Use of cell wall degrading enzymes for the recovery of lipids from microalgae

Nowadays, nearly 80% of world energy demand is satisfied by the use of fossil fuels, which provoke the increasing accumulation of CO2 in atmosphere. Thus, the capture of this gas and the prevention of its release will play a key role to prevent climate changes, which are causing adverse effects. Great efforts have been focused on the production of biodiesel, bioethanol, or biogas. The use of biofuels does not contribute to an increase in the atmospheric CO2 concentration, since the carbon released has previously been taken from atmosphere by photosynthesis. In this scenario, the utilization of microalgae cultures can contribute to CO2 capture and storage, converting it into a biomass rich in valuable products, such as carotenoids, aminoacids and lipids for biodiesel production. Another advantage is that the conversion efficiency of solar energy into biomass for algal cultures and the productivity per hectare are much greater than those obtainable with traditional crops. Moreover, algal cultures do not compete for fertile land, do not require pesticides and can be grown in seawaters or wastewater from where microalgae take nutrients that they transform in biomass. One of the factors hindering the large-scale use of microalgae as a lipid source is the high energy consumption associated with the recovery of lipids. In fact, in most microalgal species, lipids are located inside the cell, which must be disrupted to allow their extraction. The hardness of algal walls and their organization into a complex multi-layered structure make disruption an energy-intensive process. Common methods of cell disruption involve the use of mechanical (e.g., ultrasonication, high-pressure homogenization, bead beating) or chemical (e.g., alkali, acid, detergent) means. In addition to their request for a large consumption of energy or chemicals, these treatments may cause damage to the most easily degradable algal components, such as proteins and carotenoids, which could be coextracted with lipids in a biorefinery perspective. In this context, the primary objective of this doctoral thesis was the development of an enzyme-assisted lipid extraction method using unpurified low cost industrial enzyme preparations. Enzymatic treatment of microalgae is an attractive, but still little explored method of cell wall disruption. It is based on the selective degradation of cell wall components by specific enzymes. This method has the potential to facilitate the recovery of lipids or the post-extraction use of the algal biomass and to preserve the most labile compounds. However, the choice of suitable enzymes is strictly related to the characteristics of the algal wall which, in turn, depend on the microalgae species, the growth conditions and the harvesting and dewatering steps. Unfortunately, microalgal cell walls are still poorly characterized. The first part of the work focused on the selection and the screening of enzyme preparations on the basis of the characteristics of the cell wall of Nannochloropsis sp., the microalga that was used as model organism. This microalga is of great industrial interest because of its ability to accumulate large amounts of lipids and other valuable components, such as the carotenoids astaxanthin and zeaxanthin and the omega-3 polyunsaturated fatty acid EPA. However, it shows unusual resistance towards mechanical and chemical treatments, which seems to be at least partly due to the presence in the outer cell layer of the aliphatic biopolymer algaenan. This makes the extraction of intracellular algal components a challenging and energy-consuming process. Subsequently, an enzyme-assisted extraction processes based on the development of optimized cell wall-degrading enzyme cocktail was developed by the use of the statistical methodology of mixture design. The influence of the most important process parameters (pretreatment time, enzymes to biomass ratio, pH and temperature of pre-treatment) on the yield of lipid extraction was then analyzed using the pretreatment mixture of optimized composition. A factorial design has been identified with the purpose to analyze their effect on the yield of lipid extraction. Numerical optimization of the obtained model allowed identifying the optimal pretreatment conditions, in terms of extraction yield and cost of the pretreatment. The recovery of the enzyme pre-treatment solution for the re-use in subsequent cycles was also evaluated to lower the operating cost. Moreover, a characterization of the untreated and enzymatically treated biomass was performed through the use of instrumental methods, such as Fourier transform infrared spectroscopy (FTIR), X-ray diffraction spectrometry (XRD), thermogravimetry (TGA/DTG) and scanning and transmission electron microscopy (SEM and TEM) in order to understand the action of each enzyme and, consequently, to properly design the process. Finally, the exhausted algal biomass after lipid extraction process has been studied to assess its implementation as a low cost absorbent material for textile azo-dyes, in order to find new application that could allow a further decrease of the cost of the entire biodiesel production chain from microalgae.