Optimization of Enzymatic Transesterification of Acid Oil for Biodiesel Production Using a Low-Cost Lipase: The Effect of Transesterification Conditions and the Synergy of Lipases with Different Regioselectivity.

This study describes the enzymatic production of second-generation biodiesel using low-quality acid oil as a substrate. Biolipasa-R, a commercially available and low-cost lipase, was employed for enzymatic transesterification. Response surface methodology was applied to optimize the enzymatic transesterification process. The optimal conditions for biodiesel production, which comprised 42% lipase concentration (per weight of oil), 32% water content (per weight of oil), a methanol to oil molar ratio of 3:1, pH 7.0 and reaction temperature 30°C, resulted in the highest fatty acid methyl ester (FAME) content (71.3%). Subsequently, the synergistic effect of two lipases with different regioselectivities under the optimum transesterification conditions was studied, aiming at the enhancement of process efficiency. The transesterification efficiency of immobilized Biolipasa-R was determined and compared to that of Biolipasa-R in its free form. The results revealed a good performance on FAME content (66.5%), while the recycling of immobilized lipase resulted in a decrease in transesterification efficiency after three consecutive uses.

A Novel Bio-Purification Process Employing an Engineered E. coli Strain for Downstream Processing of Lactic Acid Solutions from the Fermentation of Agro-Industrial by-Products.

This study aims to integrate a novel bio-purification process employing an engineered E. coli strain in the downstream processing of lactic acid (LA) fermentation broths from low-cost renewable biological feedstocks. Fermentation broth of candy waste and digestate mixture was used as a real biological feedstock. An engineered E. coli strain that selectively catabolize impurities without catabolizing LA was initially adapted on the biological feedstock, followed by shake flask experiments to prove the bio-purification concept. Scale-up and validation in a bench-scale bioreactor followed, before developing a semi-continuous membrane bioreactor (MBR) bio-purification process. The MBR bio-purification was assessed with biological feedstocks which simulated ultrafiltration or nanofiltration permeates. Incomplete removal of impurities and increased fouling was observed in the case of the ultrafiltration permeate. Contrarily, the nanofiltration permeate was successfully treated with MBR bio-purification, since low membrane fouling, 100% maltose and acetic acid removal, and no LA catabolism was achieved. MBR bio-purification as a post-treatment step in the downstream processing of LA was demonstrated as a promising technology for increasing the purity of LA solutions.

Development of a hybrid bio-purification process of lactic acid solutions employing an engineered E. coli strain in a membrane bioreactor.

BACKGROUND: A potential alternative to lactic acid production through sugar fermentation is its recovery from grass silage leachate. The separation and purification of lactic acid from fermentation broths remain a key issue, as it amounts to up to 80% of its industrial production cost. In this study, a genetically engineered E. coli strain (A1:ldhA), that cannot catabolize lactic acid, has been used to selectively remove impurities from a synthetic medium comprising typical components (i.e., glucose and acetic acid) of green grass silage leachate. A systematic approach has been followed to provide a proof-of-concept for a bio-purification process of lactic acid solutions in a membrane bioreactor operating in semi-continuous mode. RESULTS: The synthetic medium composition was initially optimized in shake-flasks experiments, followed by scale-up in bench-scale bioreactor. Complete (i.e., 100%) and 60.4% removal for glucose and acetic acid, respectively, has been achieved in batch bioreactor experiments with a synthetic medium comprising 0.5 g/L glucose and 0.5 g/L acetic acid as carbon sources, and 10 g/L lactic acid; no lactic acid catabolism was observed in all batch fermentation tests. Afterwards, a hybrid biotechnological process combining semi-continuous bioreactor fermentation and ultrafiltration membrane separation (membrane bioreactor) was applied to in-situ separate purified medium from the active cells. The process was assessed under different semi-continuous operating conditions, resulting in a bacteria-free effluent and 100% glucose and acetic acid depletion, with no lactic acid catabolism, thus increasing the purity of the synthetic lactic acid solution. CONCLUSIONS: The study clearly demonstrated that a bio-purification process for lactic acid employing the engineered E. coli strain cultivated in a membrane bioreactor is a technically feasible concept, paving the way for further technological advancement.

Novel evolved Yarrowia lipolytica strains for enhanced growth and lipid content under high concentrations of crude glycerol.

