Non-agitated Conditions Research Articles

Production of mycelial biomass, proteases and protease inhibitors by Ganoderma lucidum under different submerged fermentation conditions.

Ganoderma lucidum is a medicinal mushroom widely recognized as a source of biomolecules with pharmacological properties, however, little is known about the factors that influence the synthesis of bioactive proteins by this fungus when cultivated under submerged fermentation. The objective of this work was to evaluate the production of mycelial biomass and intracellular proteases and protease inhibitors by G. lucidum cultivated under different submerged fermentation conditions. The cultivation was carried out in a medium composed of glucose (10 or 20 g.L-1), soy peptone (2.5 or 5 g.L-1) and yeast extract (5 g.L-1), with incubation under agitation (120 rpm) and non-agitation, totaling 8 experimental conditions. Biomass production was determined from the dry weight, while glucose consumption was estimated by quantification of reducing sugars. The proteins were extracted in NaCl (0.15 M), and the protein extracts were submitted to protein quantification by the Bradford method, total proteolytic activity using azocasein, caseinolytic and fibrinolytic activity in Petri dishes, activity of serine (trypsin and chymotrypsin) and cysteine (papain) protease inhibitors. Cultivation in agitated condition showed higher biomass production with a maximum value of 7 g.L-1, in addition to higher activities of trypsin, chymotrypsin and papain inhibitors, with 154 IU.mg-1, 153 IU.mg-1 e 343 IU.mg-1 of protein, respectively. The non-agitated condition showed a greater potential for obtaining proteins, total proteases, caseinolytic and fibrinolytic enzymes, with maximum values of 433 mg.g-1 of extract, 71 U.mL-1 of extract, 63.62 mm2 and 50.27 mm2, respectively. Thus, a medium composed of soy peptone, yest extract and glucose in a 1:2:4 proportion is recommended, under agitation to produce protease inhibitors, and the non-agitated condition when the target is, mainly caseinolytic and fibrinolytic enzymes.

Development of an exposure protocol for toxicity test (FEET) for a marine species: the European sea bass (Dicentrarchus labrax).

Regulatory assessment of the effects of chemicals requires the availability of validated tests representing different environments and organisms. In this context, developing new tests is particularly needed for marine species from temperate environments. It is also important to evaluate effects that are generally poorly characterized and seldom included in regulatory tests. In this study, we designed an exposure protocol using European sea bass (Dicentrarchus labrax) larvae. We examined classical toxicological values (LCx) as well as behavioral responses. By comparing different hatching and breeding strategies, we defined the optimal conditions of exposure as non-agitated conditions in 24- or 48-well microplates. Our exposure protocol was then tested with 3,4-dichloroaniline (3,4-DCA), a recommended reference molecule. Based on our results, the 96 h LC50 for 3,4-DCA corresponded to 2.04 mg/L while the 168 h LC50 to 0.79 mg/L. Behavioral analyses showed no effect of 3,4-DCA at low concentration (0.25 mg/L). In conclusion, the present work established the basis for a new test which includes behavioral analysis and shows that the use of sea bass is suitable to early-life stage toxicity tests.

Effects of Agitation Speed and Kinetic Studies on Probiotication of Pomegranate Juice with Lactobacillus casei.

The issues of lactose intolerance and vegetarianism have encouraged the introduction of non-dairy fermented food into the market. Therefore, this study aims to evaluate the effect of agitation speed on the bioactive compounds and functional characteristics of probioticated pomegranate juice. Pomegranate juice was fermented with Lactobacillus casei at different agitation speeds ranging from 0 (microaerophilic) to 150 rpm at 37 °C. The functional properties of probioticated pomegranate juice were evaluated in terms of growth (biomass), lactic acid production, antioxidant activity, total phenolic content, and key metabolites using LC-MS/MS. The growth kinetics of fermentation was monitored at the optimal condition using one factor at a time method. High cell growth (3.58 × 1010 cfu/mL or 7.9 gL−1) was observed for L. casei probioticated pomegranate juice agitated at 0 rpm. The findings of this study reveal the potential of pomegranate juice as a medium for L. casei cultivation without nutrient supplementation. The improvement of antioxidant activity in the probioticated juice could be due to the increment of quercetin-3-glucoside. Therefore, L. casei grew well in pomegranate juice with a high cell viability and antioxidant activity at a non-agitated condition. Probioticated pomegranate juice is a potentially functional drink.

