Development of microbial and/or enzymatic systems for the valorization of lignocellulosic wastes

Abstract

The future trend of white biotechnology is the seek of renewable resources for the production of goods, traditionally obtained from petrol. Shifting society’s dependence away from petroleum to renewable biomass resources is generally viewed as an important contribute to the development of a sustainable industrial society and the reduction of greenhouse gas emissions. Among the available resources that are exploitable for biofuel production, there are the residues coming from different human activities such as agriculture (i.e.: cereals straws), food processing-industry, forestry, green and organic fractions of urban wastes. Their exploitation can contribute to the reduction of the price of the bioethanol production process, raw materials being responsible of almost half of the total production cost. A further element contributing to soaring biofuels costs, is constituted by enzymes. Their use roughly doubles the cost of cellulosic ethanol production and lessens the economic advantages of using waste materials. A valide alternative to purified/commercial enzymes may be the direct production of the enzymes of interest on the lignocellulosic material to be converted through its microbial fermentation. At this purpose, a powerful technique is solid state fermentation (SSF). This fermentative technique reproduces conditions really close to the natural environment in which many higher filamentous fungi have evolved. These microorganisms can be exploited both for wastes pretreatment, for the removal of lignin and hemicellulose hydrolysis, and for the production of industrially relevant enzymes (oxidative as well as hydolytic activities) for further applications. As a matter of fact, lignocellulosic wastes may contain significant concentrations of soluble carbohydrates and inducers of enzyme synthesis ensuring efficient production of lignino-cellulolytic enzymes. In the present study the residues from tomato and apple processing were chosen as raw materials. On the other side, as conversion microorganisms, the two white rot fungi Pleurotus ostreatus and Trametes versicolor, were selected. Culture conditions were set up, allowing waste colonization and transformation. This study showed the good potential of tomato pomace as substrate for laccases production by P. ostreatus and T. versicolor SSF, considering that significant enzyme activity levels were achieved without any optimization of culture conditions, neither by nutrient addition nor by O2 enrichment. Furthermore, SSFs on tomato pomace hold enormous potential for protease production, giving activity levels higher than those reported for fungi typically considered as the best protease producers. A process of fungal SSF was developed on apple waste, identifying the parameters allowing fast substrate colonization by both fungi. It was shown that apple pomace induced high levels of xylanases, with P. ostreatus secreting higher levels than T. versicolor. Both P. ostreatus and T. versicolor secreted levels of laccase activities that are lower or comparable with those obtained on tomato pomace. T. versicolor was shown to produce Manganese peroxidase, even if at low levels. On this substrate, low levels of protease activity were obtained, for both microorganisms. Furthermore, both tomato and apple pomace SSFs were shown to be better systems than liquid culture for the production of high laccases levels by P. ostreatus. Moreover, as one of the most significant results of this study, the developed P. ostreatus SSF processes provide the production of two laccase isoforms not detected in any other liquid culture conditions analysed so far. Finally, a strategy for P. ostreatus xylanase enrichment was investigated. The first trials for the identification of xylanolytic enzymes allowed the identification of an α-galactosidase. This enzyme even though not involved in xylan main chain break-down, plays its role in the removal of galactose units from both galactomannans and arabinoxylans, acting as ancillar xylanolytic enzyme.