Response of Bacillus velezensis 83 to interaction with Colletotrichum gloeosporioides resembles a Greek phalanx-style formation: A stress resistant phenotype with antibiosis capacity.

Plant growth-promoting rhizobacteria, such as Bacillus spp., establish beneficial associations with plants and may inhibit the growth of phytopathogenic fungi. However, these bacteria are subject to multiple biotic stimuli from their competitors, causing stress and modifying their development. This work is a study of an in vitro interaction between two model microorganisms of socioeconomic relevance, using population dynamics and transcriptomic approaches. Co-cultures of Bacillus velezensis 83 with the phytopathogenic fungus Colletotrichum gloeosporioides 09 were performed to evaluate the metabolic response of the bacteria under conditions of non-nutritional limitation. The bacterial response was associated with the induction of a stress-resistant phenotype, characterized by a lower specific growth rate, but with antimicrobial production capacity. About 12% of co-cultured B. velezensis 83 coding sequences were differentially expressed, including the up-regulation of the general stress response (sigB regulon), and the down-regulation of alternative carbon sources catabolism (glucose preference). Defense strategies in B. velezensis are a determining factor in order to preserve the long-term viability of its population. Mostly, the presence of the fungus does not affect the expression of antibiosis genes, except for those corresponding to surfactin/bacillomycin D production. Indeed, the up-regulation of antibiosis genes expression is associated with bacterial growth, regardless of the presence of the fungus. This behavior in B. velezensis 83 resembles the strategy used by the classical Greek phalanx formation: by sacrificing growth rate and metabolic versatility, resources can be redistributed to defense (stress resistant phenotype) while maintaining the attack (antibiosis capacity). The presented results are the first characterization of the molecular phenotype at the transcriptome level of a biological control agent under biotic stress caused by a phytopathogen without nutrient limitation.

Laccase-luminol chemiluminescence system: an investigation of substrate inhibition.

Chemiluminescence (CL) reactions are widely used for the detection and quantification of many types of analytes. Laccase has previously been proposed in CL reactions; however, its light emission behaviour has not been characterized. This study was conducted to characterize the laccase-luminol system, determine its kinetic parameters, and analyze the effects of protein and OH- concentration on the CL signal. Laccase from Coriolopsis gallica was combined with different concentrations of luminol (125 nM to 4 mM), and the enzyme kinetics were evaluated using diverse kinetic models. The laccase-luminol system was able to produce CL without an intermediate molecule, but it exhibited substrate-inhibition behaviour. A two-site random model was used and suggested that when the first luminol molecule was bound to the active site, laccase affinity for the second luminol molecule was increased. This inhibition effect could be avoided using a low luminol concentration. At 5 µM luminol concentration, 1 mg/ml (0.13 U) laccase is needed to achieve nearly 90% of the maximum CL signal, suggesting that the available luminol could not bind to all active sites. Furthermore, the concentration of NaOH negatively affected the CL signal. The laccase-luminol system represents an alternative to existing CL systems, with potential uses in molecular detection and quantification.

Bacillus velezensis 83 increases productivity and quality of tomato (Solanum lycopersicum L.): Pre and postharvest assessment.

Bacillus spp. are well known plant growth promoting bacteria (PGPB) and biological control agents (BCA) due to their capacity to synthesize a wide variety of phytostimulant and antimicrobial compounds. B. velezensis 83 is a strain marketed in Mexico as a foliar biofungicide (Fungifree AB™) which has been used for biological control of five different genera of phytopathogenic fungi (Colletotrichum, Erysiphe, Botrytis, Sphaerotheca, Leveillula) in crops of agricultural importance such as mango, avocado, papaya, citrus, tomato, strawberry, blueberry, blackberry and cucurbits, among others. In this work, the potential of plant growth promotion of B. velezensis 83 was evaluated on different phenological stages of tomato plants as well as the biocontrol efficacy of B. velezensis 83 formulations (cells and/or metabolites) against B. cinerea infection on leaves and postharvest fruits. Greenhouse grown tomato plants inoculated with a high concentration (1 × 108 CFU/plant) of B. velezensis 83 yielded 254 tons/Ha•year of which the 64% was first quality tomato (≥100 g/fruit), while the control plants produced less than 184 tons/Ha•year with only 55% of first quality tomato. Additionally, in vitro assays carried out with leaves and fruits, shown that the B. velezensis 83 cells formulation had an efficacy of control of B. cinerea infection of ∼31% on leaves and ∼89% on fruits, while the metabolites formulation had an efficacy of control of less than 10%. Therefore, it was concluded that spores (not the metabolites) are the main antagonism factor of Fungifree AB™. The high effectivity of B. cinerea control on fruits by B. velezensis 83, opens the possibility for a postharvest use of this biofungicide.

