, cellulases, polygalacturonases, chitinases, esterases and lipases predominating the secretome. Time course analysis revealed a total of 196, 166, 172 and 182 proteins on day 1, 3, 7 and 14 respectively. The fungus used 20% of the xylan and cellulose by day 7 and 30% by day 14. Cellobiose dehydrogenase, feruloyl esterases, and CAZy family 61 endoglucanases, all of which are thought to reduce the recalcitrance of biomass to hydrolysis, were found in high abundance.

Conclusions

Our results show that A. nidulans secretes a wide array of enzymes to degrade the major polysaccharides and lipids (but probably not lignin) by 1?day of growth on sorghum. The data suggests simultaneous breakdown of hemicellulose, cellulose and pectin. Despite secretion of most of the enzymes on day 1, changes in the relative abundances of enzymes over the time course indicates that the set of enzymes secreted is tailored to the specific substrates available. Our findings reveal that A. nidulans is capable of degrading the major polysaccharides in sorghum without any chemical pre-treatment.     

Enzymology of Phanerochaete chrysosporium with respect to the degradation of recalcitrant compounds and xenobiotics

The archetypal white-rot fungus Phanerochaete chrysosporium has been shown to degrade a variety of persistent environmental pollutants. Many of the enzymes responsible for pollutant degradation, which are normally involved in the degradation of wood, are extracellular. Thus, P. chrysosporium is able to degrade toxic or insoluble chemicals more efficiently than other microorganisms. P. chrysosporium has a range of oxidative and reductive mechanisms and uses highly reactive, nonspecific redox mediators which increase the number of chemicals that can be effectively degraded. This review gives an overview of the enzymes that are believed to be important for bioremediation and briefly discusses the degradation of some individual chemicals. Received: 25 April 2000 / Received revision: 05 June 2000 / Accepted: 04 July 2000     

Decolorization of Victoria blue by the white rot fungus, <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Synthetic textile dyes are among the most dangerous chemical pollutants released in industrial wastewater streams. Recognizing the importance of reducing the environmental impact of these dyes, the ability of the white rot fungus Phanerochaete chrysosporium to decolorize various textile dyes was investigated. This fungus decolorized 6 of the 14 structurally diverse dyes with varying efficiency (between 14% and 52%). There was no discernable pattern of decolorization even among dyes of the same chemical class, suggesting that attack on the dyes is relatively non-specific. Among the three dyes which showed >40% decolorization, Victoria Blue B (VB) was chosen for further analysis because the ability of the fungus to decolorize VB was nearly independent over a relatively broad concentration range. Blocking lignin peroxidase (LiP) and manganese peroxidase (MnP) production by the fungus did not substantially affect VB decolorization. Inhibition of laccase production by adding various inhibitors to shaken cultures reduced VB decolorization significantly suggesting a role for laccase in VB decolorization. When sodium azide and aminotriazole were used to inhibit endogenous catalase and cytochrome P-450 oxygenase activities, there was 100% and 70% reduction in VB decolorization, respectively. Adding benzoate to trap hydrogen peroxide-derived hydroxyl radicals resulted in 50% decolorization of VB. Boiling the extracellular fluid (ECF) for 30 min resulted in approximately 50% reduction in VB decolorization. Collectively, these data suggest that laccase, and/or oxygenase/oxidase and a heat-stable non-enzymatic factor, but not Lip and MnP, play a role in VB decolorization by P. chrysosporium.     

Postnatal development of morphological features and vocalization in the pomona leaf-nosed bat <Emphasis Type="Italic">Hipposideros pomona</Emphasis>

Based on mark-recapture data, we studied the postnatal development of morphological features and vocalization of the pomona leaf-nosed bat (Hipposideros pomona). Morphological changes indicated that body mass and length of forearm followed a linear pattern of growth until 13 days of age at mean growth rates of 0.14 g/day and 1.08 mm/day, respectively, and thereafter, growth rates slowed. The length of the total epiphyseal gap of the fourth metacarpal–phalangeal joint showed a linear increase for up to 10 days, followed by a linear decrease until day 40 at a mean rate of 0.09 mm/day. Together, two equations permitted estimation of the age of H. pomona pups between 1 and 40 days. The logistic equation provided the best fit to the empirical curves for body mass and length of forearm. Studies of vocal development showed that the precursors of echolocation calls were not emitted until day 7 after birth. As the pups grew, the dominant frequency (DF) of isolation calls increased and number of harmonics (NH) decreased, whereas the duration remained relatively stable. The DF and BFM₂ (the bandwidth of the terminal frequency-modulated sweep from the second harmonic) of the early echolocation calls increased; however, the NH and duration decreased.     

