ABSTRACT
Abstract Production of lignocellulolytic enzymes by filamentous fungi have a great potential at industrial level due to their widespread applications. Mixed fungal cultures and particularly mixed fungal biofilms constitute a promising fermentation system for an enhanced enzyme production. However, it has not been addressed how much of this enhancement depends on the mixed biomass proportion. In this sense, the aim of this study was to develop a method to specifically and accurately quantify mixed fungal biomass. For this purpose, mixed biofilm cultures composed of Aspergillus niger and Trichoderma reesei, two filamentous fungi used industrially for cellulase production, were collected from 48 to 120 h of growth; mycelia were pulverized, and DNA was extracted for qPCR assays with specific primers for each fungus. Primers were designed from non-conserved regions of sequences of actin and β-tubulin genes of both A. niger and T. reesei. Specificity of these primers was tested in silico and experimentally. A statistically significant correlation was obtained between qPCR-calculated biomass and dry weight biomass data. By this method, it was possible to detect changes on mycelia proportions in biofilms over time, suggesting a competitive interaction between these two fungi. In conclusion, this method allows a specific and accurate quantification of mixed fungal biomass and could be also applied to different mixed culture systems for studying microbial interactions.
Resumen La producción de enzimas lignocelulolíticas por hongos filamentosos tiene un gran potencial a nivel industrial debido a sus diversas aplicaciones. Los cultivos fúngicos mixtos y particularmente las biopelículas fúngicas mixtas constituyen un sistema de fermentación prometedor para una mayor producción enzimática. Sin embargo, no se ha abordado cuánto de esta mejora depende de la proporción de biomasa mixta. En este sentido, el objetivo de este estudio fue desarrollar un método para cuantificar de forma específica y precisa la biomasa fúngica mixta. Para este propósito, se recolectaron cultivos mixtos de biopelículas de 48 a 120 h de crecimiento compuestos por Aspergillus niger y Trichoderma reesei, dos hongos filamentosos utilizados industrialmente para la producción de celulasas; el micelio se pulverizó y el ADN se extrajo para ensayos de qPCR con cebadores específicos para cada hongo. Los cebadores se diseñaron a partir de regiones no conservadas de las secuencias de los genes de actina y β-tubulina de A. niger y T. reesei. La especificidad de estos cebadores se probó in silico y experimentalmente. Se obtuvo una correlación estadísticamente significativa entre la biomasa calculada mediante qPCR y los datos de biomasa en peso seco. Mediante este método, fue posible detectar cambios en las proporciones de los micelios en las biopelículas a lo largo del tiempo, lo que sugiere una interacción competitiva entre estos dos hongos. En conclusión, este método permite una cuantificación específica y precisa de la biomasa fúngica mixta y también podría aplicarse a diferentes sistemas de cultivo mixto para estudiar interacciones microbianas.
ABSTRACT
Alkaline cellulases are demanded by the textile industry for several purposes but commercial preparations showing activity at alkaline conditions are very scarce. Aim: To characterize a Penicillium strain isolated form soils of a Peruvian rainforest showing alkaline cellulase activity that may be useful for the textile industry. Methodology: The molecular identification was based on the DNA sequence of its ITS region using ITS1 and ITS4 primers after PCR amplification. Cellulase production was evaluated in shaken flasks by using either lactose or microcrystalline cellulose. Total cellulase (as FPA) and endoglucanase activities were evaluated by the standard methods at several pH levels. Also, the cellulase activity of culture filtrates was tested for antipilling activity as compared to a commercial neutral cellulase preparation. Results: After raw data of ITS DNA sequence was processed, multiple alignment and phylogenetic analysis confirmed that our strain can be named as Penicillium mallochii LMB-HP37. Higher activity was attained for neutral total cellulase on lactose (3371±108 U/l at pH 7.4) and alkaline cellulases attained similar activity levels than the acid cellulase (2978±151 U/l at pH 8.4 and 2910±42 U/l at pH 9.4). FPA and endoglucanase activities were produced at high volumetric (46.8±1.5 and 13.5±1.0 U/l.h, respectively) and specific (32.9±1.1 and 9.5±0.7 U/gbiomass.h, respectively) productivities at the same pHs which indicate that this strain may be suitable for commercial development. The enzyme of P. mallochii LMB-HP37 had slightly better results than the commercial enzyme as an anti-pilling agent even though is a crude preparation. Conclusion: Penicillium mallochii LMB-HP37 produced high total cellulase activity on lactose which compares to well-known cellulase producers but at neutral to alkaline pH levels. Data obtained reveal that the crude enzyme is suitable for anti-pilling process (biopolishing) and may be also useful for biostoning.