Active heat sink with piezoelectric translational agitators, piezoelectric synthetic jets, and micro pin fin arrays

Air-cooled heat sinks are widely used for electronics cooling. Active and passive cooling components can be added to enhance the performance of the air-cooled heat sinks. In this paper, piezoelectric translational agitators and synthetic jets are integrated as active cooling components while micro pin fins are adopted as a passive cooling scheme. The heat transfer performance of the active heat sink system that combines these active and passive cooling components along with a suction fan is experimentally investigated. The piezoelectric translational agitators installed within cooling channels are operating at 222 Hz ∼ 820 Hz with a peak-to-peak displacement of 1.0 mm ∼ 1.4 mm, depending on the attached carbon fiber blade type. The piezoelectric synthetic jet array provides impingement flow into the cooling channels with an inclined configuration, that has been successfully integrated into the system without interference with other components by using a wedge platform. The jet operates at 720 Hz with the jet velocity of up to 39 m/s. Micro pin fins are fabricated onto both surfaces of each heat sink channel walls by a double-sided microfabrication technique. They have diameter, height, and spacing of 500 μm, 250 μm, and 1500 μm, respectively. The experimental results indicate that the micro pin fins are most efficient among the employed active and passive cooling components, reducing thermal resistance up to 38%, compared to plain heat sink performance. The piezoelectric translational agitator reduces system thermal resistance by 22%, compared to the non-agitated condition, at the same through-flow rate. The synthetic jet shows weaker cooling capability in the setting tested, compared to enhancement by the agitator plates or pin fins. The active heat sink with the micro pin fins, agitators, and jets provides a thermal resistance of 0.064 °C/W at 70 CFM (33 L/sec) through-flow of air, about a 48% reduction from that of the non-agitated, plain heat sink under the same operating conditions. The results demonstrate how more effective the active heat sink system is compared to traditional air-cooled heat sinks.

Hydrogen gas generation from metal aluminum-water interaction in municipal solid waste incineration (MSWI) bottom ash

In the present research, municipal solid waste incineration (MSWI) bottom ash (BA) residues from three incinerators (N, K, and R) in Japan were collected for hydrogen gas generation purpose. The samples were split into four particle size fractions: (1) d≤0.6, (2) 0.6≤d≤1.0, (3) 1.0≤d≤2.0, and (4) 2.0≤d≤4.75mm for the characterization of metal aluminum, the relationship between the present metal aluminum and hydrogen gas production, and the influence of external metal aluminum on the enhancement of hydrogen gas. The batch experiments were performed for each BA fraction under agitated (200rpm) and non-agitated conditions at 40°C for 20days. The highest amount of hydrogen gas (cumulative) was collected under agitation condition that was 39.4, 10.0, and 8.4 L/kg of dry ash for N2, R2, and K2 (all fraction 2), respectively. To take the benefit of the BA high alkalinity (with initial pH over 12), 0.1 and 1g of household aluminum foil were added to the fractions 2 and 3. A Significantly larger amount of hydrogen gas was collected from each test. For 0.1g of aluminum foil, the cumulative amount of gas was in the range of 62 to 78 L/kg of dry ash and for 1g of aluminum foil the cumulative amount of hydrogen was in the range of 119–126 L/kg of dry ash. This indicated that the hydrogen gas yield was significantly a function of supplementary aluminum and the intrinsic alkaline environment of the BA residues rather than ash source or particle size.

Enhanced Chondrocyte Proliferation in a Prototyped Culture System with Wave-Induced Agitation

Abstract One of the actual challenges in tissue engineering applications is to efficiently produce as high of number of cells as it is only possible, in the shortest time. In static cultures, the production of animal cell biomass in integrated forms (i.e. aggregates, inoculated scaffolds) is limited due to inefficient diffusion of culture medium components observed in such non-mixed culture systems, especially in the case of cell-inoculated fiber-based dense 3D scaffolds, inside which the intensification of mass transfer is particularly important. The applicability of a prototyped, small-scale, continuously wave-induced agitated system for intensification of anchorage-dependent CP5 chondrocytes proliferation outside and inside three-dimensional poly(lactic acid) (PLA) scaffolds has been discussed. Fibrous PLA-based constructs have been inoculated with CP5 cells and then maintained in two independent incubation systems: (i) non-agitated conditions and (ii) culture with wave-induced agitation. Significantly higher values of the volumetric glucose consumption rate have been noted for the system with the wave-induced agitation. The advantage of the presented wave-induced agitation culture system has been confirmed by lower activity of lactate dehydrogenase (LDH) released from the cells in the samples of culture medium harvested from the agitated cultures, in contrast to rather high values of LDH activity measured for static conditions. Results of the proceeded experiments and their analysis clearly exhibited the feasibility of the culture system supported with continuously wave-induced agitation for robust proliferation of the CP5 chondrocytes on PLA-based structures. Aside from the practicability of the prototyped system, we believe that it could also be applied as a standard method offering advantages for all types of the daily routine laboratory-scale animal cell cultures utilizing various fiber-based biomaterials, with the use of only regular laboratory devices.

