Amelioration of ligninolytic enzyme production by Phanerochaete chrysosporium in airlift bioreactors

Maximum activities of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) in free cultures of Phanerochaete chrysosporium (ATCC 24725) were 258 U l^–1 and 103 U l^–1, respectively, in an airlift bioreactor. Immobilisation of the fungus on an inert carrier as well as several design modifications of the bioreactor employed gave MnP activities around 500–600 U l^–1 during 9 days' operation. The continuous operation of the latter led to MnP and LiP activities about 140 U l^–1 and 100 U l^–1, respectively, for two months, without operational problems. Furthermore, the extracellular liquid secreted decolourised the polymeric dye Poly R-478 about 56%.     

Extracellular ligninolytic enzyme production by Phanerochaete chrysosporium in a new solid-state bioreactor

The production of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) by the fungus Phanerochaete chrysosporium (ATCC 24725) in a new bioreactor, the Immersion Bioreactor, which grows cells under solid-state conditions, was studied. Maximum MnP and LiP activities were 987 U l^–1 and 356 U l^–1, respectively. The polymeric dye, Poly R-478, was degraded at 2.4 mg l^–1 min^–1 using the extracellular culture filtrate.     

Decolorization of synthetic dyes and production of manganese-dependent peroxidase by new fungal isolates

Two yeasts, Debaryomyces polymorphus, Candida tropicalis, and two filamentous fungi, Umbelopsis isabellina, Penicillium geastrivorus, could completely decolorize 100 mg Reactive Black 5 (RB 5) l^–1 within 16–48 h. Manganese-dependent peroxidase (MnP) activities between 60 and 424 U l^–1 were detected in culture supernatants of three of these organisms indicating the color removal by enzymatic biodegradation but with P. geastrivorus there was no ligninolytic enzyme activity in its culture and the decolorization was mainly due to biosorption to mycelium. Extensive decolorization by D. polymorphus (69–94%) and C. tropicalis (30–97%) was obtained with five other azo dyes and one anthraquinone dye. Except for Reactive Brilliant Blue KNR and Reactive Yellow M-3R, the four azo dyes, Reactive Red M-3BE, Procion Scharlach H-E3G, Procion Marine H-EXL and Reactive Brilliant Red K-2BP, induced D. polymorphus to produce MnP (105–587 U l^–1). However, MnP activities of 198–329 U l^–1 were only detected in the culture of C. tropicalis containing Reactive Red M-3BE and Reactive Brilliant Red K-2BP, respectively.     

Optimization of manganese peroxidase and laccase production in the South American fungus<Emphasis Type="Italic"> Fomes sclerodermeus</Emphasis> (Lév.) Cke

Fomes sclerodermeus produces manganese peroxidase (MnP) and laccase as part of its ligninolytic system. A Doehlert experimental design was applied in order to find the optimum conditions for MnP and laccase production. The factors studied were Cu²⁺, Mn²⁺ and asparagine. The present model and data analysis allowed us not only to define optimal media for production of both laccase and MnP, but also to show the combined effects between the factors. MnP was strongly influenced by Mn²⁺, which acts as an inducer. Under these conditions Cu²⁺ negatively affected MnP activity. At 13 days of growth 0.75 U ml^–1 were produced in the optimized culture medium supplemented with 1 mM MnSO₄ and 4 g l^–1 asparagine. The laccase titer under optimized conditions reached maximum values at 16 days of growth: 13.5 U ml^–1 in the presence of 0.2 mM CuSO₄, 0.4 mM MnSO₄ and 6 g l^–1 asparagine. Mn²⁺ promoted production of both enzymes. There were important interactions among the nutrients evaluated, the most significant being those between Cu²⁺ and asparagine.     

Degradation of tributyltin by the filamentous fungus Cunninghamella elegans, with involvement of cytochrome P-450

Cunninghamella elegans degraded tributyltin (TBT) at 20 mg l^–1 when grown in Sabouraud medium. Above this concentration, growth was inhibited. After 7 d 70% TBT (added at 10 mg l^–1) was converted to less toxic derivatives: dibutyltin and monobutyltin. TBT metabolism was totally blocked by cytochrome P-450 inhibitors, metyrapone and proadifen. Only in medium with 1-aminobenzotriazole, was dibutyltin (0.42 mg l^–1) found after 7 d of culturing. It is postulated that the significant resistance of C. elegans to TBT is associated with the capacity of the fungus to metabolise TBT.     

