Biological tools to improve biogas production from microalgae biomass

Resumen

Great efforts are undertaken to develop biofuel-based microalgae biomass as replacements to non-renewable fossil fuels. According to the European directive on Renewable Energy (2009/28/EC) (RED, 2009), special emphasis should be placed on the production of renewable energy by stating that 10% of energy in transport should be renewable by 2020. Out of the possible energy forms, biogas production is envisaged as a feasible route to sustainable energy production. Biogas production has strong potential over other energy forms since its needs less concentrated biomass and no macromolecular extraction. However, due to the hard cell wall and low C/N ratio of microalgae biomass, its practical application is prevented by the low biogas yields obtained. Several possibilities for overcoming those limitations were investigated in this Doctoral Thesis in order to enhance microalgae anaerobic biodegradability. Even though some strains exhibit no cell wall, strains with hard cell wall prevail in outdoor culture conditions. Thus, strains possessing rigid cell walls are resistant to anaerobic digestion and the application of suitable pretreatments are highly required. In this Doctoral Thesis, autohydrolysis and biological pretreatments have been applied in different microalgae biomass (namely, Chlorella vulgaris, Scenedesmus sp. and Chlamydomonas reinhardtii). The pretreatments were evaluated with regard to organic matter, protein, carbohydrate solubilisation as well as methane production enhancement. Autohydrolysis induced by low temperature application resulted in low organic matter solubilisation (6-16%) and low carbohydrates solubilisation (15-31%) and accordingly, methane production was enhanced to minor extent with C. vulgaris biomass (only 10% higher compared to raw biomass, 127 mL CH4 g COD in-1) or even negligible in the case of Scenedesmus biomass. In this context, it was concluded that autohydrolysis was unable to break down efficiently the complex cell wall of microalgae biomass and alternative approaches were studied to maximize biomass hydrolysis efficiency and justify the pretreatments costs. Since carbohydrates have been traditionally recognized as the polymers responsible for the low microalgae digestibility and microalgae biomass exhibit typically a high protein content, microalgae biomass was subjected to two groups of biocatalysts, namely carbohydrases and proteases for microalgae hydrolysis prior to anaerobic digestion. Out of the several carbohydrases tested (Pectinase, Viscozyme and Celluclast), Viscozyme addition resulted in the highest carbohydrates solubilisation (85%, 96% and 36% with C. vulgaris, C. reinhardtii and Scenedesmus sp., respectively). Despite of the high carbohydrates solubisation, the increase on methane yield was low (14-16% with C. vulgaris and Scenedesmus sp. and negligible with C. reinhardtii). In the case of C. reinhardtii biomass, the application of biocatalysts did not report significant enhancement since the raw biomass was already easily digestible (263 mL CH4 g COD in-1). Even though different microalgae biomass used herein exhibited different cell wall composition and/or different macromolecular distribution, protease addition resulted in highest organic matter solubilisation (47-57%) and highest methane yield (1.5-1.7-fold higher compared to raw biomass) for two of the most robust microalgae biomass, namely C. vulgaris and Scenedesmus sp. Carbohydrases and protease were also combined to verify potential synergetic effect on biomass hydrolysis but the results showed methane yield enhancement similar to that attained with protease pretreated biomass. Different experiments were conducted in order to optimize protease hydrolysis of C. vulgaris biomass. The attempt of decreasing the enzymatic dosages (from 0.585 down to 0.146 AU g DW-1) diminished hydrolysis efficiency (from 50 to 41% in COD terms) concomitantly with a decreased methane yield enhancement (from 256 down to 224 mL CH4 g COD in-1). The best result was achieved with protease dosage at 0.585 AU g DW-1 by enhancing methane yield 1.73-fold. Additionally, increasing biomass loads from 16 up to 65 g L-1 did not affect markedly the hydrolysis efficiency (51%) nor methane yield enhancement (1.55-fold) when using protease dosage of 0.585 AU g DW-1. Since the proteolytic enzyme gave promising results in batch mode anaerobic digestion, further investigation using this biocatalyst as pretreatment of C. vulgaris was assessed in semicontinuously fed reactors (CSTR). The CSTR was operated at organic loading rate (OLR) of 1.5 g COD L-1 d-1 and hydraulic retention time (HRT) of 20 days. In contrast to that attained in batch anaerobic digestion, feeding the CSTR with protease pretreated biomass showed volatile fatty acids (VFA) accumulation as a result of ammonium inhibition (1.9 g N-NH4+ L-1). Therefore, a stepwise reduction in methane production rate and yield was observed throughout the digestion time. To overcome this issue, the protein content of microalgae biomass was reduced by cultivating the microalgae under nutrients limited conditions (wastewater was used a sole nutrient source). More specifically, C. vulgaris protein content decreased from 65% to 33% (VSS basis). In response to that macromolecular profile change, no VFAs accumulation nor ammonium inhibition were registered in the CSTR fed with carbohydrate-rich C. vulgaris and a steady methane production rate was displayed along digestion. In addition, this carbohydrate rich biomass was also pretreated by using carbohydrases and fed to the CSTR, nevertheless, the results evidenced an increase in methane yield (3-fold compared to raw biomass 25 mL CH4 g COD in-1) but still lower than the enhancement attained with protease pretreated biomass (5-fold). Once circumvented the high ammonium concentration in the digesters and attained a stable methane production with protease pretreated carbohydrate-rich C. vulgaris biomass, the OLR was doubled (3 Kg COD m-3 day-1) and the HRT shortened (15 days) to verify anaerobic digestion stability. The data obtained in the later experimental conditions denoted not only the same stability but even higher methane production was achieved (6.3- fold higher methane yield compared to raw biomass). Overall, this Thesis highlights the crucial role of microalgae proteins on their anaerobic digestion. Biomass protein content in C. vulgaris and Scenedesmus sp. hampered the anaerobic digestion in two ways, firstly as a polymer embedded in microalgae cell wall decreasing the access of anaerobes for degradation and secondly, by causing methanogens inhibition due to the high ammonium concentration reached in the anaerobic digester when feeding protease pretreated biomass. The possible solutions (pretreatment with proteases and enrichment of biomass in carbohydrate fraction) to overcome the negative effects of high protein content of this microalgae biomass were deeply discussed and evaluated throughout the Thesis.