Aluminum and aluminum alloys in municipal solid waste incineration (MSWI) bottom ash: A potential source for the production of hydrogen gas

Bottom ash residues from three incinerators (K, N, and R) in Japan were used to characterize the metal aluminum and aluminum alloys and their potential for hydrogen gas generation. The samples were sieved into four fractions: (1) d ≤ 0.6 mm, (2) 0.6 ≤ d ≤ 1.0 mm, (3) 1.0 ≤ d ≤ 2.0 mm, and (4) 2.0 ≤ d ≤ 4.75 mm for characterization and hydrogen gas evolution experiments. Based on microscopic and microanalytical (SEM-EDX) examinations, the metal aluminum alloys with high purity (>90 wt% Al in most occurrences) and various sizes were randomly distributed in the ash particles. X-ray diffraction (XRD) analysis was carried out to identify the primary and secondary mineral phases in the ash residues before and after the hydrogen gas generation experiments. Batch experiments were performed to evaluate the hydrogen generation potential in each bottom ash fraction under agitated (200 rpm) and non-agitated conditions at 40 °C for 20 days. The highest amount of hydrogen gas (cumulative) under agitation was 39.4, 10.0, and 8.4 l/kg of dry ash for N2, R2, and K2, respectively. Under non-agitated condition, N2, R2, and K2 also yielded the highest amount of hydrogen, which was 38.3, 6.5, and 6.8 l/kg of dry ash, respectively. As a result of these experiments, the metal aluminum aggregates considerably converted to gel-like and crystalline Al-rich hydrate phases. The inherent alkalinity of the ash environment (pH ≥ 12) and the existence of metal aluminum were considered the key parameters to trigger and maintain the hydrogen generation reactions.

Wavelet-based multiscale analysis of bioimpedance data measured by electric cell-substrate impedance sensing for classification of cancerous and normal cells.

The paper presents a study to differentiate normal and cancerous cells using label-free bioimpedance signal measured by electric cell-substrate impedance sensing. The real-time-measured bioimpedance data of human breast cancer cells and human epithelial normal cells employs fluctuations of impedance value due to cellular micromotions resulting from dynamic structural rearrangement of membrane protrusions under nonagitated condition. Here, a wavelet-based multiscale quantitative analysis technique has been applied to analyze the fluctuations in bioimpedance. The study demonstrates a method to classify cancerous and normal cells from the signature of their impedance fluctuations. The fluctuations associated with cellular micromotion are quantified in terms of cellular energy, cellular power dissipation, and cellular moments. The cellular energy and power dissipation are found higher for cancerous cells associated with higher micromotions in cancer cells. The initial study suggests that proposed wavelet-based quantitative technique promises to be an effective method to analyze real-time bioimpedance signal for distinguishing cancer and normal cells.

An innate immune system-mimicking, real-time biosensing of infectious bacteria.

An animal cell-based biosensor was investigated to monitor bacterial contamination in an unattended manner by mimicking the innate immune response. The cells (RAW 264.7 cell line) were first attached onto the solid surfaces of a 96-well microtiter plate and co-incubated in the culture medium with a sample that might contain bacterial contaminants. As Toll-like receptors were present on the cell membrane surfaces, they acted as a sentinel by binding to pathogen-associated molecular patterns (PAMPs) of any contaminant. Such biological recognition initiates signal transmission along various pathways to produce different proinflammatory mediators, one of which, tumor necrosis factor-α (TNF-α) was measured using an immunosensor. To demonstrate automated bacterium monitoring, a capture antibody specific for TNF-α was immobilized on an optical fiber sensor tip and then used to measure complex formation in a label-free sensor system (e.g., Octet Red). The sensor response time depended significantly on the degree of agitation of the culture medium, controlling the biological recognition and further autocrine/paracrine signaling by cytokines. The response, particularly under non-agitated conditions, was also influenced by the medium volume, revealing a local gradient change of the cytokine concentration and also acidity, caused by bacterial growth near the bottom surfaces. A biosensor system retaining 50 μL medium and not employing agitation could be used for the early detection of bacterial contamination. This novel biosensing model was applied to the real-time monitoring of different bacteria, Shigella sonnei, Staphylococcus aureus, and Listeria monocytogenes. They (<100 CFU mL(-1)) could be detected automatically within the working time. Such analysis was carried out without any manual handling regardless of the bacterial species, suggesting the concept of non-targeted bacterial real-time monitoring. This technique was further applied to real sample testing (e.g., with milk) to exemplify, for example, the food quality control process without using any additional sample pretreatment such as magnetic concentration.