Induction of functional cytochrome P450 and its involvement in degradation of benzoic acid by <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

The white rot fungus Phanerochaete chrysosporium has the largest cytochrome P450 contingent known to date in fungi, but the study on the function of these P450s is limited. In this study, induction of functional P450 in P. chrysosporium was first shown and P450-mediate degradation of benzoic acid was demonstrated in this fungus. Carbon monoxide difference spectra indicated significant induction of P450 by benzoic acid, m-chlorobenzoic acid, p-chlorobenzoic acid and n-hexane, and showed the effect of inducer concentration and nutrient condition on the induction of P450. The high contents of P450 in the microsomal fractions facilitated the study on the function of P450. While the n-hexane-induced P450 could not interact with benzoic acid, the microsomal P450 induced by benzoic acid produced type I substrate binding spectra upon the addition of benzoic acid. The benzoic acid degradation by the microsomal P450 was NADPH-dependent at a specific rate of 194 ± 14 min⁻¹, and significantly inhibited by piperonyl butoxide (a P450 inhibitor). However, inhibition of benzoic acid degradation by piperonyl butoxide was slight or not detectable in the cultures of this fungus, suggesting presumable involvement of other enzyme in benzoic acid degradation. The extracellular ligninolytic enzymes, lignin peroxidase and manganese-dependent peroxidase, were not involved in initial metabolism of benzoic acid under the test conditions.     

Limonene bioconversion to high concentrations of a single and stable product, perillic acid, by a solvent-resistant Pseudomonas putida strain

The white-rot basidiomycete Phanerochaete chrysosporium BKM-F-1767 was tested for its capacity to degrade dehydroabietic acid (DHA). In anaerobic treatment, this molecule is the most recalcitrant member of the resin acid group, which is known to cause operational problems to anaerobic reactors treating pulp and paper industry wastewaters. In this study the effect of DHA on different parameters, such as growth, ligninolytic enzyme activity, extracellular protein production as well as both glycerol and ammonium consumption by the fungus, was determined. Although the above parameters were affected by the addition of DHA, the results show that the fungus could still produce significant titres of ligninolytic enzymes. The fungus removed 47% of the DHA initially present in the static culture, after 10 days of incubation. Anaerobic toxicity assays showed that the treatment of DHA with P. chrysosporium reduced the methanogenesis and acetogenesis inhibition caused by DHA and allowed improved methane production by the anaerobic bacteria. Received: 10 June 1997 / Received revision: 6 January 1998 / Accepted: 24 January 1998     

Ligninolytic enzymes of selected white rot fungi cultivated on wheat straw

Ligninolytic enzymes of the white rot fungiCoriolopsis polyzona, Phanerochaete chrysosporium, andTrametes versicolor growing on wheat straw under nearly natural conditions were investigated. Manganese peroxidase (MnP), secreted as early as on day 3, was dominant over other activities during the initial phase (the first 10 days). Its activity profile was similar in all the three fungi. Lignin peroxidase (LIP) was not detected in the extracellular enzyme extracts ofC. polyzona andP. chrysosporium cultures.T. versicolor secreted LIP after 10 d of growth. Another, recently described, enzyme activity of manganese-independent peroxidase (MIP) was detected in all the three fungi tested and it appeared on about day 5 (later than MnP and earlier than LIP); it was the dominant activity after day 10. Laccase activity appeared at basal levels without any significant changes. Pyranose 2-oxidase was probably the major extracellular H₂O₂-generating activity (with all the three fungi) that appeared contemporarily with MnP, increased with time, peaking on day 17–18. Glyoxal oxidase could not be detected with any of the fungi.     

