Effect of carbon and nitrogen limitation on lignin peroxidase and manganese peroxidaseproduction by Phanerochaete flavido-alba

The ligninolytic enzymes lignin peroxidase (LiP) and manganese dependent peroxidase(MnP), were detected in extracellular fluids of Phanerochaete flavido-alba FPL 106507cultures under carbon or nitrogen limitation. MnP activities were found to be higher than LiPactivities under all growth conditions tested. Higher titres of both peroxidases were obtainedunder carbon limitation in excess nitrogen. Isoelectric points (pIs) observed after FPLC and IEFof concentrated extracellular fluids revealed more acidic pIs for LiP enzymes obtained innitrogen-limited cultures than those in carbon-limited cultures. However, the change in thelimiting growth factor does not significantly affect MnP pIs.     

Production of the ligninolytic enzymes by immobilized <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> in an air atmosphere

Summary The production of the ligninolytic enzymes by Phanerochaete chrysosporium immobilized on polyurethane foam cubes in air was investigated by adopting different sizes and amounts of the carriers, different medium C/N ratios and different glucose-feeding strategies. No lignin peroxidase (LiP) activity was observed under nitrogen limitation (C/N ratio, expressed as glucose/NH₄⁺, 56/2.2 mM) with two sizes and three amounts of the carriers, while comparable levels of manganese peroxidase (MnP) activities were detected only in non-immersed cultures with two sizes of the carriers. A non-immersed state also stimulated LiP formation under carbon limitation (C/N ratio 28/44 mM). High peak activities of LiP, 197 and 164 U/l, were obtained in non-immersed cultures under carbon limitation at the C/N ratios of 28/44 and 56/44 mM, respectively, the occurrence of the activities coinciding with the complete consumption of glucose. A very low level of MnP was measured at the C/N ratio of 28/44 mM compared with the similar activities at 56/2.2 and 56/44 mM. An addition of 2 g glucose/l after its complete depletion improved both the production of LiP and MnP markedly in non-immersed culture at the initial C/N ratio of 28/44 mM, whereas a replenishment of 5 g/l, still enhancing the formation of MnP, inhibited the production of LiP first before the later reactivation. It is suggested that non-immersed liquid culture under carbon limitation reinforced by a suitable glucose feeding strategy is one potential way to realize high production of the ligninolytic enzymes by P. chrysosporium in air.     

Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium

The decline of lignin peroxidase (LiP) activity observed after day 6 in cultures of Phanerochaete chrysosporium was found to be correlated with the appearance of idiophasic extracellular protease activity. Daily addition of glucose started on day 6 resulted in low protease levels and in turn in stable LiP levels. Addition of cycloheximide to day 6 cultures resulted in virtually no change of LiP activity and extracellular protein and negligible levels of protease activity, indicating that this protease is synthesized de novo. LiP activity was found to be stable upon removal of the fungal pellets on day 6 and incubation of the extracellular fluid alone. An almost complete disappearance of LiP activity and LiP proteins and high levels of protease activity were observed upon incubation of 6-day extracellular fluid in the presence of fungal pellets. Moreover, incubation of crude or purified LiP isoenzymes with protease-rich extracellular fluid of day 11 or 11-day cell extracts resulted in a marked loss of activity. In contrast, incubation of crude LiP with boiled and clarified extracellular fluid of day 11 cultures resulted in virtually no loss of activity. These results indicate that protease-mediated degradation of LiP proteins is a major cause for the decay of LiP activity during late secondary metabolism in cultures of P. chrysosporium.     