Bacillus velezensis 83 a bacterial strain from mango phyllosphere, useful for biological control and plant growth promotion.

Bacillus velezensis 83 was isolated from mango tree phyllosphere of orchards located in El Rosario, Sinaloa, México. The assessment of this strain as BCA (biological control agent), as well as PGPB (plant growth-promoting bacteria), were demonstrated through in vivo and in vitro assays. In vivo assays showed that B. velezensis 83 was able to control anthracnose (Kent mangoes) as efficiently as chemical treatment with Captan 50 PH™ or Cupravit hidro™. The inoculation of B. velezensis 83 to the roots of maize seedlings yielded an increase of 12% in height and 45% of root biomass, as compared with uninoculated seedlings. In vitro co-culture assays showed that B. velezensis 83 promoted Arabidopsis thaliana growth (root and shoot biomass) while, under the same experimental conditions, B. velezensis FZB42 (reference strain) had a suppressive effect on plant growth. In order to characterize the isolated strain, the complete genome sequence of B. velezensis 83 is reported. Its circular genome consists of 3,997,902 bp coding to 3949 predicted genes. The assembly and annotation of this genome revealed gene clusters related with plant-bacteria interaction and sporulation, as well as ten secondary metabolites biosynthetic gene clusters implicated in the biological control of phytopathogens. Despite the high genomic identity (> 98%) between B. velezensis 83 and B. velezensis FZB42, they are phenotypically different. Indeed, in vitro production of compounds such as surfactin and bacillomycin D (biocontrol activity) and γ-PGA (biofilm component) is significantly different between both strains.

Efficient removal of azo-dye Orange II by fungal biomass absorption and laccase enzymatic treatment.

In this study, the exact contribution of T. versicolor fungal biomass and laccase in the removal of the Orange II dye from liquid culture was determined. Biomass and laccase were produced with three different carbon sources [bran flakes (BF), wheat bran (WB) and wheat flour (WF)]. The contribution of the biomass and the laccase enzyme in the removal of the Orange II dye was assessed as follows: (A) in vivo treatment with fungal biomass; in vivo treatment with fungal biomass and inhibited laccase (using 0.6 mM sodium azide); and (B) in vitro treatment with crude laccase. The results of fungal biomass production were similar for all the carbon sources evaluated, while laccase volumetric activities were different. The highest enzyme production was obtained with WB, followed by BF and WF. In the in vivo treatment with fungal biomass-laccase, dye removal was over 84% for all the carbon sources. Dye adsorption by fungal biomass varied from 1.5-2%, presenting enzymatic activities ranging from 62 to 163 U L-1. In the in vivo treatment with fungal biomass-inhibited laccase, the removal of the dye varied from 30 to 72%. In this case, the percentage of dye adsorption by fungal biomass was significantly increased and ranged from 18 to 53%. In the in vitro treatment with laccase, the removal ranged from 80 to 84%. The best treatment for dye removal involved the use of both fungal biomass and laccase. The carbon source for biomass and laccase production had an impact on dye removal.

Improving the heterologous expression of human ß-defensin 2 (HBD2) using an experimental design.

At present, expressing antimicrobial peptides in bacterial models is considered a routine lab bench work. However, low expression yields of these types of proteins are usually obtained. In this work, the antimicrobial peptide human ß-defensin 2 (HBD2) was obtained in low expression yields in Escherichia coli BL21(DE3). To improve the expression yields of HBD2, some variables such as cell density, temperature, and length of induction, as well as the inducer concentration, were investigated using a 24-factorial design of experiments (DoE). This approach allowed us to identify the identification of critical variables (main effects and interactions among factors) affecting bacterial HBD2 expression. After the evaluation of 19 different combination, the best condition to express HBD2 had a pre-induction temperature of 37 °C, a cell density of 1.0 U (600 nm), an induction temperature of 20 °C and a 0.1 mM of gene expression inducer (IPTG) over four hours. Under such conditions, the expression yield of the HBD2 peptide was one order of magnitude higher than the peptide expression performed initially.

Glucose limitation and glucose uptake rate determines metabolite production and sporulation in high cell density continuous cultures of Bacillus amyloliquefaciens 83.