The tropical white rot fungus, <Emphasis Type="Italic">Lentinus squarrosulus</Emphasis> Mont.: lignocellulolytic enzymes activities and sugar release from cornstalks under solid state fermentation

Lentinus squarrosulus Mont., a high temperature tolerant white rot fungus that is found across sub-Saharan Africa and many parts of Asia, is attracting attention due to its rapid mycelia growth and potential for use in food and biodegradation. A solid state fermentation (SSF) experiment with L. squarrosulus (strain MBFBL 201) on cornstalks was conducted. The study evaluated lignocellulolytic enzymes activity, loss of organic matter (LOM), exopolysaccharide content, and the release of water soluble sugars from degraded substrate. The results showed that L. squarrosulus was able to degrade cornstalks significantly, with 58.8% LOM after 30 days of SSF. Maximum lignocellulolytic enzyme activities were obtained on day 6 of cultivation: laccase = 154.5 U/L, MnP = 13 U/L, peroxidase = 27.4 U/L, CMCase = 6.0 U/mL and xylanase = 14.5 U/mL. L. squarrosulus is a good producer of exopolysaccharides (3.0–5.13 mg/mL). Glucose and galactose were the most abundant sugars detected in the substrate during SSF, while fructose, xylose and trehalose, although detected on day zero of the experiment, were absent in treated substrates. The preference for hemicellulose over cellulose, combined with the high temperature tolerance and the very fast growth rate characteristics of L. squarrosulus could make it an ideal candidate for application in industrial pretreatment and biodelignification of lignocellulosic biomass.     

Nutritional study on <Emphasis Type="Italic">Embellisia astragali</Emphasis>, a fungal pathogen of milk vetch (<Emphasis Type="Italic">Astragalus adsurgens</Emphasis>)

Embellisia astragali is a strong, virulent pathogen that develops within milk vetch (Astragalus adsurgens). In order to determine nutrient requirements, the fungus was cultured on 9 carbon sources, 9 nitrogen sources, and 13 growth media in the dark at 25°C. Growth rates and sporulation capacity were measured after 4 and 12 weeks. All carbon sources supported growth, but only soluble starch, inulin, and dextrose supported sporulation. In general, better growth was obtained on disaccharides and polysaccharides than on monosaccharides. Compared with no growth on NH₄ ⁺-N and urea, the fungus grew little on all NO₃ ⁻-N, amino-N, and other organic-N such as peptone. There was no sporulation or very sparse conidia on almost all nitrogen sources with supplied dextrose or soluble starch as sole carbon source. The better growth and sporulation on most of the semidefined media than on defined media indicates that some components in plant or animal material may be vital to the fungus. Sporulation was positively correlated with growth rate in N source experiment at 12 weeks and in growth media experiment at 4 and 12 weeks. The fungus favors grow within agar with growth rate less than 1.18 mm day⁻¹.     

Bio-remediation of colored industrial wastewaters by the white-rot fungi Phanerochaete chrysosporium and Pleurotus ostreatus and their enzymes

The effect of Phanerochaete chrysosporium and Pleurotus ostreatus whole cells and their ligninolytic enzymes on models of colored industrial wastewaters was evaluated. Models of acid, direct and reactive dye wastewaters from textile industry have been defined on the basis of discharged amounts, economic relevance and representativeness of chemical structures of the contained dyes. Phanerochaete chrysosporium provided an effective decolourization of direct dye wastewater model, reaching about 45% decolourization in only 1 day of treatment, and about 90% decolourization within 7 days, whilst P. ostreatus was able to decolorize and detoxify acid dye wastewater model providing 40% decolourization in only 1 day, and 60% in 7 days. P. ostreatus growth conditions that induce laccase production (up to 130,000 U/l) were identified, and extra-cellular enzyme mixtures, with known laccase isoenzyme composition, were produced and used in wastewater models decolourization. The mixtures decolorized and detoxified the acid dye wastewater model, suggesting laccases as the main agents of wastewater decolourization by P. ostreatus. A laccase mixture was immobilized by entrapment in Cu-alginate beads, and the immobilized enzymes were shown to be effective in batch decolourization, even after 15 stepwise additions of dye for a total exposure of about 1 month.     

Improved simultaneous production of mycelial biomass and polysaccharides by submerged culture of <Emphasis Type="Italic">Hericium erinaceum</Emphasis>: optimization using a central composite rotatable design (CCRD)

The aim of this study was to optimize the culture medium used for the mycelial growth and production of intracellular polysaccharides (IPS) and exopolysaccharides (EPS) in a submerged culture of Hericium erinaceum. Of the various factors examined, including carbon and nitrogen sources, vitamins, mineral elements, and initial pH, those that proved to have a significant effect were then tested using a 2⁴ central composite rotatable design (CCRD). Under the optimal culture conditions, the maximal yield of biomass reached 14.24 ± 0.45 g l⁻¹ and was 1.85-fold higher than in the basal medium. The kinetics of EPS biosynthesis in a bioreactor showed that although the highest yield of EPS (2.75 ± 0.27 g l⁻¹) could be obtained on day 8, the process of biosynthesizing high molecular weight polysaccharides proceeded until the depletion of the carbon source in the medium (after 14 days of cultivation). Our results could be very helpful in the large-scale production of bioactive polysaccharides from H. erinaceum.     