Selective production of bikaverin in a fluidized bioreactor with immobilized Gibberella fujikuroi

The best culture medium composition for the production of bikaverin by Gibberella fujikuroi in shake-flasks, i.e. 100 g glucose l^–1; 1 g NH₄Cl l^–1; 2 g rice flour l^–1; 5 g KH₂PO₄ l^–1 and 2.5 g MgSO₄ l^–1, was obtained through a fractional factorial design and then scaled-up to a fluidized bioreactor. The effects of carbon and nitrogen concentrations, inoculum size, aeration, flow rate and bead sizes on batch bikaverin production using immobilized G. fujikuroi in a fluidized bioreactor were determined by an orthogonal experimental design. Concentrations of up to 6.83 g bikaverin l^–1 were obtained when the medium contained 100 g glucose l^–1 and 1 g NH₄Cl l^–1 with an inoculum ratio of 10% v/v, an aeration rate of 3 volumes of air per volume of medium min^–1, and a bead size of 3 mm. Based on dry weight, the bikaverin production was 30–100 times larger than found in submerged culture and approximately three times larger than reported for solid substrate fermentation.     

Micropropagation of zedoary (<Emphasis Type="Italic">Curcuma zedoaria</Emphasis> Roscoe) – a valuable medicinal plant

Tissue culture propagation system was developed for zedoary (Curcuma zedoaria Roscoe), a valuable medicinal plant, using rhizome sprout cultures. Shoots were induced from rhizomes on basal MS medium containing 20 g l^–1 sucrose and 5 g l^–1 agar, supplemented with 20 (v/v) coconut water (CW) and benzylaminopurine (BA) concentrations from 0.5 to 5.0 m g l^–1. The excised shoots were subcultured on Murashige-Skoog (MS) medium with 20 (v/v) CW and different concentrations of BA and kinetin (Kin), either alone or in combination with indolebutyric acid (IBA) or naphthaleneacetic acid (NAA). MS medium with 20 (v/v) CW, 3 mg l^–1 BA, and 0.5 mg l^–1 IBA resulted in a multiplication rate per shoot; 5.6 shoots per explant were obtained on average after 30 days of culture. Well-developed shoots (30–40 mm in length) were rooted on MS medium containing 20 g l^–1 sucrose and 8 g l^–1 agar, supplemented with 20 (v/v) CW and 2 mg l^–1 NAA. More than 95 of the rooted plants were established in pots after hardening.     

Continuous cultures of Clostridium acetobutylicum: culture stability and low-grade glycerol utilisation

Continuous cultures of two strains of Clostridium acetobutylicum were stable for over 70 d when grown on glucose/glycerol mixtures. Butanol was the major fermentation end-product, accounting for 43 to 62% (w/w) of total products. Low-grade glycerol [65% (w/v) purity] could replace commercial glycerol [87% (w/v) purity], leading to a similar fermentation pattern: a butanol yield of 0.34 (mol/mol), a butanol productivity of 0.42 g l^–1 h^–1 and a 84% (w/w) glycerol consumption were attained when cultures were grown at pH 6 and D = 0.05 h^–1; butanol accounted for 94% (w/w) of total solvents. These values are among the highest reported in literature for C. acetobutylicum simple chemostats.     

Impact of enzymatic pre-hydrolysis on batch activated sludge systems dealing with oily wastewaters

Dairy wastewater containing different oil and grease contents was treated in batch activated sludge systems with and without (control) an enzymatic pre-hydrolysis stage [with 0.2% (w/v) of fermented babassu cake containing Penicillium restrictum lipases]. When the oil and grease concentration in the control bioreactor was increased (400, 600 and 800 mg l^–1), the COD removal efficiency fell (86%, 75% and 0%). However, in the reactor fed with pre-hydrolysed wastewater, COD removal efficiency was maintained (93%, 92% and 82%). At an oil and grease concentration of 800 mg l^–1, the control bioreactor presented final volatile suspended solids (VSS) values ten times greater (2225 mg l^–1) than those obtained for bioreactor fed with pre-hydrolysed wastewater (200 mg l^–1).     