Bacillus amyloliquefaciens spores have been used as the principal ingredient of biocontrol products. However, during the process of spore production, wild-type strains produce poly-γ-glutamic acid (γ-PGA), an undesirable byproduct that increases broth viscosity and hinders recovery and drying. This work examined the influence of specific glucose uptake rates (qGluc) in glucose-controlled overflow metabolism. Diverse scenarios, from glucose limitation to glucose sufficiency, were evaluated in continuous cultures to control qGluc. Cell yields of glucose were higher at low qGluc, while the opposing trend was found for γ-PGA and other overflow metabolic byproducts yields. However, γ-PGA production was still detectable in cultures with the highest glucose limitation (D = 0.06 h-1), even though high sporulation incidence was observed in these cultures. Indeed, in such conditions, nonsporulating vegetative cells seem to maintain glucose overflow metabolism, allowing limited γ-PGA production. These findings can be used to establish fed-batch culture strategies for high cell density Bacillus amyloliquefaciens cultures where γ-PGA production (and apparent viscosity) is significantly reduced. This is the first time that the dependence of qGluc on growth, sporulation and carbon overflow metabolism of a spore and biofilm producer, Bacillus amyloliquefaciens strain, has been reported.

Improved production, purification, and characterization of biosurfactants produced by Serratia marcescens SM3 and its isogenic SMRG-5 strain.

In this study, the biosurfactants (Bs) production of two Serratia marcescens strains (SM3 and its isogenic SMRG-5 strain) was improved and the tenso-active agents were purified and characterized. A 23 factorial design was used to evaluate the effect of nitrogen and carbon sources on the surface tension (ST) reduction and emulsion index (EI24 ) of the produced Bs. Optimum Bs production by SM3 was achieved at high concentrations of carbon and nitrogen, reducing ST to 26.5 ± 0.28 dynes/cm, with an EI24 of 79.9 ± 0.2%. Meanwhile, the best results for SMRG-5 were obtained at low concentrations, reducing the ST to 25.2 ± 0.2 dynes/cm, with an EI24 of 89.7 ± 0.28%. The optimal conditions for Bs production were scaled up in a 2-L reactor, yielding 4.8 and 5.2 g/L for SM3 and SMRG-5, respectively. Gas Chromatography-Mass Spectrometry (GC-MS) analysis revealed the presence of two different lipopeptides (hidrofobic fractions: octadecanoic and hexadecanoic acid for SM3 and SMRG5, respectively). Both strains were capable of benzo [a] pyrene removal (59% after 72 H of culture).

Diffusional and transcriptional mechanisms involved in laccases production by Pleurotus ostreatus CP50.

The independent effects of hydrodynamic stress (assessed as the Energy Dissipation/Circulation Function, EDCF) and dissolved oxygen tension (DOT) on the growth, morphology and laccase production by Pleurotus ostreatus CP50 were studied using a 3(2) factorial design in a 10L reactor. A bell-shape function for fungus growth between 8 and 22% DOT was observed, as well as a significant negative effect on laccase production and the expression of poxc, the gene encoding for the most abundant laccase produced by P. ostreatus CP50. Increasing EDCF from 1 to 21 kW/m(3)s, had a positive effect on fungus growth, whereas no effect on poxc gene expression was observed. However, the increase in EDCF favored the specific laccase production due to the generation of smaller pellets with less diffusional limitations and increased metabolically active biomass. The results show, for the first time, that hydrodynamic effects on growth and laccase production are mainly physical and diffusional, while the influence of the dissolved oxygen is at transcriptional level.

Impact of Meyerozyma guilliermondii isolated from chickens against Eimeria sp. protozoan, an in vitro analysis.

BACKGROUND: Avian coccidiosis is a disease caused worldwide by several species of parasite Eimeria that causes significant economic losses. This disease affects chickens development and production, that most of times is controlled with anticoccidial drugs. Although efforts have been made to address this disease, they have been made to control Eimeria sporozoites, although enteric stages are often vulnerable, however; the parasite oocyst remains a problem that must be controlled, as it has a resistant structure that facilitates dispersion. Despite some commercial products based on chemical compounds have been developed as disinfectants that destroy oocysts, the solution of the problem remains to be solved. RESULTS: In this work, we assessed in vitro anticoccidial activity of a compound(s) secreted by yeast isolated in oocysts suspension from infected chickens. The yeast was molecularly identified as Meyerozyma guilliermondii, and its anticoccidial activity against Eimeria tenella oocysts was assessed. Here, we report the damage to oocysts walls caused by M. guilliermondii culture, supernatant, supernatant extract and intracellular proteins. In all cases, a significant decreased of oocysts was observed. CONCLUSIONS: The yeast Meyerozyma guilliermondii secretes a compound with anticoccidial activity and also has a compound of protein nature that damages the resistant structure of oocyst, showing the potential of this yeast and its products as a feasible method of coccidiosis control.