The kinetics and physiology of stipitatic acid and gluconate production by carbon sufficient cultures of Penicillium stipitatum growing in continuous culture

The yield from glucose of ammonia-grown carbon-limited continuous cultures of Penicillium stipitatum was ca. 20% higher than that of nitrate-grown cultures at all growth rates examined. However, the yield from oxygen was similar during growth on both nitrogen sources. Under phosphate limitation the specific rate of gluconic acid and stipitatic acid production increased with growth rate, but the former product accounted for virtually 100% of the excreted carbon. Stipitatic acid was not produced under nitrogen limitation, and glucose supplied to the culture in excess of that required for growth was virtually quantatively converted into gluconic acid. Productivities of 11.4 g gluconic acid/L/h were stably maintained in continuous culture. Under conditions of glucose excess the enzyme glucose oxidase was excreted into the culture. The specific activity of this extracellular enzyme increased when the input glucose concentration to the culture was progressively increased. The excretion of a protein under nitrogen limitation suggests that this enzyme plays an important role under these conditions. Indeed, it was demonstrated that nitrogen-limited cultures did not overmetabolize gluconate at either pH 6.5 or 3.5, although up to 29 g/L gluconate was present in the culture. The Y(gluconate) and YO(2) of C- and N-limited gluconate-grown cultures were similar indicating that the rapid conversion of glucose to gluconate probably affords a means of regulating carbon flow in this organism. Nitrogen-limited cultures of P. stipitatum overmetabolized glucose to a much greater extent than acetate, fructose, or gluconate.     

Protease-mediated degradation of lignin peroxidase in liquid cultures of Phanerochaete chrysosporium.

The decline of lignin peroxidase (LiP) activity observed after day 6 in cultures of Phanerochaete chrysosporium was found to be correlated with the appearance of idiophasic extracellular protease activity. Daily addition of glucose started on day 6 resulted in low protease levels and in turn in stable LiP levels. Addition of cycloheximide to day 6 cultures resulted in virtually no change of LiP activity and extracellular protein and negligible levels of protease activity, indicating that this protease is synthesized de novo. LiP activity was found to be stable upon removal of the fungal pellets on day 6 and incubation of the extracellular fluid alone. An almost complete disappearance of LiP activity and LiP proteins and high levels of protease activity were observed upon incubation of 6-day extracellular fluid in the presence of fungal pellets. Moreover, incubation of crude or purified LiP isoenzymes with protease-rich extracellular fluid of day 11 or 11-day cell extracts resulted in a marked loss of activity. In contrast, incubation of crude LiP with boiled and clarified extracellular fluid of day 11 cultures resulted in virtually no loss of activity. These results indicate that protease-mediated degradation of LiP proteins is a major cause for the decay of LiP activity during late secondary metabolism in cultures of P. chrysosporium.     

Lignin Peroxidases, Manganese Peroxidases, and Other Ligninolytic Enzymes Produced by Phlebia radiata during Solid-State Fermentation of Wheat Straw

The white rot fungus Phlebia radiata 79 (ATCC 64658) produces lignin peroxidase (LiP), manganese peroxidase (MnP), glyoxal oxidase (GLOX), and laccase in the commonly used glucose low-nitrogen liquid medium. However, the enzymes which this fungus utilizes for selective removal of lignin during degradation of different lignocellulosic substrates have not been studied before. Multiple forms of LiP, MnP, GLOX, and laccase were purified from P. radiata culture extracts obtained after solid-state fermentation of wheat straw. However, the patterns of extracellular lignin-modifying enzymes studied were different from those of the enzymes usually found in liquid cultures of P. radiata. Three LiP isoforms were purified. The major LiP isoform from solid-state cultivation was LiP2. LiP3, which has usually been described as the major isoenzyme in liquid cultures, was not expressed during straw fermentation. New MnP isoforms have been detected in addition to the previously reported MnPs. GLOX was secreted in rather high amounts simultaneously with LiP during the first 2 weeks of growth. GLOX purified from P. radiata showed multiple forms, with pIs ranging from 4.0 to 4.6 and with a molecular mass of ca. 68 kDa.     

Tween 80 enhanced TNT mineralization by Phanerochaete chrysosporium

The effect of a nonionic surfactant (Tween 80) on 2,4,6-trinitrotoluene (TNT) mineralization by the white-rot fungus Phanerochaete chrysosporium strain BKM-F-1767, was investigated in a liquid culture at 20, 50, and 100 mg TNT.L-1. The presence of 1% (w/v) Tween 80, at 20 mg.L-1 TNT, added to a 4-d-old culture, allowed the highest TNT mineralization level, that is 29.3% after 24 d, which is two times more than the control culture, without Tween 80 (13.9%). The mineralization of TNT resumed upon additional Tween 80 supplementation, consequently, 39.0% of the TNT was respired on day 68. Orbital agitation of the fungal culture was found detrimental to TNT mineralization, with or without Tween 80 in the culture medium. The surfactant also stimulated the growth of P. chrysosporium without any notable effect on either the glycerol consumption rate or the extracellular LiP and MnP activity levels. Respirometric assays highlighted some differences between the oxygen uptake rate of the fungal culture supplemented with or without Tween 80.     