Biodegradation of environmental pollutants by the white rot fungus Phanerochaete chrysosporium: Involvement of the lignin degrading system

The white-rot fungus Phanrochaete chrysosporium has the ability to degrade a wide variety of structurally diverse organic compounds, including a number of environmentally persistent organopollutants. The unique biodegradative abilities of this fungus appears to be dependent upon its lignin-degrading system. The non-specific and partially extracellular nature of this system suggests that it may be useful as a supplementary means to treat organochemical wastes.     

Novel evidence of cytochrome P450-catalyzed oxidation of phenanthrene in <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> under ligninolytic conditions

The presence of cytochrome P450 and P450-mediated phenanthrene oxidation in the white rot fungus Phanerochaete chrysosporium under ligninolytic condition was first demonstrated in this study. The carbon monoxide difference spectra indicated induction of P450 (130 pmol mg⁻¹ in the microsomal fraction) by phenanthrene. The microsomal P450 degraded phenanthrene with a NADPH-dependent activity of 0.44 ± 0.02 min⁻¹. One of major detectable metabolites of phenanthrene in the ligninolytic cultures and microsomal fractions was identified as phenanthrene trans-9,10-dihydrodiol. Piperonyl butoxide, a P450 inhibitor which had no effect on manganese peroxidase activity, significantly inhibited phenanthrene degradation and the trans-9,10-dihydrodiol formation in both intact cultures and microsomal fractions. Furthermore, phenanthrene was also efficiently degraded by the extracellular fraction with high manganese peroxidase activity. These results indicate important roles of both manganese peroxidase and cytochrome P450 in phenanthrene metabolism by ligninolytic P. chrysosporium.     

Biodegradability of lignin—Polypropylene composite films

Biological degradation of composite lignin-polypropylene films containing 4% organocell lignin was confirmed by treatment with lignin-degrading enzymes produced by the white-rot fungusPhanerochœte chrysosporium. The kinetics ofP. chrysosporium culture in the presence of lignin-containing and lignin-free polypropylene films show that the fungus produced lignin-degrading enzymes into the liquid medium during incubation with the lignin-polypropylene film. The degree of biodegradation of both types of film was followed by monitoring their mechanical properties. Correlation was found between the decrease of elongation at break and the amount of released lignin fragments into the extracellular fluid in the course of microbial treatment. The incorporation of lignin into polyolefins represents a new way of using wastes from pulp and paper industry to reduce the environmental impact factor of waste plastics.     

Arsenal of plant cell wall degrading enzymes reflects host preference among plant pathogenic fungi

Background  

The discovery and development of novel plant cell wall degrading enzymes is a key step towards more efficient depolymerization of polysaccharides to fermentable sugars for the production of liquid transportation biofuels and other bioproducts. The industrial fungus Trichoderma reesei is known to be highly cellulolytic and is a major industrial microbial source for commercial cellulases, xylanases and other cell wall degrading enzymes. However, enzyme-prospecting research continues to identify opportunities to enhance the activity of T. reesei enzyme preparations by supplementing with enzymatic diversity from other microbes. The goal of this study was to evaluate the enzymatic potential of a broad range of plant pathogenic and non-pathogenic fungi for their ability to degrade plant biomass and isolated polysaccharides.     

Establishment of the white rot fungus Phanerochaete chrysosporium on unsterile straw in solid substrate fermentation systems intended for degradation of pesticides

The effects of different inoculum-loading rates and pre-treatment of wheat straw with formic acid and hot water (50 °C) on the establishment of Phanerochaete chrysosporium on unsterile straw were studied in laboratory scale and in a 1.5-m³ bioreactor. The establishment of P. chrysosporium on unsterile straw was satisfactory. Phanerochaete chrysosporium and other fungi, which developed simultaneously, were able to produce the activity necessary to degrade two herbicides, bentazon and MCPA (4-chloro-2-methylphenoxyacetic acid) in 20 days (65 and 75%, respectively). The decrease of both herbicides coincided with the presence of the activity of the lignin-degrading enzymes lignin peroxidase and manganese peroxidase/laccase. Extensive growth of P. chrysosporium or other lignin-degrading fungi on unsterile straw would be excellent for inexpensive solid substrate systems intended for degradation of pesticides.     