Microbial ecology in anaerobic digestion at agitated and non-agitated conditions.

To investigate the distribution and dynamics of microbial community in anaerobic digestion at agitated and non-agitated condition, 454 pyrosequencing of 16s rRNA was conducted. It revealed the distinct community compositions between the two digesters and their progressive shifting over time. Methanogens and syntrophic bacteria were found much less abundant in the agitated digester, which was mainly attributed to the presence of bacterial genera Acetanaerobacterium and Ruminococcus with relatively high abundance. The characterization of the microbial community corroborated the digestion performance affected at the agitated condition, where lower methane yield and delayed methane production rate were observed. This was further verified by the accumulation of propionic acid in the agitated digester.

Effects of heat, pH, antioxidant, agitation and light on betacyanin stability using red-fleshed dragon fruit (Hylocereus polyrhizus) juice and concentrate as models.

Red-fleshed dragon fruit (Hylocereus polyrhizus) is rich in antioxidants. The aim of this study was to determine the effects of heat pasteurization, pH adjustment, ascorbic acid addition as well as storage under agitation and light or dark condition on betacyanin content in red-fleshed dragon fruit (Hylocereus polyrhizus) juice and concentrate. The concentrate was produced by concentrating clarified red-fleshed dragon fruit juice in a rotary evaporator at 40°C. UV-Visible spectrophotometer was used for analyzing betacyanin content. Addition of 0.25% ascorbic acid, pH4.0, and pasteurization at 65°C for 30min were selected as the best processing conditions to retain betacyanin content in red-fleshed dragon fruit juice. Storage at the agitation speed of 220rpm showed that the concentrated samples had higher betacyanin stability compared to juice, while both juice and concentrate had almost similar betacyanin stability when tested for storage in the presence of light. In summary, ascorbic acid stabilized betacyanin in both juice and concentrate at agitated or non-agitated conditions. In contrast, light degraded betacyanin in both juice and concentrate models.

Different morphology of amyloid fibrils originating from agitated and non-agitated conditions

In vitro amyloid formation has been suggested to be a common property of any polypeptide chain depending on particular environmental conditions although in vivo amyloid fibril formation can be promoted by point mutations or triplet expansions. Here, we explored the influence of agitation on fibril formation of amyloidogenic alanine segments fused to Cold Shock Protein B (CspB) of Bacillus subtilis. While without agitation fibril formation was clearly dependent on the presence of an amyloidogenic alanine segment, fibril formation was independent of the amyloidogenic segment under agitation. Agitation even led to fibrillation of native CspB lacking the amyloidogenic segment. Furthermore, agitation not only influenced the kinetics of fibril formation, but also resulted in completely different fibril morphologies. These results indicate that experimental conditions can alter the region that undergoes a conformational change during in vitro fibrillation. Moreover, the data show that deductions from in vitro assays on in vivo fibril formation mechanisms are afflicted with a certain degree of uncertainty and therefore need to be cautiously discussed.