BACKGROUND: Yarrowia lipolytica is a well-studied oleaginous yeast known for its ability to accumulate and store intracellular lipids, while growing on diverse, non-conventional substrates. Amongst them, crude glycerol, a low-cost by-product of the biodiesel industry, appears to be an interesting option for scaling up a sustainable single-cell oil production process. Adaptive laboratory evolution (ALE) is a powerful tool to force metabolic adaptations endowing tolerance to stressful environmental conditions, generating superior phenotypes with industrial relevance. RESULTS: Y. lipolytica MUCL 28849 underwent ALE in a synthetic medium with increasing concentration of pure or crude glycerol as a stressing factor (9-20% v/v) for 520 generations. In one case of pure glycerol, chemical mutagenesis with ethyl methanesulfonate (EMS) was applied prior to ALE. Growth profile, biomass production and lipid content of 660 evolved strains (EVS), revealed 5 superior isolates; exhibiting from 1.9 to 3.6-fold increase of dry biomass and from 1.1 to 1.6-fold increase of lipid concentration compared to the parental strain, when grown in 15% v/v crude glycerol. NGS for differential gene expression analysis, showed induced expression in all EVS affecting nucleosomal structure and regulation of transcription. As strains differentiated, further changes accumulated in membrane transport and protein transport processes. Genes involved in glycerol catabolism and triacylglycerol biosynthesis were overexpressed in two EVS. Mismatches and gaps in the expressed sequences identified altered splicing and mutations in the EVS, with most of them, affecting different components of septin ring formation in the budding process. The selected YLE155 EVS, used for scale-up cultivation in a 3L benchtop bioreactor with 20% v/v crude glycerol, achieved extended exponential phase, twofold increase of dry biomass and lipid yields at 48 h, while citric acid secretion and glycerol consumption rates were 40% and 50% lower, respectively, compared to the parental strain, after 24 h of cultivation. CONCLUSION: ALE and EMS-ALE under increasing concentrations of pure or crude glycerol generated novel Y. lipolytica strains with enhanced biomass and lipid content. Differential gene expression analysis and scale-up of YLE155, illustrated the potential of the evolved strains to serve as suitable "chassis" for rational engineering approaches towards both increased lipid accumulation, and production of high-added value compounds, through efficient utilization of crude glycerol.

The Effect of Heat Sterilization on Key Filtration Performance Parameters of a Commercial Polymeric (PVDF) Hollow-Fiber Ultrafiltration Membrane.

Membrane processes can be integrated with fermentation for the selective separation of the products from the fermentation broth. Sterilization with saturated steam under pressure is the most widely used method; however, data concerning heat sterilization applicability to polymeric ultrafiltration (UF) membranes are scarcely available. In this study, the effect of the sterilization process on the filtration performance of a commercial polyvinylidene difluoride (PVDF) hollow fiber UF membrane was evaluated. Membrane modules were constructed and sterilized several times in an autoclave. Pure water flux tests were performed, to assess the effect of heat sterilization on the membrane's pure water permeance. Dextran rejection tests were performed for the characterization of membrane typical pore size and its fouling propensity. Filtration performance was also assessed by conducting filtration tests with real fermentation broth. After repeated sterilization cycles, pure water permeance remained quite constant, varying between approx. 830 and 990 L·m-2·h-1·bar-1, while the molecular weight cut-off (MWCO) was estimated to be in the range of 31.5-98.0 kDa. Regarding fouling behavior, the trans-membrane pressure increase rate was stable and quite low (between 0.5 and 7.0 mbar/min). The results suggest that commercial PVDF UF membranes are a viable alternative to high-cost ceramic UF membranes for fermentation processes that require heat sterilization.

A Novel Application of Recycled Ultrafiltration Membranes in an Aerobic Membrane Bioreactor (aMBR): A Proof-of-Concept Study.

The use of recycled ultrafiltration (r-UF) membranes, originating from end-of-life reverse osmosis membranes, as submerged flat-sheet membranes in an aerobic membrane bioreactor (aMBR) system is described herein for the first time. A feasibility study of this new approach was performed in a laboratory-scale aMBR system. The r-UF membrane performance was evaluated in terms of permeability, fouling behavior, and permeate quality using a widely used commercial flat sheet microfiltration membrane (c-MF) as a reference. Tests were conducted under steady-flux operation (at 12 and 14 L·m-2·h-1) and a variable trans-membrane pressure. Synthetic wastewater simulating urban wastewater characteristics with approx. 0.4-0.5 g/L COD concentration was used as the feed. The obtained results showed that the rejection performance of the r-UF membrane was similar to the performance of the commercial flat sheet microfiltration membrane (c-MF) under comparable operating conditions. Moreover, concerning fouling behavior, the r-UF membrane exhibited higher fouling resistance compared with the c-MF membrane, although the permeability decline rate was lower. Both membranes had comparable fouling mechanisms behavior, with cake layer fouling resistance accounting for approx. 60% of the total fouling resistance. Finally, a preliminary economic assessment pointed out the potential competitiveness of using r-UF membranes for aMBRs (5.9-10.9 EUR·m-2) and the scaling-up challenges toward industrial applications.

Hybrid life cycle assessment of agro-industrial wastewater valorisation.

Wastewater from food processing facilities can have high nutrient valorisation potential. Valorising sugar-rich agro-industrial wastewater may have significant impacts on sustainability of wastewater treatment plant (WWTP). The objective of this study is to evaluate the environmental, economic and social impacts of a novel wastewater valorisation technology. This technology is designed to produce single cell protein (SCP) from wastewater of a fruit juice processing facility. To evaluate the comprehensive sustainability impacts on WWTP and overall background economy, a hybrid life cycle assessment model was developed by combining the multi-regional input-output database (Exiobase) with process-based life cycle inventories of conventional and AgroCycle WWTP. The results indicated the upstream impacts of wastewater could have significant influence on sustainability of WWTP with nutrient valorisation. Therefore the 'zero burden assumption' should not be adopted for upstream wastewater. For the sustainability performance, valorising nutrients from WWTP with AgroCycle technology can improve the environmental performance of WWTP. However, the positive social-economic impacts were directly associated with WWTP system, not the whole background economy. The production of SCP could reduce the Gross Value Added (GVA) and employment in the 'oil seeds sector'. In order to improve the social-economic impacts and promote a circular bioeconomy model in the fruit juice sector, further development is required to improve valorisation productivity and create a better value chain for valorised products.