Toward an understanding of the effects of agitation and aeration on growth and laccases production by Pleurotus ostreatus.

Mycelial growth and laccase production by Pleurotus ostreatus CP50 cultured in a 10-L mechanically agitated bioreactor were assessed through a 2(3) factorial experimental design. The main effects and interactions of three factors (agitation, aeration and copper induction) over five responses (µ, αLacc, ßLacc, maximal volumetric laccase activity and maximal biomass concentration) were analyzed. P. ostreatus growth was significantly improved when culturing was conducted with high agitation (5.9kW/m(3)s) and aeration flow (0.5vvm) rates. Under the experimental conditions evaluated, no evidence of hydrodynamic stress affecting fungal growth was observed. However, the high agitation and aeration conditions were detrimental for the growth-associated laccase production constant (αLacc), leading to a very complex optimization of the process. The maximal laccase volumetric activity (1.2 and 3.8U/ml for non-induced and copper-induced cultures, respectively) was observed when the culturing was performed at a low agitation rate (0.9kW/m(3)s) and a high aeration flow rate (0.5vvm). Laccase proteolysis may explain the complex interactions observed between agitation and aeration and the effects of these factors on the laccase volumetric activity observed in the cultures.

Induction of laccases in Trametes versicolor by aqueous wood extracts.

The induction of laccase isoforms in Trametes versicolor HEMIM-9 by aqueous extracts (AE) from softwood and hardwood was studied. Samples of sawdust of Pinus sp., Cedrela sp., and Quercus sp. were boiled in water to obtain AE. Different volumes of each AE were added to fungal cultures to determine the amount of AE needed for the induction experiments. Laccase activity was assayed every 24 h for 15 days. The addition of each AE (50 to 150 µl) to the fungal cultures increased laccase production compared to the control (0.42 ± 0.01 U ml(-1)). The highest laccase activities detected were 1.92 ± 0.15 U ml(-1) (pine), 1.87 ± 0.26 U ml(-1) (cedar), and 1.56 ± 0.34 U ml(-1) (oak); laccase productivities were also significantly increased. Larger volumes of any AE inhibited mycelial growth. Electrophoretic analysis revealed two laccase bands (lcc1 and lcc2) for all the treatments. However, when lcc2 was analyzed by isoelectric focusing, inducer-dependent isoform patterns composed of three (pine AE), four (oak AE), and six laccase bands (cedar AE) were observed. Thus, AE from softwood and hardwood had induction effects in T. versicolor HEMIM-9, as indicated by the increase in laccase activity and different isoform patterns. All of the enzymatic extracts were able to decolorize the dye Orange II. Dye decolorization was mainly influenced by pH. The optimum pH for decolorization was pH 5 (85%), followed by pH 7 (50%) and pH 3 (15%). No significant differences in the dye decolorizing capacity were detected between the control and the differentially induced laccase extracts (oak, pine and cedar). This could be due to the catalytic activities of isoforms with pI 5.4 and 5.8, which were detected under all induction conditions.

Nitrogen limitation in Neochloris oleoabundans: a reassessment of its effect on cell growth and biochemical composition.

The aim of this work was to reassess the effect of nitrogen limitation (from 0 to 1 mM nitrate), on the growth and the biochemical composition of Neochloris oleoabundans cultures, where only the CO2 available in the air was provided. Slight differences in the initial nitrate concentration, even minimal increments of 0.2 mM, significantly modify the microalgal response towards nitrogen limitation. This stress condition reduced cell proliferation, but increased cell mass values due to the simultaneous accumulation of two storage compounds: lipids, which contained up to a 55.9 % of total fatty acids; and carbohydrates, which may be primarily composed by starch. The highest biomass and lipid productivities of 98.24 and 43.24 mg/l/day, respectively, were attained at an initial nitrate concentration of 0.6 mM. The theoretical annual projection, based on these productivities, allowed the estimation of the liquid fuel energy yields, which are comparable or even higher than those calculated for several biomass feedstocks such as corn, oil palm, sugarcane, or even fast growing grasses, confirming the potential of nitrogen-limited N. oleoabundans biomass as an appropriate feedstock for biofuel purposes.

