Hydrodynamics, Fungal Physiology, and Morphology.

Filamentous cultures, such as fungi and actinomycetes, contribute substantially to the pharmaceutical industry and to enzyme production, with an annual market of about 6 billion dollars. In mechanically stirred reactors, most frequently used in fermentation industry, microbial growth and metabolite productivity depend on complex interactions between hydrodynamics, oxygen transfer, and mycelial morphology. The dissipation of energy through mechanically stirring devices, either flasks or tanks, impacts both microbial growth through shearing forces on the cells and the transfer of mass and energy, improving the contact between phases (i.e., air bubbles and microorganisms) but also causing damage to the cells at high energy dissipation rates. Mechanical-induced signaling in the cells triggers the molecular responses to shear stress; however, the complete mechanism is not known. Volumetric power input and, more importantly, the energy dissipation/circulation function are the main parameters determining mycelial size, a phenomenon that can be explained by the interaction of mycelial aggregates and Kolmogorov eddies. The use of microparticles in fungal cultures is also a strategy to increase process productivity and reproducibility by controlling fungal morphology. In order to rigorously study the effects of hydrodynamics on the physiology of fungal microorganisms, it is necessary to rule out the possible associated effects of dissolved oxygen, something which has been reported scarcely. At the other hand, the processes of phase dispersion (including the suspended solid that is the filamentous biomass) are crucial in order to get an integral knowledge about biological and physicochemical interactions within the bioreactor. Digital image analysis is a powerful tool for getting relevant information in order to establish the mechanisms of mass transfer as well as to evaluate the viability of the mycelia. This review focuses on (a) the main characteristics of the two most common morphologies exhibited by filamentous microorganisms; (b) how hydrodynamic conditions affect morphology and physiology in filamentous cultures; and (c) techniques using digital image analysis to characterize the viability of filamentous microorganisms and mass transfer in multiphase dispersions. Representative case studies of fungi (Trichoderma harzianum and Pleurotus ostreatus) exhibiting different typical morphologies (disperse mycelia and pellets) are discussed.

Production of laccases by Pleurotus ostreatus in submerged fermentation in co-culture with Trichoderma viride.

AIMS: To evaluate the production and stability of laccases by Pleurotus ostreatus in liquid co-cultures with Trichoderma viride as a function of infection time and agitation rate. METHODS AND RESULTS: Pleurotus ostreatus cultures were infected with T. viride spores at 30 and 48 h. Maximal laccase volumetric activity was seen after 48 h (control cultures) or 72 h (co-cultures) of cultivation time. Only the cultures infected at 30 h showed an increased laccase volumetric activity compared to control cultures. After maximal laccase volumetric activity value was reached, a sharp decrease in it was observed in control cultures. Co-cultures exhibited a comparatively lower loss of activity. The influence of P. ostreatus and/or T. viride on the stability of laccase volumetric activity and isoenzyme pattern was evaluated. Trichoderma viride induced changes in the laccase isoenzyme pattern. Agitated cultures increased biomass growth and specific productivity threefold and sevenfold, respectively, to the static cultures. CONCLUSIONS: The laccase volumetric activity is very likely the result of the balance between biosynthesis and degradation/biotransformation rates occurring during the cultures. The individual presence of P. ostreatus or T. viride in the culture negatively affected the volumetric laccase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The evaluation of culture parameters that could influence Trichoderma-basidomycetes interaction and laccase production during submerged fermentation has not been reported. This study showed how laccase production in co-cultures of P. ostreatus and T. viride was influenced by the infection time and agitation/oxygenation conditions.

Selection of Trichoderma strains capable of increasing laccase production by Pleurotus ostreatus and Agaricus bisporus in dual cultures.

