Modelling in-vitro growth of filamentous fungi in phenanthrene presence.

ROMERO M.C.; URRITIA M.I.; REYNALDI F. J.; REINOSO E. H.

Mar del Plata

Congreso; IV Encuentro Iberoamericano de Biometrìa y XVIII Reuniòn Científica del grupo Argentino de Biometría; 2013

Grupo Argentino de Biometría

ABSTRACT Cellular aggregation and pellets formation in Fusarium solani cultures were increased by polycyclic aromatic hydrocarbon presence, as sole carbon source. In phenanthrene flasks, two pellet sub-populations were found; moreover, dissolved oxygen levels as well as phenanthrene determined the pellet stability. The hyphal growth and the biofloculants excretion incremented the cell wall hydrophobic. Hyphae brought together by adhesive forces stimulated by phenanthrene particles, us this compound is un adverse factorthat required enzymatic adaptation. A mathematical model was developed based on the rheology properties to predict the size of the pellets formed in fungal cultures in the presence of aromatic hydrocarbons. Further the model was validated by studying the effect of various parameters on the cellular aggregation and pellet size such as viscosity, surface/interfacial tension, density difference between pellet forming and cultural components. Cellular aggregation and pellets were prepared in assays with different phenanthrene levels. The surface/interfacial tension, density difference between the aggregates and column of the liquid-culture were found to be the most important factors affecting pellet size. The mathematical model developed was found to successfully predict the cellular aggregation that led to pellets, with an average error of 3.32% for the different tested models. Keywords: cellular aggregation, filamentous fungi, pellets, phenanthrene, mathematical models, morphology Introduction Control of mycelial morphology is often a prerequisite for industrial application, as in the case of Penicillium chrysogenum and Aspergillus niger to produce penicillin and citric acid, respectively. The factors influencing cellular aggregation are inoculum size, age, bioflocculants, polymers biosysthesis, surfactants and chelators presence, temperature, medium composition and viscosity. It is difficult to define the mechanism for pellets formation, and often more than one parameter is the adjusted variable. A number of process parameters, such as inoculums conditions, growth rate, medium composition, dissolved oxygen level in the assay and mixing intensity affect cell morphology and consequently have a profound influence on the culture rheology, which, was responsible for mass, heat and other parameters variations during in-vitro culture. Mixing and aeration supplied oxygen to the fungi and removed carbon dioxide, and cooling, which leads away the metabolic heat, having a strong influence on the productivity and efficiency of the entire process [1,2]. Different mathematical models expressed the behaviour of the fungal growth depending on the factors that dominated the culture, most of them showed Newtonian behaviour, but others responded to the Power, Bingham or Casson model [3,4]. Materials and methods Filamentous fungi were isolated from sediments of the YPF-Refinery effluents, La Plata, Argentina, in a agar-mineral medium with phenanthrene as sole carbon source. The fungi was cultured in the same liquid medium, to evaluate the mycelial pellet formation with and without (control flask) phenanthrene. Pellet size was analyzed by sieving mycelial flocs, bore sizes 0.2, 0.5, 0.8, 1.0, 1.4, 1.6, 2.0 µm, and bed volume of each fraction was measured. Results and discussion Fusarium solani formed spherical pellets of varying sizes in phenanthere presence. At low inoculum sizes (106 spores/ m3), most of the pellets were larger than 2mm, the cellular yield was 0.3-0.6 g dry wt/ml. In contrast, with higher inoculums the fungi yield was around a constant value c.a. 1.6 g dry wt/m). So, the relation of the inoculums size with the mass yiels responded to an hyperbolic curve. Average pellet sizes fitted to the relation: S av =  Si (Vi /  Vi ), where Si was the arithmetical mean of bore size (upper to lower sieve, µm), Vi was the volume of settle bed of lets minus voidage. The pellet numbers, N, was obtained from: N = Vi / vi , where vi was the volume of sphere whose diameter was equal to Si. Cellular flocculation was evaluated by measuring the time required to reduce to a half of the initial turbidity of mycelial suspension. The surfactant effects were evaluated by adding them to cell suspension, in the cuvette previous to turbidity measurements. This phenomenon was related to poor growth rate due to oxygen limitation inside large pellets and to the hydrocarbon presence. At 1010 spores/m3 in shake flasks and 1012 spores/ m3 in nonshaked flasks, small unstable pellets were formed during the 2nd day culture, that disappeared later. With the increase in inoculum level, pulp growth was the prevalent development. The average pellet size seemed to be inversely proportional to the inoculum sizes; however, it appeared that within large inoculum ranges the average pellet size reached a plateau, remaining close to 1 µm. Analysis of pellet size distributions showed that this was a result of bimodal relation. In phenanthrene flasks, two pellet sub-populations was found, one either large to  2.5 µm or small to  0.4 µm, and a symmetric one with median pellet size of 1.4 µm. On the contrary, in the control flasks, only one population was observed with average pellet size = 1.0 µm. The ability of hyphae to form pellets and the pellet numbers were determined by the cultural conditions. At harvest time of each pellet was 10 times higher than the pellet yields/per spore at the same inoculation size, but the distribution was different, as one populations was obtained with 0.7 µm as dominant size. The smaller sizes and larger pellet units may be explained by phenanthrene presence, dissolved oxygen levels (DO), as the stability of the pellets was limited by both factors. The OD limitation determined that cells seemed to adapt by forming smaller flocs, resulting greater surface area per unit cell volume, and hence in increased oxygen transfer to cells. Moreover, an increment in growth rate was correlated not only with the decreased in pellet size, but also with the hydrophobicity of cell walls, as expressed in contact angles of cells with water. Different conditions yielding smaller pellets reducing the cell wall hydrophobicity; thus contact angles demonstrated that larger pellets were more hydrophobic. The hydrophobicity depended upon soluble factors, since washed cells were considerable less hydrophobic [7,8]. The addition of surfactants induced larger pellets formation; with Pluronic-F68 the correlation between the size and hydrophobicity was obtained after the removal of F68. These results suggested that pellet size might be regulated by release of bioflocculants in the hydrocarbon presence that modified the cell wall hydrophobicity. Under oxygen limitation their release was reduced and smaller pellets resulted. In conclusion, the cell age did not influence the aggregation; washing and pressure lessened the surfactant effect. Regardless of pellet initiation, continuation of pellet formation can not be explained by mechanical factors. Hyphae brought together by adhesive forces determined by their surface properties due to the presence of phenanthrene particles. Thus, hydrophobic exclusion may be the driving force of hyphal interaction and pellet formation in phenanthrene cultures of Fusarium solani. Bibliography [1] Cheboyina S, O'Haver J, Wyandt CM. (2006). A mathematical model to predict the size of the pellets formed in freeze pelletization techniques: parameters affecting pellet size. J. Pharm Sci.95 (1): 167-180. 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