Impact of laminar flow velocity of different acids on enamel calcium loss

The aim of the study was to evaluate the impact of flow velocity under laminar flow conditions of different acidic solutions on enamel erosion. A total of 240 bovine enamel specimens were prepared and allocated to 30 groups (n = 8 each). Samples of 18 groups were superfused in a flow chamber system with laminar flow behavior using 1 ml of citric acid or hydrochloric acid (HCl) of pH 2.0, 2.6 or 3.0. Flow rates in the sample chamber were adjusted to 10, 60 or 100 μl/min. To simulate turbulent flow behavior, samples of six groups were immersed in 1 ml of the respective solution, which was vortexed (15 min, 600 rpm). For simulating non-agitated conditions, specimens of the remaining six groups were immersed in 1 ml of the respective solution without stirring. Calcium in the solutions, released from the enamel samples, was determined using Arsenazo III method. For acidic solutions of pH 2.6 and 3.0, erosive potential of citric acid was equivalent to that of HCl at a flow of 100 μl/min. The same observation was made for the samples subjected to turbulent conditions at pH 3. At all other conditions, citric acid induced a significantly higher calcium loss than HCl. It is concluded that under slow laminar flow conditions, flow rate variations lead to higher erosive impact of citric acid compared to hydrochloric acid at pH 2.0, but not at pH ≥ 2.6 and increasing laminar flow or turbulent conditions. Erosive enamel dissolution under laminar flow conditions is a complex issue influenced by flow rate and acidic substrate.

Pretreatment processing of fabrics by alkalothermophilic xylanase from Bacillus stearothermophilus SDX

A highly active alkalothermophilic cellulase free xylanase (3446 U/g of dry substrate) has been produced from newly isolated strain of Bacillus stearothermophilus under solid-state fermentation using wheat bran (2.5%, w/v) pH 7.0 and at 37 °C. The xylanase was stable over broad range of pH (6.0–12.5) and temperature (37–85 °C) and hence was used in the bioprocessing of fabrics as an alternative to conventional chemical method. The desizing and bioscouring treatments were most efficient at 70 °C after 90 and 180 min of incubation respectively using 5 g/l of xylanase dose in the medium of pH 9.5. Enzymatically desized fabrics when subjected to bioscouring resulted in 0.91%, 0.88% weight loss for micropoly and 0.83%, 0.80% for cotton respectively under agitated (50 ± 2 rpm) and non-agitated conditions. Consequently Whiteness index for cotton (11.81%) and micropoly (52.15%) fabrics was increased. Induction in the release of reducing sugars was also observed for cotton (8.37%) and micropoly (16.79%) fabric over conventional method. Enzymatically processed fabric samples possessed 1.12% and 1.95% more tensile strength than reference. Tearness value also increased for bio-treated cotton (1.57%) and micropoly (2.37%) fabric samples as compared to control.

Evaluation of the optical sulfite oxidation method for the determination of the interfacial mass transfer area in small-scale bioreactors

The optical sulfite oxidation method was recently used by Hermann et al. [R. Hermann, M. Lehmann, J. Büchs, Characterization of gas–liquid mass transfer phenomena in microtiter plates, Biotechnol. Bioeng. 81 (2003) 178–186] and Kensy et al. [F. Kensy, H.F. Zimmermann, I. Knabben, T. Anderlei, H. Trauthwein, U. Dingerdissen, J. Büchs, Oxygen transfer phenomena in 48-well microtiter plates: determination by optical monitoring of sulfite oxidation and verification by real-time measurement during microbial growth, Biotechnol. Bioeng. 89 (2005) 698–708.] for determination of interfacial mass transfer area a in a microtiter plate at different shaking intensities. They have determined relative increase of a liquid surface area due to the shaking as a ratio of average specific oxygen absorption rates N ave in 0.5 M sulfite solution with 10 −4 M CoSO 4 under shaken and unshaken conditions. In this paper it is shown that the method is based on the assumption that the profiles of instantaneous oxygen flux Φ during the whole experiments are the same for the shaken and nonshaken solutions. Our experiments performed in a stirred cell did not confirm validity of this assumption. Monitoring of pH and sulfite concentration during the oxidation of sulfite solutions revealed lower values of Φ into nonagitated solution compared to agitated one. The value of N ave in the nonagitated (nonshaken) sulfite solution was by 30% lower than the rate in the agitated one. Consequently, the mass transfer areas measured by the optical sulfite oxidation method are overestimated seriously, cca. 1.4 times. The lower oxygen transfer rate in the nonagitated solution is caused by a local drop of the sulfite concentration near the interface which is due to insufficiently fast transport of sulfite ions from the bulk of a liquid phase to interface under nonagitated (nonshaken) conditions. It is shown that the optical method for measuring of a is fundamentally flawed and should not be used in the presented form.