The pH mediated effects of initial glucose concentration on the transitory occurrence of extracellular metabolites, gas exchange and growth yields of aerobic batch cultures of Klebsiella pneumoniae

The changes in growth kinetics in aerobic batch cultures of Klebsiella pneumoniae were followed by measurements of extracellular metabolites, rates of gas exchange, dissolved oxygen tension, pH, and carbon balance at all stages of growth. When the initial growth-limiting glucose concentration in media without pH control was increased from 1.0 g carbon L(-1) to 2.2 g carbon L(-1), the number of different, mainly acidic, extracellular metabolites of glucose at the end of exponential growth increased, while the proportion of acetate decreased. During the postexponential growth phase, the extracellular metabolites were oxidized, resulting in an increasing complexity of changes in pH, gas exchange, and dissolved oxygen tension with increasing initial substrate concentration. All these parameters showed concomitant stepwise changes. This pattern was independent of the dissolved oxygen tension in the range 30-200 muM. When pH was kept constant, the number, slope, and relative magnitude of the steps in gas exchange and dissolved oxygen tension were pH-dependent, being most complex at low pH. Detailed carbon balances showed that 20% of the initial glucose was converted into extracellular metabolites at the end of exponential growth at neutral pH. In the postexponential phase, pyruvate (2%) was reoxidized first followed by acetate (13%). The observed molar growth yield coefficient (Y(ATP)) was 8.4 if the transitory occurrence of pyruvate and acetate was accounted for, and 6.4 if it was neglected. The corrected observed molar growth yield coefficient (Y'(ATP)) was 9.4 and compared well with the true molar growth yield coefficient (Y(Max) (ATP)), which was found to be 11.0. Specific in situ respiration rates of the exponential growth phase of cultures grown at different controlled pH values compared well with in situ values for energy-limited chemostat grown cells at the same growth rates, suggesting that growth in the batch culture was energy-limited throughout the exponential growth phase. This view was supported by low levels of intracellular glycogen and exopolysaccharides of all cultures, by the value of Y'(ATP) of 9.4, and by a constant specific production rate of the extracellular metabolites throughout exponential growth. It was concluded that even under strictly aerobic conditions, control of pH is as important as control of dissolved oxygen tension during growth of enterobacteriaceae in batch cultures.     

Endoglucanase activity and relative expression of glycoside hydrolase genes of Fibrobacter succinogenes S85 grown on different substrates

The endoglucanase activity of cells and extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, soluble polysaccharides (beta-glucan, lichenan) and intact plant polysaccharides, was compared. The specific activity of cells grown on cellulose or forages was 6- to 20-fold higher than that of cells grown on soluble substrates, suggesting an induction of endoglucanases by the insoluble substrates. The ratios of cells to extracellular culture fluid endoglucanase activities measured in cultures grown on sugars or insoluble polysaccharides suggested that the endoglucanases induced by the insoluble polysaccharides remained attached to the cells. The mRNA of all the F. succinogenes glycoside hydrolase genes sequenced so far were then quantified in cells grown on glucose, cellobiose or cellulose. The results show that all these genes were transcribed in growing cells, and that they are all overexpressed in cultures grown on cellulose. Endoglucanase-encoding endB and endA(FS) genes, and xylanase-encoding xynC gene appeared the most expressed genes in growing cells. EGB and ENDA are thus likely to play a major role in cellulose degradation in F. succinogenes.     