AIMS: To select Trichoderma strains for enhanced laccase production in Pleurotus ostreatus or Agaricus bisporus cultures. METHODS AND RESULTS: Laccase production by P. ostreatus and A. bisporus was evaluated in liquid (axenic) and solid (dual cultures) malt extract medium. Oxidation of ABTS, DMP and syringaldazine was evaluated in order to assess the potential of Trichoderma strains to enhance laccase production by basidiomycetes. Selected Pleurotus-Trichoderma interactions yielded higher increases in laccase volumetric activity and an additional laccase isoform was produced. By contrast, Agaricus-Trichoderma interactions lead to smaller increases on laccase volumetric activity, probably as result of repression (or degradation) towards one of the laccases isoforms. CONCLUSIONS: The strains of P. ostreatus and A. bisporus assessed in this work showed good potential as laccase producers. The Trichoderma-mediated biological stimulation of laccase production by P. ostreatus and A. bisporus is relevant in order to develop highly productive processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Extracellular laccases from basidiomycetes are produced only in small amounts. It is therefore important to increase process productivity for potential industrial applications. The results from this study enable the selection Trichoderma strains capable of increasing laccase production by P. ostreatus or A. bisporus in dual cultures.

A fermentation system designed to independently evaluate mixing and/or oxygen tension effects in microbial processes: development, application and performance.

In order to evaluate the independent effects of hydrodynamic conditions and/or oxygen tension on culture physiology and productivity, a fermentation system designed to control dissolved oxygen at constant power drawn (P/V) was developed. The system included a fully instrumented 14 l bioreactor coupled to a PC for data acquisition and control. Power drawn was measured (using a commercial torquemeter coupled to the shaft) and maintained constant by varying the agitation speed; while gas blending was used to control dissolved oxygen concentration. To validate the system, rheological-complex fermentations involving xanthan gum production and filamentous fungal cultivation (using Xanthomonas campestris and Trichoderma harzianum) were developed. In both cases, and despite the changing environmental conditions (due to increased broth viscosities and microbial respiration), both variables were controlled at the desired set points. Through such a system, a rigorous evaluation of the hydrodynamic conditions and/or oxygen tension on culture physiology and productivity is now feasible.

Production and biotransformation of 6-pentyl-alpha-pyrone by Trichoderma harzianum in two-phase culture systems.

The final concentration of 6-pentyl-a-pyrone (6PP) produced in cultures of Trichoderma spp. is limited by the fact that inhibition of biomass growth occurs at 6PP concentrations as low as 100 mg/l. The aim of this work was to evaluate liquid-liquid extractive fermentation systems as an alternative to overcome the toxicity problems and to increase the production of 6PP by this fungus. Two alkanes (n-decane and n-hexadecane) and two dicarboxylic esters (dibutyl phthalate and dioctyl phthalate) were evaluated in shake flask cultures. The highest 6PP production (173 ppm) was achieved when n-hexadecane was used, being 3.5-fold the maximum 6PP concentration of a culture without the solvent. Cultivation of Trichoderma harzianum in a 10-1 bioreactor with n-hexadecane yielded 6PP production ninefold higher than that from control cultures. However, 6PP production in the bioreactor (83 ppm) was lower than in shake flasks. Differences in the power drawn to the fluid at each scale could account for such behavior. Even in the presence of the solvent, 6PP content decreased after reaching its maximal concentration.

The potential application of aqueous two-phase systems for in situ recovery of 6-pentyl-infinity-pyrone produced by Trichoderma harzianum.

Commercial production of aroma compounds by de novo microbial biosynthesis has been principally limited by the low productivity so far achieved. Production of 6-pentyl-alpha-pyrone (6PP), a coconut-like aroma compound, by Trichoderma harzianum has been limited by the toxic effect that occurs even at low concentration (<100 ppm). This work evaluated the feasibility of the use of aqueous-two phase systems (ATPS), as in situ extraction systems, in order to overcome the toxic effects of 6PP and to improve culture productivity. The partition behaviour of 6-pentyl-alpha-pyrone and Trichoderma harzianum mycelium in polyethylene glycol (PEG)-salt and PEG-dextran two-phase systems was investigated and it is reported for the first time. The evaluation of system parameters such as PEG molecular mass, concentration of PEG as well as salt, volume ratio (Vr) and dextran molecular mass, was carried out to determine under which conditions the 6PP partitions to the opposite phase that mycelium does. PEG-dextran systems proved to be unsuitable for the in situ recovery of 6PP because either 6PP and biomass partitioned to the same phase or a large extraction phase was required for the process. ATPS extraction comprising Vr = 0.26, PEG 1450 (7.2% w/w) and sulphate (16.6% w/w) provided the best conditions for the maximum accumulation of the biomass into the bottom phase and concentrated the 6PP in the opposite phase (i.e. 86% of biomass and 56% of 6PP of the total amount loaded from the fermentation extract into the ATPS) for ex situ bioseparation. However, this system caused complete inhibition of the growth of the microorganism during the in situ bioseparation, probably as a consequence of the high ionic strength resulting from the salt concentration. Consequently, two ATPS PEG 8000-sulphate (12%/7% and 6%/14%) were evaluated and proved to be more suitable in the potential application for the in situ recovery of 6PP.