The challenges of introducing a new biofungicide to the market: a case study

Background: Scientific literature contains many reports documenting the isolation and antagonist-testing of microorganisms with the potential for biological control in agriculture, however, very few have addressed all aspects involved in the long process that occurs before a potential strain, found and tested in laboratory, can reach commercialization. Results: After a multi-institutional and multi-disciplinary effort to develop a biological control agent with remarkable technical characteristics and performance, the fungicide Fungifree AB® was recently introduced in Mexican market. This product that has no chemical residues and is environmentally friendly which can be used by mango, avocado, papaya and citrus fruits (and other crops in the short term) producers to increase crops quality, and therefore, to access world markets. Conclusions: The successful introduction of Fungifree AB® in the Mexican market has been the result of a wide variety of factors: remarkable product technical characteristics, researchers' high scientific level, mango producers and exporters' interest in testing the product and commercial companies interested in its distribution.

Increasing Pleurotus ostreatus laccase production by culture medium optimization and copper/lignin synergistic induction.

Laccases have great biotechnological potential in diverse industries as they catalyze the oxidation of a broad variety of chemical compounds. Production of laccases by basidiomycetes has been broadly studied as they secrete the enzymes, grow on cheap substrates, and they generally produce more than one isoenzyme (constitutive and/or inducible). Laccase production and isoenzyme profile can be modified through medium composition and the use of inducers. The objective of this work was to increase laccase production by Pleurotus ostreatus CP-50 through culture medium optimization and the simultaneous use of copper and lignin as inducers. Increased fungal growth was obtained through the use of a factorial fractional experimental design 26⁻² where the influence of the nature and concentration of carbon and nitrogen sources was assessed. Although specific laccase production (U/mg biomass) decreased when malt extract medium was supplemented with carbon and nitrogen sources, fungal growth and laccase volumetric activity increased four and sixfold, respectively. The effect of media supplementation with copper and/or lignin on laccase production by P. ostreatus CP-50 was studied. A positive synergistic effect between copper and lignin was observed on laccase production. Overall, the use of an optimized medium and the simultaneous addition of copper and lignin improved growth, laccase volumetric activity, and process productivity by 4-, 60-, and 10-fold, respectively.

Análisis digital de imágenes para la caracterización microscópica de parámetros críticos en la producción fermentativa de metabolitos secundarios / Digital image analysis for microscope characterisation of the critical parameters involved in the fermentative production of secondary metabolites

Los metabolitos secundarios, y particularmente los antibioticos, se encuentran entre el grupo de farmacos de mayor relevancia en el mercado mundial, y son producidos, en su mayoria, mediante el cultivo sumergido de hongos filamentosos. Desde el punto de vista hidrodinamico, estos procesos involucran fundamentalmente la dispersion de hasta cuatro fases diferentes: agua-aceite-aire-hongo. Aun cuando se sabe que la hidrodinamica del cultivo determina la eficiencia global del proceso de fermentacion, poco se ha logrado en cuanto al entendimiento de las relaciones hidrodinamica-dispersion-fisiologia-productividad. Esto se debe en parte a la falta de metodologias que permitan cuantificar y caracterizar con precision tanto la viabilidad del hongo (ya que en buena medida determina el rendimiento y la productividad del metabolito de interes), como el tamaño de las burbujas de gas y de gotas de aceite (ya que de ello depende la eficiencia de transporte de nutrientes como el oxigeno y los acidos grasos del aceite). En este trabajo se resume y revisa el desarrollo de las metodologias que nuestro grupo ha publicado, basadas en el procesamiento y el analisis digital de imagenes aplicadas al estudio del cultivo de Trichoderma harzianum, hongo que produce antimicoticos de alta potencia (como la 6-pentil-alfa-pirona). Con el uso de estas metodologias hemos generado conocimiento basico de los fenomenos que ocurren en el fermentador, lo que nos ha permitido establecer estrategias para incrementar la productividad de este tipo de procesos.

The influence of circulation frequency on fungal morphology: a case study considering Kolmogorov microscale in constant specific energy dissipation rate cultures of Trichoderma harzianum.