Roles of Lignin Peroxidase and Manganese Peroxidase from Phanerochaete chrysosporium in the Decolorization of Olive Mill Wastewaters

The relative contributions of lignin peroxidase (LiP) and manganese peroxidase (MnP) to the decolorization of olive mill wastewaters (OMW) by Phanerochaete chrysosporium were investigated. A relatively low level (25%) of OMW decolorization was found with P. chrysosporium which was grown in a medium with a high Mn(II) concentration and in which a high level of MnP (0.65 (mu)M) was produced. In contrast, a high degree of OMW decolorization (more than 70%) was observed with P. chrysosporium which was grown in a medium with a low Mn(II) concentration but which resulted in a high level of LiP activity (0.3 (mu)M). In this culture medium, increasing the Mn(II) concentration resulted in decreased levels of OMW decolorization and LiP activity. Decolorization by reconstituted cultures of P. chrysosporium was found to be more enhanced by the addition of isolated LiP than by the addition of isolated MnP. The highest OMW decolorization levels were obtained at low initial chemical oxygen demands combined with high levels of extracellular LiP. These data, plus the positive effect of veratryl alcohol on OMW decolorization and LiP activity, indicate that culture conditions which yield high levels of LiP activity lead to high levels of OMW decolorization.     

Influence of glucose and dissolved oxygen concentrations on yields of Escherichia coli B in dialysis culture.

Yields of Escherichia coli B grown on glucose were determined in dialysis and non-dialysis culture. The molar growth yields were compared under conditions of excess glucose and oxygen as well as glucose- and oxygen-limiting conditions. The molar growth yields on glucose (YG) were determined for different periods during growth in non-dialysis cultures. A rapid decrease of YG was observed and growth ceased even in the presence of high concentrations of glucose and dissolved oxygen in the culture liquid. The decrease in YG was delayed in dialysis cultures where a high YG could be maintained at very high cell concentrations. The inhibition of growth depended on the accumulation of end-products of fermentative degradation of glucose. These products interfered with the oxidative phosphorylation. A large proportion of the glucose was fermented even in the presence of high concentrations of dissolved oxygen in the culture liquid. A decrease in the growth yield per g glucose was also observed.     

Ligninolytic System Formation by Phanerochaete chrysosporium in Air

This study characterizes the effect of oxygen concentration on the synthesis of ligninolytic enzymes by Phanerochaete chrysosporium immobilized on polyurethane foam cubes in a nonimmersed liquid culture system and maintained under different carbon-to-nitrogen (C/N) ratios and levels. Lignin peroxidase (LIP) activity was obtained in cultures exposed to air when the C/N ratio was low (7.47), i.e., when nitrogen levels were high (C/N = 56/45 mM) or carbon levels were low (C/N = 5.6/4.5 mM). At the low C/N ratio, the fungus was carbon starved and did not produce extracellular polysaccharides. At a high C/N ratio (153), i.e., under conditions of excess carbon (nitrogen limitation) (C/N = 56/2.2 mM), cultures exposed to air produced large amounts of polysaccharide, and LIP activity was detected only in cultures exposed to pure oxygen. Under high-nitrogen conditions, LIP production was 1,800 U/liter in cultures exposed to pure oxygen and 1,300 U/liter in cultures exposed to air, with H1 and H2 being the main isoenzymes. The oxygen level did not significantly alter the isoenzyme profile, nor did low-carbon conditions. The formation of manganese peroxidase was generally less affected by the oxygen level than that of LIP but was considerably reduced by a low C/N ratio. The effects of oxygen level and C/N ratio on the synthesis of glyoxal oxidase paralleled their effects on LIP synthesis except in the case of high nitrogen, which totally suppressed glyoxal oxidase activity.     

Strain of Pseudomonas aeruginosa--producer of bioPAV

A Pseudomonas aeruginosa strain producing an extracellular surfactant (biosurfactant) was isolated. The growth of this strain, referred to as 50.3, on a mineral glycerol-containing medium produces an emulsifying activity (60%) and decreases the surface tension of the culture liquid by a factor of 2.8 (to 25 mN/m). The optimum conditions for its growth and production of biosurfactants: intense aeration, pH 7.0-8.0, and the presence of Mg2+. The optimum biosurfactant properties were achieved when glucose was used as the only source of carbon and energy and NH4Cl was used as a source of nitrogen. The biosurfactant was isolated from the culture liquid by extraction and precipitation.     