Enzymatic pre-hydrolysis applied to the anaerobic treatment of effluents from poultry slaughterhouses

A pool of hydrolases with 21.4 U g⁻¹ lipase activity was produced through solid-state fermentation of the fungus Penicillium restrictum in waste from the Orbignya oleifera (babassu) oil processing industry. Enzymatic hydrolysis and anaerobic biodegradability tests were conducted on poultry slaughterhouse effluents with varying oil and grease contents (150–1200 mg l⁻¹) and solid enzymatic pool concentrations (0.1–1.0% w/v). Enhanced anaerobic treatment efficiency relative to raw effluent was achieved when a 0.1% concentration of enzymatic pool was used in the pre-hydrolysis stage with 1200 mg oil and grease l⁻¹ (chemical oxygen demand (COD) removal efficiency of 85% vs. 53% and biogas production of 175 ml vs. 37 ml after 4 d).     

Optimization of thermostable lipase production from a thermophilic Geobacillus sp. using Box-Behnken experimental design

Thermostable lipase production by Geobacillus thermoleovorans was optimized in shake-flask cultures using Box-Behnken experimental design. An empirical model was developed through response surface methodology to describe the relationship between tested variables (Tween 80, olive oil, temperature and pH) and enzyme activity. Maximum enzyme activity (495 U l^–1) was attained with Tween 80 at 5 g l^–1; olive oil at 60 g l^–1; 70 °C and pH 9. Experimental verification of the model showed a validation of 95%, which is more than 4-fold increase compared to the basal medium.     

Tannase production by Bacillus licheniformis

Bacillus licheniformis KBR 6 produced maximum extracellular tannase activity at 0.21 U ml^–1 with 1.5% (w/v) tannic acid either in the absence or presence of glucose (1 g l^–1) after 18–21 h growth though the organism did not attain maximum growth until 36 h.     

Invertase production by Aspergillus niger in submerged and solid-state fermentation

Three Aspergillus nigerstrains were grown in submerged and solid state fermentation systems with sucrose at 100 g l^–1. Average measurements of all strains, liquid vs solid were: final biomass (g l^–1), 11 ± 0.3 vs 34 ± 5; maximal enzyme titres (U l^–1) 1180 ± 138 vs 3663 ± 732; enzyme productivity (U l^–1h^–1) 20 ± 2 vs 87 ± 33 and enzyme yields (U/gX) 128 ± 24 vs 138 ± 72. Hence, better productivity in solid-state was due to a better mould growth.     

Color removal ability of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> in relation to lignin peroxidase and manganese peroxidase produced in molasses wastewater

Decolorization of molasses wastewater (MWW) from an ethanolic fermentation plant by Phanerochaete chrysosporium was studied. By diluting MWW properly (10%v/v) and incubating it with an appropriate concentration of the spores (2.5 × 10⁶/ml), extensive decolorization occurred (75%) on day 5 of the incubation. The colour removal ability was found to be correlated to the activity of ligninolytic enzyme system: lignin peroxidase (LiP) activity was 185 U/l while manganese peroxidase (MnP) activity equaled 25 U/l. Effects of some selected operating variables were studied: manganese(II), veratryl alcohol (VA), glucose as a carbon source and urea and ammonium nitrate, each as a source of nitrogen. Results showed that the colour reduction and LiP activity were highest (76% and 186 U/l, respectively) either when no Mn(II) was added or added at the lowest level tested (0.16 mg/l to provide 0.3 mg/l). Activity of MnP was highest (25 U/l) when Mn(II) added to the diluted MWW at the highest level (100 ppm) while activity of LiP was lowest (7.1 U/l) at this level of added Mn(II). The colour reduction in the presence of the added VA was shown to be little less than in its absence (70 vs. 75%). When urea as an organic source of nitrogen for the fungus, was added to the MWW, the decolorizing activity of P. chrysosporium decreased significantly (15 vs. 75%) and no activities were detected for LiP and MnP. Use of ammonium nitrate as an inorganic source of nitrogen did not show such a decelerating effects, although no improvements in the metabolic behavior of the fungus (i.e., LiP and MnP activities) deaccelerating was observed. Effects of addition of glucose was also discussed.     

Biotransformation of [ring-U-14C]4-n-nonylphenol by Agrostemma githago cell culture in a two-liquid-phase system

The biotransformation of [¹⁴C]4-n-nonylphenol (5 mg l^–1; 10 mg l^–1) by Agrostemma githago cell suspensions was studied using a batch two-liquid-phase system (medium/n-hexadecane 200:1, v/v). The highly lipophilic 4-n-nonylphenol was applied via n-hexadecane phase. After 7 d of incubation, more than 85% of applied 4-n-nonylphenol was absorbed by the cells, and 40% was transformed to 10 side-chain monohydroxylated metabolites (two with additional double bond at side-chain). The primary metabolites were analyzed by GC-EIMS. In the cells, the monohydroxylated products and residual 4-n-nonylphenol were present as glycosides. The method proved to be suitable for the production of primary metabolites of 4-n-nonylphenol on a larger scale for identification purposes and for metabolic profiling of the compound.     