The energy dissipation/circulation function (EDCF) is the product of the specific energy dissipation rate in the impeller swept volume (P/kD(3)) and the frequency of particle circulation (1/t(c)) through that volume. A direct relationship between mycelial fragmentation and EDCF has been reported. However, and although hyphal fragmentation is assumed to occur by hyphae-eddy interaction, Kolmogorov microscale (lambda) has not been shown to determine, at least directly, fungal morphology. In this work we studied the influence of lambda and EDCF evolution, as well as the individual effects of P/kD(3) and 1/t(c), on Trichoderma harzianum cultures in an attempt to elucidate the mechanistic interactions between parameters. T. harzianum cultures, conducted at equivalent yielding P/kD(3) conditions, were developed using two different Rushton turbines diameter sets. For the studied conditions, 1/t(c) had a greater effect over mycelial clump size and growth rate than P/kD(3). Consequently, broth viscosity, and hence Kolmogorov microscale, was a function of impeller diameter, even among cultures operated at equivalent specific energy dissipation rates. The latter could partially explain why Kolmogorv's theory has not been able to fully correlate morphological data, and highlights the importance of 1/t(c) on fungal bioprocesses. A theoretical approach to monitor lambda in large-scale bioreactors is proposed.

Direct comparison between ion-exchange chromatography and aqueous two-phase processes for the partial purification of penicillin acylase produced by E. coli.

A direct comparison of a chromatography and an aqueous two-phase system (ATPS) processes for the partial purification of penicillin acylase (PA) produced by a recombinant strain of E. coli, was performed. An established chromatography process for the recovery of PA was selected as a model system and characterised for comparison with a developed ATPS prototype process. PEG-phosphate systems were selected for the recovery of PA over PEG-citrate systems, since higher enzyme recovery and increased purity was obtained. ATPS proved to be suitable to process highly concentrated disrupted extract (35%, w/w) and maintain a high top phase enzyme recovery. In the direct comparison of the processes, the superiority of the ATPS approach was highlighted since a reduction of the number of unit operations from 7 to 4 was achieved. An outline economic analysis based on the cost of separation agent of the processes favour the ATPS process, in which a gross cost reduction of 37% (from 0.47 to 0.30 US dollars) was achieved. Such result was obtained considering a potential re-use of up to 100 times of the resin used in the chromatography process. Additionally, by assuming that all the unit operations are equivalent in investment and operating cost, further reduction of approximately 43% of the respectively involved cost can be obtained when the ATPS process is used. Overall, the proposed ATPS process comprising of PEG1450-phosphate, tie-line length (TLL) of 48.5% (w/w), volume ratio (Vr) of 1.0, pH of 7.0 and 35% (w/w) PA crude extract loaded into the system proved to be more efficient, recovering 97% of PA at the top phase (PEG rich phase) with a purity factor of 3.5. It is clear that the results reported herein raise the consideration for the potential substitution of the chromatography process for PA recovery from E. coli as a first step for the development of an optimised and economic process with evident commercial application.

6-pentyl-alpha-pyrone production by Trichoderma harzianum: the influence of energy dissipation rate and its implications on fungal physiology.

The influence of the agitation conditions on biomass growth, morphology, carbon metabolism, viability, and 6-pentyl-alpha-pyrone (6PP) production by Trichoderma harzianum were studied in an extractive fermentation system. Batch spore-inoculated cultures developed at dissolved oxygen concentrations above 35% of air saturation were carried out in a 14 L bioreactor. The effect of energy dissipation rate over culture performance was assessed using two sets of three Rushton turbines (having different diameters) operated at different agitation speeds. Higher mechanical stress enhanced cellular differentiation (i.e., sporulation), while yielding lower specific growth rates and increased specific CO(2) production rates (CPRs) at relatively constant specific glucose consumption rates. In addition, fungal viability and clump mean diameter decreased gradually at higher energy dissipation rates. 6PP biosynthesis was growth associated and its specific productivity showed a bell-shaped relationship with the energy dissipation rate. T. harzianum physiology was, therefore, strongly influenced by the prevailing hydrodynamic conditions as it triggered cellular metabolism and differentiation shifts.

Rhizoctonia solani, an elicitor of 6-pentyl-alpha-pyrone production by Trichoderma harzianum in a two liquid phases, extractive fermentation system.

6-pentyl-alpha-pyrone (6PP) production by Trichoderma harzianum, in an extractive fermentation system, was elicitated by Rhizoctonia solani. The extent of 6PP elicitation was related to the state of Rhizoctonia and to the Trichoderma inoculum type. The use of non-viable Rhizoctonia solani mycelium in mycelium-inoculated Trichoderma harzianum culture, yielded the maximal 6PP production (474 mg l(-1)) compared to control cultures (147 mg l(-1)) and decreased the process time from 192 to 96 h.