Production of bioplastic precursors (polyhydroxyalkanoates) from macroalgae

Abstract

In recent years, there has been increasing interest in the use of macroalgae to obtain high value-added products through biotechnological processes. This natural biomass has many advantages, such as the fact that they do not need land or fresh water to be cultivated. Moreover, they are not energy crops, so they do not compete directly with those that are a food resource, such as maize or wheat. Another aspect that makes them interesting is that around half of their dry weight are carbohydrates, such as cellulose or laminarin in the case of brown algae, in addition to other glucose polysaccharides that can be found depending on the species. These unconventional carbon sources can be used as substrates in fermentation processes to produce compounds of industrial interest, such as polyhydroxyalkanoates (PHAs), precursors of bioplastics, and thus contribute to reducing the impact of conventional plastics on the environment. However, due to the integral nature of the macroalgae structure, the accessibility of microorganisms to organic matter is restricted, so it is necessary to study the most appropriate way to solubilise it. Studies on the solubilisation of organic matter in macroalgae are scarce and very recent, so methodologies described for lignocellulosic matter, which have been widely studied, are usually applied. Among the techniques used, the ones that offer the best yields include a chemical pretreatment stage and an enzymatic hydrolysis or saccharification stage. The former is used to weaken the structure of the biomass and facilitate accessibility to the carbohydrates of which it is composed. In the second, the bioconversion of polysaccharides to fermentable monosaccharides takes place. In this Doctoral Thesis, the invasive species Rugulopteryx okamurae was used as algal biomass, a brown alga whose origin is in the coasts of East Asia, but which has reached the Mediterranean coast and is causing serious environmental problems, affecting native species, and having great repercussions on the local economy. To minimise its impact, it was evaluated the use of this macroalga as a raw material to produce PHAs, by fermentation with a pure culture of Cupriavidus necator, using as substrate sugar hydrolysates obtained from the alga without pretreatment, and previously pretreated with acid dilutions and heat input. Firstly (article 1), the dietary fibre content of the biomass without pretreatment was analysed and compared with that of 10 other species. It was found that R. okamurae contained the richest protein fraction among the species compared, with 49.05 % (w/w), and a hydrolysable polysaccharide content of 27.29 % (w/w), a high value compared to the algae compared. Thus, the enzymatic hydrolysis stage was studied in order to obtain the highest amount of fermentable sugars. For this purpose, the main variables in the saccharification process were evaluated, in batch mode, and using algae without pretreatment. Different ratios of biomass loading, 6, 8 and 10 % (w/v), enzyme doses, 18, 30 and 50 FPU/g biomass, and stirring rates, 150 and 250 rpm, were tested. Furthermore, the condition at which the highest concentration of fermentable sugars was achieved was tested in Fed-Batch mode. For a better comparison of the results, a new kinetic model was developed based on the conventional model, which considers first order kinetics for the enzymatic reaction. A new term was added to the proposed model to account for the effect of enzyme diffusion through the inert solid biomass. The proposed model showed a better fit to the experimental data, compared to the conventional model, especially for prolonged hydrolysis times, obtaining the highest sugar concentration (13.7 g/L) after 165 hours of hydrolysis, in the condition: 10 % (w/v) biomass loading, an enzyme dose of 50 FPU/g dry biomass, and a stirring rate of 250 rpm, in Batch mode. Secondly (article 2), a study of the effect of hydrothermal acid pretreatment on the enzymatic saccharification process was carried out. The acid (HCl) concentrations studied were 0.05 N, 0.1 N and 0.2 N, for reaction times of 15, 30 and 60 minutes. In order to determine the most suitable pretreatment conditions among those analysed, the amount of reducing sugars (RS) generated after the saccharification process was evaluated, taking into account the severity of the pretreatment. Thus, the optimum pretreatment conditions were found when 0.2 N HCl was used for 15 minutes, achieving 20.7 g/L RS, of which 92.6 % was glucose. In addition, the severity of pretreatment on dietary fibre composition was analysed, and it was observed that increasing the severity resulted in an impoverishment of the protein fraction and an enrichment of the hydrolysable polysaccharide fraction. The latter is positive from the point of view of its application in biorefinery, as it favours the fractionation of the biomass and the macroalgae can therefore be better utilised. Finally (article 3), fermentations were carried out to evaluate the capacity of the R. okamurae algal biomass to be used as feedstock for the production of PHAs. The hydrolysates obtained from the non-pretreated algae, and pretreated under optimal conditions, were used as culture medium for fermentation to PHAs with C. necator, and the profiles of substrate consumption, biomass generation and biopolymer production were compared with those obtained with fermentations carried out in synthetic medium. Thus, those fermentations that were carried out with hydrolysate of pretreated algae showed the best values of yields (0.774 g PHB/g RS), and percentage of PHB accumulation (89.8 % CDW), reaching values comparable to the highest values reported in the scientific literature so far. From the results obtained in this Doctoral Thesis it was possible to estimate the amount of PHB that could be produced using macroalgae as feedstock. The estimation was based on a real case of R. okamurae disposal, specifically on the coast of Ceuta (Spain), where, in 2016, more than 5,000 tonnes of algal waste deposited along 15 km of coastline had to be removed. It was calculated, that up to 9.3 tons of PHB could be produced using such amount of residual biomass and applying the pretreatment, saccharification and fermentation conditions proposed in this Doctoral Thesis. Thus, based on the results obtained in this Doctoral Thesis, it would be interesting to carry out a technical-economic analysis for the production of PHB from R. okamurae, since the production costs depend to a large extent on the raw material, and for this case, the process could be cost-effective.