Transformation of arachidonic acid to 19-hydroxy- and 20-hydroxy-eicosatetraenoic acids using Candida bombicola

Candida bombicola (ATCC 22214) and C. apicola (ATCC 96134), grown on glucose (100 g l^–1) and arachidonic acid (5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid; AA), 1.25 g l^–1, synthesized sophorolipid up to 0.93 g l^–1. Acid hydrolysis of sophorolipid yielded 19-hydroxy-5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid (19-HETE) and 20-hydroxy-5Z, 8Z, 11Z, 14Z-eicosatetraenoic acid (20-HETE) which were identified by TLC and GC-MS; the ratio of synthesis was 73:27, respectively. Conversion of AA by immobilized Candida bombicola, suspended in beads of 2% (w/v) calcium alginate for 96 h, gave an 83% conversion of 1 g AA l^–1 to 19- and 20-HETE. There was no significant loss in the efficiency of the immobilized cells after ten uses.     

Production of a thermostable extracellular lipase by Kluyveromyces marxianus

Kluyveromyces marxianus was grown in submerged culture in a complex medium with several potential inducers of lipolytic activity (triacylglycerols, fatty acids). The highest extracellular lipolytic enzyme production (about 80 U ml^–1 in 3 d) was obtained when the medium was supplemented with 2 g urea l^–1 plus 5 g tributyrin l^–1. Addition of surfactants (1 g l^–1) did not improve production. The lipase had a high thermal stability in aqueous solution (73% residual activity after 9 d at 50 °C, 16 min half-life time at 100 °C). It was also stable at acidic pH and showed good tolerance to organic solvents (70% residual activity after 2 d in n-hexane of cyclohexane).     

Lactulose production by β-galactosidase in permeabilized cells of<Emphasis Type="Italic"> Kluyveromyces lactis</Emphasis>

Lactulose production from lactose and fructose was investigated with several commercial -galactosidases. The enzyme from Kluyveromyces lactis exhibited the highest lactulose productivity among the -galactosidases tested. The reaction conditions for lactulose production were optimized using cells that had been permeabilized by treatment with 50% (v/v) ethanol: cell concentration, 10.4 g l^–1; concentration of substrates, 40% (w/v) lactose and 20% (w/v) fructose; temperature, 60^°C; pH 7.0. Under these conditions, the permeabilized cells produced approximately 20 g l^–1 lactulose in 3 h with a lactulose productivity of 6.8 g l^–1 h^–1. These results represent 1.3- and 2.1-fold increases in lactulose concentration and productivity compared with untreated washed cells. This is the first reported trial of enzymatic synthesis of lactulose using permeabilized yeast cells.     

Laccase and Manganese Peroxidase Activities of Phellinus Robustus and Ganoderma Adspersum Grown on Food Industry Wastes in Submerged Fermentation

Phellinus robustus produced both laccase (700–4,000 U l⁻¹) and manganese peroxidase (MnP) (1,000–11,300 U l⁻¹) in fermentation of nine food wastes, whereas Ganoderma adspersum produced only laccase (600–34,000 U l⁻¹). Glucose provided high laccase and MnP activity of P. robustus but repressed enzyme production by G. adspersum. Ammonium sulphate and ammonium tartrate increased the P. robustus laccase yield (3-fold), whereas the accumulation of MnP was not enhanced by additional nitrogen.     

Phototrophic bacterial production of oleic acid in an illuminated upflow anaerobic sludge blanket reactor

Phototrophic bacterial cells in the effluent from a lighted upflow anaerobic sludge blanket reactor supplied with a medium containing 142 mg S (as SO₄ ^2–) l^–1 accumulated a 6.8% w/w oleic acid content in cells and 19 mg cell-bound oleic acid l^–1 in the effluent. Pure cultures of Rhodopseudomonas palustris and Blastochloris sulfoviridis isolated from the effluent also accumulated 5.1 and 6.4% w/w oleic acid contents in cells, respectively. The oleic acid content in the cells recovered from the LUASB reactor effluent was related to the phototrophic bacterial population in the LUASB reactor. The inverse relationship was observed in the LUASB reactor between phototrophic bacterial growth and sulfate concentration in the influent.