Kinetics of MCM-41 Synthesis under Nonagitated and Agitated Conditions

MCM-41 has been synthesized under both agitated and nonagitated conditions. The optimum conditions of MCM-41 synthesis and the effect of various ions on the crystal yield and synthesis time have been studied. The parameters have been reported in molar ratios to make the studies usable during scale-up. The kinetics of MCM-41 synthesis has been studied under both nonagitated and agitated conditions, and the activation energy and preexponential factor have been determined. The kinetics was found to be first order. The crystal growth rate is found to increase linearly, and the total synthesis time decreases linearly with the Si/CTAB molar ratio for all temperatures. The total time of MCM-41 synthesis under agitated conditions is found to be about one-third, with only a minor decrease in the crystal yield. The acidity of the samples synthesized under agitated conditions is found to be higher than those synthesized under nonagitated conditions. The minimum surfactant concentration for the formation of MCM-41 is 5 times in agitated as compared to nonagitated conditions.

Culture conditions for jasmonic acid and biomass production by Botryodiplodia theobromae in submerged fermentation

Jasmonic acid (JA) is a plant growth regulator produced by Botryodiplodia theobromae in submerged fermentation. Eight strains of B. theobromae isolated from Cuban orange waste peel were screened for JA production. Strain 2434 was selected for its higher JA production (914 mg/l) and productivity (91.4 mg/l day). Studies carried out at different temperatures showed that the highest JA production (925 mg/l) was obtained at 30–32°C. Although biomass production was enhanced under agitation (up to 100 rpm), JA production was negatively affected when the agitation was increased. Maximum JA production (900 mg/l) was obtained under non-agitated conditions. Studies concerning the effect of different carbon and nitrogen sources showed that fructose, glucose and sodium nitrate were the best sources for JA production.

Preparation of calcium silicate absorbent from recycled glass

AbstractCalcium silicate hydrates were prepared from hydrated lime and post‐consumer recycled glass in an aqueous slurry. These high surface area solids have shown promise as alkaline sorbents for environmental gas cleaning applications. Surface area was monitored at a variety of reaction conditions. The rate of surface area formation was found to be directly proportional to the initial surface area of the glass. At 92°C, the addition of gypsum to the system had a significant positive effect, forming solids with surface areas up to 125 m2/g over long reaction times. Increasing the temperature from 92°C to 120°C increased the initial rate of surface area formation, however the rate decreased over time and the ultimate surface area was higher at 92°C. The addition of gypsum or calcium chloride to the reaction at 120°C increased the ultimate surface area, but not to the extent of the 92°C product. Up to a surface area of ∼100 m2/g, sorbents formed at 92°C with gypsum were not affected by agitation, nor by solids content between 20–50% under non‐agitated conditions. At reaction times after this point, surface area increased slightly with water content and more significantly with agitation. In addition, the dissolution of silica from glass was measured and was found to be faster than the rate of sorbent formation.

Characterization of apatite layer formation on P2O5-CaO, P2O5-CaO-Na2O, and P2O5-CaO-Na2O-Al2O3 glass hydroxyapatite composites.

P2O5-based glass hydroxyapatite (HA) composites denoted HA-2 oxide, HA-3 oxide, and HA-4-oxide, were immersed in Hank's balanced salt solution for a period of 1, 2, 3, and 4 weeks at 37 degrees C in nonagitated condition for in vitro evaluation. A surface layer was precipitated on the composites that was analyzed using scanning electron microscopy (SEM), X-ray photoelecton spectroscopy, and thin film X-ray diffractometry (TF-XRD). SEM micrographs showed complete coverage of the composite surface by crystallites after immersion of 1 week in the solution, which grew thicker with respect to immersion time. The binding energies measured by XPS indicated apatite formation and the presence of carbonate on the composite surface, showing the newly formed layer was a carbonated apatite. Confirmation of the formed apatite layer was obtained by TF-XRD.

Strategies for production of high ligninase activities by Phanerochaete chrysosporium

Phanerochaete chrysosporium was activated by veratryl alcohol to produce high ligninase activities. In non-agitated conditions with lignocellulose activities up to 180 U ml −1 were obtained. Maximal enzyme yields in agitated nitrogen limited cultures were 118 U ml −1 with a specific activity of 12 U μg −1 protein, and in carbon-limited agitated cultures 670 U ml −1, the specific activity being 36 U μg −1 protein. In N-limited conditions the major proteins were two 39000 dalton ligninases with apparent molecular weights of 42 000 and isoelectric points of 3.5 and 3.1. In C-limited conditions the major proteins were two 39000 dalton ligninases with isoelectric points of 4.5 and 4.7. The high activity with C-limited pellets was obtained only after 60 h of growth. The activity level was dependent on the concentration of veratryl alcohol, on the size and concentration of pellets and on the oxygen partial pressure.