The relationship between lignin peroxidase and manganese peroxidase production capacities and cultivation periods of mushrooms

Mushrooms are able to secrete lignin peroxidase (LiP) and manganese peroxidase (MnP), and able to use the cellulose as sources of carbon. This article focuses on the relation between peroxidase-secreting capacity and cultivation period of mushrooms with non-laccase activity. Methylene blue and methyl catechol qualitative assay and spectrophotometry quantitative assay show LiP secreting unvaryingly accompanies the MnP secreting in mushroom strains. The growth rates of hyphae are detected by detecting the dry hyphal mass. We link the peroxidase activities to growth rate of mushrooms and then probe into the relationship between them. The results show that there are close relationships between LiP- and/or MnP-secretory capacities and the cultivation periods of mushrooms. The strains with high LiP and MnP activities have short cultivation periods. However, those strains have long cultivation periods because of the low levels of secreted LiP and/or MnP, even no detectable LiP and/or MnP activity. This study provides the first evidence on the imitate relation between the level of secreted LiP and MnP activities and cultivation periods of mushrooms with non-laccase activity. Our study has significantly increased the understanding of the role of LiP and MnP in the growth and development of mushrooms with non-laccase activity.     

Manganese peroxidases of the white rot fungus Phanerochaete sordida.

The ligninolytic enzymes produced by the white rot fungus Phanerochaete sordida in liquid culture were studied. Only manganese peroxidase (MnP) activity could be detected in the supernatant liquid of the cultures. Lignin peroxidase (LiP) and laccase activities were not detected under a variety of different culture conditions. The highest MnP activity levels were obtained in nitrogen-limited cultures grown under an oxygen atmosphere. The enzyme was induced by Mn(II). The initial pH of the culture medium did not significantly affect the MnP production. Three MnP isozymes were identified (MnPI, MnPII, and MnPIII) and purified to homogeneity by anion-exchange chromatography followed by hydrophobic chromatography. The isozymes are glycoproteins with approximately the same molecular mass (around 45 kDa) but have different pIs. The pIs are 5.3, 4.2, and 3.3 for MnPI, MnPII, and MnPIII, respectively. The three isozymes are active in the same range of pHs (pHs 3.0 to 6.0) and have optimal pHs between 4.5 and 5.0. Their amino-terminal sequences, although highly similar, were distinct, suggesting that each is the product of a separate gene.     

Oxygen- and glucose-dependent expression of Trhxt1, a putative glucose transporter gene of Trichoderma reesei

The filamentous fungus Trichoderma reesei is adapted to nutrient-poor environments, in which it uses extracellular cellulases to obtain glucose from the available cellulose biomass. We have isolated and characterized Trhxt1, a putative glucose transporter gene, as judged by the glucose accumulation phenotype of a DeltaTrhxt1 mutant. This gene is repressed at high glucose concentrations and expressed at micromolar levels and in the absence of glucose. The gene is also induced during the growth of T. reesei on cellulose when the glucose concentration generated from the hydrolysis of cellulose present in the culture medium is in the micromolar range. We also show that oxygen availability controls the expression of the Trxht1 gene. In this regard, the gene is down-regulated by hypoxia and also by the inhibition of the flow of electrons through the respiratory chain using antimycin A. Intriguingly, anoxia but not hypoxia strongly induces the expression of the gene in the presence of an otherwise repressive concentration of glucose. These results indicate that although the absence of repressing concentrations of glucose and an active respiratory chain are required for Trhxt1 expression under normoxic conditions these physiological processes have no effect on the expression of this gene under an anoxic state. Thus, our results highlight the presence of a novel coordinated interaction between oxygen and the regulatory circuit for glucose repression under anoxic conditions.     