Aroma compounds recovery from mycelial cultures in aqueous two-phase processes.

This paper presents the evaluation of the potential use of aqueous two-phase systems (ATPS) for the recovery of 6-pentyl-alpha-pyrone (6PP) produced by Trichoderma harzianum. The partition behaviour of 6PP and Trichoderma harzianum mycelium (biomass) in polyethylene glycol (PEG)-salt (phosphate and sulphate) and PEG-dextran ATPS was investigated. The influence of defined system parameters (e.g. molecular mass of PEG and dextran, volume ratio, etc.) on the partition behaviour of 6PP and Trichoderma harzianum mycelium was evaluated to select under which conditions 6PP and mycelium partition to opposite phases. In PEG-dextran systems either large extraction phases were required or mycelium and 6PP partitioned to the same phase. ATPS comprising V(r)=0.23, PEG 8000 6.6% w/w and sulphate 14.0% w/w provided the best conditions to satisfy the process requirement of biomass accumulation into the bottom phase and 6PP concentration in the top phase.

The influence of impeller type in pilot scale xanthan fermentations

The rheological complexity of Xanthan fermentations presents an interesting problem from a mixing viewpoint, because the phenomena of poor bulk blending and low oxygen mass transfer rates inherent in highly viscous fermentations (and their consequences) can be systematically investigated, even at the pilot plant scale. This study in a 150 L fermentor compares the physical and biological performance of four pairs of impellers: a standard Rushton turbine, a large diameter Rushton turbine, a Prochem Maxflo T, and a Scaba 6SRGT. Accurate in-fermentor power measurements, essential for the comparison of impellers in relation to operating costs are also reported. It is demonstrated that the agitator performance in Xanthan fermentations is very specific and the choice of which impeller to use in bioreactors to obtain enhanced performance is dependant on the applied criterion. None of the criterion favored the use of the standard Rushton turbine, therefore suggesting that there are strong grounds for retrofitting these impellers with either large diameter impellers of similar design or with novel agitators. In addition, fluid dynamic modeling of cavern formation has clearly highlighted the importance of a well mixed and oxygenated region for providing the capacity for high microbial oxygen uptake rates which govern Xanthan productivity and quality. Copyright 1998 John Wiley & Sons, Inc.

Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-alpha-Pyrone Biosynthesis by Trichoderma Species.

The understanding of the biosynthetic pathway of 6-pentyl-alpha-pyrone in Trichoderma species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of Trichoderma harzianum and T. viride with [U-C]linoleic acid or [5-C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [C]linoleic acid was incorporated into 6-pentyl-alpha-pyrone more rapidly than that from [C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-alpha-pyrone when fungal cultures were incubated with [1-C]linoleic acid. These results suggested that beta-oxidation of linoleic acid was a probable main step in the biosynthetic pathway of 6-pentyl-alpha-pyrone in Trichoderma species.

Quantitative separation of Trichoderma lipid classes on a bonded phase column.

Bond Elut aminopropyl columns were used to purify the different lipid classes of Trichoderma harzianum and Trichoderma viride. This methodology permitted good separation of the fungal lipid classes in less time than traditional techniques. The incorporation of [1 (14)C]linoleic acid into neutral lipids, free fatty acids and phospholipids was quantified for both strains. The fatty acid profile of the different lipid classes of these fungal strains was determined as a function of culture time.