Niglio, Saverio (2018) Optimization of pretreatment/hydrolysis processes of agro-food wastes to support biorefinery developments. [Tesi di dottorato]

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Abstract

The study carried out during the present Ph.D. program aimed at investigating the use of agro-food processing wastes (AFWs) to produce fermentable sugars and/or value-added bioproducts according to the biorefinery approach. The work was carried out at the Dipartimento di Ingegneria Chimica, dei Materiale e della Produzione Industriale of the Università degli Studi di Napoli ‘Federico II’. The activities were focused on the release of sugars (pentoses and hexoses) from the AFWs investigated through different pretreatment processes, followed by enzymatic hydrolysis step. The fermentation of the produced sugar solutions was also carried out to produce biobutanol and succinic acid. Two AFWs were investigated: coffee silverskin and apple pomace. Coffee silverskin as feedstock for biorefinery applications: Coffee silverskin (CSS) are the residual thin teguments wrapping and protecting the external layer of green coffee beans. It is produced in large amount during the roasting phase of green coffee beans. It is mainly composed of carbohydrates (40% w/w) and lignin (30% w/w). The study of CSS was aimed at investigating the pretreatment, enzymatic hydrolysis and fermentation of this residue. A combination of mild alkaline solution and ultrasound (US) was applied to pretreat CSS. Biomass loading, sonication time, alkali concentration, residence time in autoclave were the parameters investigated and analysed according to a response surface methodology approach. The process was characterized in terms of sugar yield after the enzymatic hydrolysis of pretreated CSS. Alkali concentration and residence time in autoclave were the most significant parameters affecting the pretreatment process according to the statistical analysis. Under optimal operating conditions the maximum sugar yield was of 60% g/g. Moreover, the phenolic content assessed in the supernatant after CSS pretreatment was (25 mgGAE/graw_CSS) larger than that reported in the literature for similar works on CSS. The study about enzymatic hydrolysis of alkali-pretreated CSS highlighted that both biomass loading and cellulolytic enzymes loading affect sugars release. A sugar yield higher than 90% g/g was obtained with 10% w/v biomass loading and 80 FPU/gcellulose enzyme loading. The rich-in-sugars hydrolysate obtained after pretreatment and enzymatic hydrolysis of CSS was supplemented with some other nutrients and used as media for fermentation of C. acetobutylicum (ABE production) and A. succinogenes (succinic acid production). Concentration of 3.2 g/L ABE and 20.8 g/L succinic acid was obtained, at the end of each fermentation, respectively. With the proposed process (pretreatment, hydrolysis and fermentation), we were able to obtain fermentable sugars, lignin, solvents (ABE fermentation) and biochemicals (phenolic compounds and succinic acid) from CSS, stressing the potentialities of this residue to be used in biorefinery processes. Apple pomace as feedstock for biorefinery applications: Apple is a fruit widely produced and eaten all over the world. In Italy, more than 2.5 million tons of apple are produced every year. Worldwide, 20 % of fresh apple is processed into value added products: apple juice concentrate (65%), apple cider, wine, vermouth, purees, jams and dried apple products. The main residue of these processes is the apple pomace. This residue accounts for 25-35% of the dry mass of apple. The apple pomace is a good lignocellulosic candidate to be used in a biorefinery process, and it was already used as feedstock for production of butanol, ethanol and crude protein (or enzymes), citric acid, microbial colours, bio-hydrogen. Pretreatment of apple pomace was carried out by means of the ligninolytic enzyme “laccase”. The aim of this study was to investigate the possibility to carry out biological pretreatment and hydrolysis of apple pomace in a bubble column bioreactor. The proposed pretreatment/hydrolysis system aimed at the optimization of some parameters affecting the process in terms of amount of sugars released during the whole process. The enzymatic delignification was carried out using fungal laccases from Pleurotus ostreatus. Then the cellulose in the pretreated biomass was hydrolysed using Cellic CTec2® (Novozymes). The optimization of enzymatic pretreatment/hydrolysis process indicated that all the parameters investigated (biomass loading, laccase loading, gas (air) flow rate, cellulase loading) affect the performances of the process in terms of biomass composition and sugars release during the enzymatic hydrolysis. 15 %w/v biomass loading, 60 nL/h air flow rate, 30 U/gbiomass laccase concentration, and 20 FPU/gcellulose cellulase concentration were the optimal conditions for the sequential enzymatic pretreatment and hydrolysis process in the bubble column. All the experiments were carried out in a lab-scale bubble column. Under the operating conditions investigated, a maximum sugar yield of 0.34 gsugars/graw biomass was obtained, corresponding to 61% (gsugars/gsugars available) of the theoretical sugar yield obtainable from raw apple pomace. The high amount of sugars obtained, make AP one of the main feedstocks to be used in fermentative processes to produce solvents and biochemicals. Moreover, a solid residue of lignin that can be recovered and reused was obtained at the end of the process. ABE fermentation at University of Western Ontario (UWO) (Canada): The reasearch activity was focused on the ABE fermentation of an industrial product derived from corn: corn syrup. The objectives were: i) to screen four Clostridium species to identify the microorganism characterized by the best fermentative performance in terms of solvents production, yield and productivity; ii) to study the effects of substrate/product concentration on fermentation process (substrate/product inhibition effects); iii) to increase solvents production by means of a fed-batch process. The final aim of this project was to investigate the possibility of using corn syrup to produce solvents through ABE fermentation. The details regarding this activity are reported in the section Appendix. Regarding the main topic of my Ph.D. project, the work carried out at UWO have had a double purpose: 1) to improve my skills in anaerobic fermentative processes carried out by different Clostridium species in accordance to the final aim of the European project Waste2Fuels that is the bio-butanol production starting from agro-industrial residues; 2) to compare the fermentative performances of Clostridium spp. on agro-industrial products with various degrees of difficulty to ferment (in particular corn syrup and coffee silverskin).

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