Anaerobic degradation of cellulose by mixed culture

A mixed culture in which cellulose is capable of being converted to methane and carbon dioxide was obtained from an inoculum procured from a sewage-treatment plant and maintained in a synthetic medium containing tissue paper and an inorganic salt and vitamin mixture. The culture was tested for its ability to degrade 12 different paper and cotton products under batch conditions in 3-l anaerobic fermenters. This culture degraded 6-8 mmol/l per week of cellulose, expressed as glucose equivalents, with total gas yields of 0.3 m3/kg of cellulose degraded. The gas produced contained between 56 and 59% of methane. Maximum cellulose degradation occurred at chemical oxygen demand:nitrogen:phosphorus level of 80:5:1 and was adversely affected by high stirring rate. Also the presence of higher proportions of lignin in cellulose products adversely affected the ability of this culture to degrade cellulose.     

Production of cellulose from glucose by Acetobacter xylinum

Acetobacter xylinum 1FO 13693 was selected as the best cellulose-producing bacterium among 41 strains belonging to the genus Acetobacter and Agrobacterium. Cellulose was found to be produced at the liquid surface in static liquid cultivation. The rate of cellulose production depended proportionally on the surface-area of the culture medium and was unaffected by the depth and volume of the medium. The optimum pH for cellulose production was 4.0 to 6.0. Glucose, fructose and glycerol were preferred carbon sources for cellulose production. The yield of cellulose, relative to the glucose consumed, decreased with an increase in initial glucose concentration, and gluconic acid accumulated at a high initial glucose concentration. The decrease in cellulose yield could be due to some glucose being metabolized to gluconic acid. However, the accumulated gluconic acid did not affect cellulose production. The culture conditions of the bacterium for cellulose production were optimized. The maximum production rate of cellulose was 36 g/d·m², with a yield of 100% for added glucose under the optimal conditions.     

Production of ethanol directly from potato starch by mixed culture of Saccharomyces cerevisiae and Aspergillus niger using electrochemical bioreactor

When cultivated aerobically, Aspergillus niger hyphae produced extracellular glucoamylase, which catalyzes the saccharification of unliquified potato starch into glucose, but not when grown under anaerobic conditions. The Km and Vmax of the extracellular glucoamylase were 652.3 mg starch l-1 and 253.3 mg glucose l-1 min-1, respectively. In mixed culture of A. niger and Saccharomyces cerevisiae, oxygen had a negative influence on the alcohol fermentation of yeast, but activated fungal growth. Therefore, oxygen is a critical factor for ethanol production in the mixed culture, and its generation through electrolysis of water in an electrochemical bioreactor needs to be optimized for ethanol production from starch by coculture of fungal hyphae and yeast cells. By applying pulsed electric fields (PEF) into the electrochemical bioreactor, ethanol production from starch improved significantly: Ethanol produced from 50 g potato starch l-1 by a mixed culture of A. niger and S. cerevisiae was about 5 g l-1 in a conventional bioreactor, but was 9 g l-1 in 5 volts of PEF and about 19 g l-1 in 4 volts of PEF for 5 days.     

Monoclonal antibodies incorporated into Neotyphodium coenophialum fungal cultures: inhibition of fungal growth and stability of antibodies.

Three monoclonal antibodies (mAbs) produced against proteins from the tall fescue (Festuca arundinacea Schreb.) fungal endophyte Neotyphodium coenophialum hybridize exclusively to a fungal protein under denaturing conditions. The protein is approximately 88 kDa in size. These mAbs were individually incorporated into liquid medium to determine their effects on fungal growth in culture. Neotyphodium-specific mAbs inhibited fungal growth for the duration of the study. Fungal cultures grown in the presence of Neotyphodium-naive mAbs or in the absence of all mAbs grew unimpeded. Bright-field microscopy and immunohistochemical studies of cultures containing Neotyphodium-specific mAbs revealed a change in mycelia morphology with clumps exhibiting a gelatinous matrix containing sparse hyphae, while cultures receiving Neotyphodium-naive mAbs in medium demonstrated unrestricted growth with overlapping and branched hyphae. In liquid culture devoid of fungal isolates, mAbs were stable and detected throughout the experiment, but were below threshold detection levels within 15 min following inclusion in liquid cultures containing Neotyphodium spp., indicating rapid binding to fungal mycelia. Monoclonal antibodies may provide a new method to help control plant pathogenic fungi where chemical or genetic means are not feasible.