Characterization of proteome alterations in <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> in response to lead exposure

Background  

Total soluble proteome alterations of white rot fungus Phanerochaete chrysosporium in response to different doses (25, 50 and 100 μM) of Pb (II) were characterized by 2DE in combination with MALDI-TOF-MS.     

Cell-mediated immune responses in mice infected with fonsecaea pedrosoi

Time course of cellular and humoral immune responses in mice infected with Fonsecaea pedrosoi was investigated by using an antigen prepared from culture filtrate of this fungus. Mice were infected by intravenous injection with yeast-like cells of the fungus. Viable fungus was recovered from the brain of the infected mice until the 36th day after inoculation, and from the other organs examined until 14th to 16th day. Inflammatory lesions were observed in the brain, lung, heart, liver, spleen, kidney and intestine during the first 30 days after inoculation. Macrophage migration inhibition factor response in these mice was insignificant until 8 days after inoculation. A significant response was developed at day 10 and persisted until day 63. This response returned negative by 95 days after inoculation. Lymphocyte transformation response of these mice was negative until 4 days after inoculation. At day 6 blastogenic index increased to 1.5, and at day 10, 14 and 16 the indices were 1.8, 2.4 and 1.7 respectively. Precipitin response to this fungus could not be detected in these mice until 16 days after inoculation. Positive results were obtained at day 21 and lasted until 51 days after inoculation. The precipitin titers, however, did not exceed one fold in any of these mice.     

The ligninolytic activities of Lentinus edodes and Phanerochaete chrysosporium

Summary Two important lignin-degrading fungi with existing or potential applications in the production of food, feed and/or fiber products from wood are Lentinus edodes (Berk.; Sing.=Lentinula edodes [Pegler]) and Phanerochaete chrysosporium (Burds). This study discusses their relative ability to degrade lignin and the factors controlling their ligninolytic activity (synthetic ¹⁴C-lignin¹⁴CO₂). Ligninolytic activity in P. chrysosporium is known to develop after the fungus ceases vegetative growth, and to require both O₂ and an exogenous carbon source such as glucose. It has an extracellular ligninase in high titer which is assayed by the oxidation of veratryl alcohol to veratraldehyde. Here, P. chrysosporium was found to have a high capacity for lignin degradation (it was not easily saturated with lignin). Certain inorganic elements, including Fe²⁺, Ca²⁺ and Mo⁶⁺, were found to stimulate its ligninolytic activity. Calcium addition was required, with 40 ppm Ca²⁺ giving the highest activity. As in P. chrysosporium, ligninolytic activity in L. edodes was found to require both O₂ and an exogenous carbon source. However, in contrast to P. chrysosporium, L. edodes was only moderately ligninolytic, had a lower capacity for lignin degradation (was more easily saturated with lignin), and showed maximal activity only during the vegetative growth period. Also in contrast to P. chrysosporium, ligninolytic activity in L. edodes was not stimulated by Ca²⁺. Instead, manganese was required, with 10 ppm Mn²⁺ giving optimal activity. An extracellular ligninase capable of oxidizing veratryl alcohol to veratraldehyde was not detected in L. edodes.     

Enzymatic degradation of granular potato starch by <Emphasis Type="Italic">Microbacterium aurum</Emphasis> strain B8.A

Microbacterium aurum strain B8.A was isolated from the sludge of a potato starch-processing factory on the basis of its ability to use granular starch as carbon- and energy source. Extracellular enzymes hydrolyzing granular starch were detected in the growth medium of M. aurum B8.A, while the type strain M. aurum DSMZ 8600 produced very little amylase activity, and hence was unable to degrade granular starch. The strain B8.A extracellular enzyme fraction degraded wheat, tapioca and potato starch at 37 °C, well below the gelatinization temperature of these starches. Starch granules of potato were hydrolyzed more slowly than of wheat and tapioca, probably due to structural differences and/or surface area effects. Partial hydrolysis of starch granules by extracellular enzymes of strain B8.A resulted in large holes of irregular sizes in case of wheat and tapioca and many smaller pores of relatively homogeneous size in case of potato. The strain B8.A extracellular amylolytic system produced mainly maltotriose and maltose from both granular and soluble starch substrates; also, larger maltooligosaccharides were formed after growth of strain B8.A in rich medium. Zymogram analysis confirmed that a different set of amylolytic enzymes was present depending on the growth conditions of M. aurum B8.A. Some of these enzymes could be partly purified by binding to starch granules.