Bilian Chen

Liquid lipases for enzymatic concentration of n-3 polyunsaturated fatty acids in monoacylglycerols via ethanolysis: Catalytic specificity and parameterization

This work examined catalytic specificity and fatty acid selectivity of five liquid lipases C. antarctica lipase A and B (CAL-A/B), and lipase TL (T. lanuginosus), Eversa Transfrom and NS in ethanolysis of fish oil with the aim to concentrate n-3 PUFAs into monoacylglycerols (MAGs) products. Lipase TL, Eversa Transform & NS entail a much faster reaction and produce higher MAGs yield (>30%); whereas CAL-A obtains the highest concentration of n-3 PUFAs/DHA/EPA into MAGs products (88.30%); followed by lipase NS (81.02%). 13C NMR analysis indicates that CAL-B and lipase TL are sn-1,3 specific; but CAL-A and lipase Eversa Transform are non-regiospecific or weak sn-2 specific; which plausibly explains high enrichment effect of the latter two lipases. All liquid lipases are observed reusable for a certain times (lipase Eversa Transform up to 12 times), demonstrating their competitive advantage over immobilized form for industrial application because of their higher activity and cheaper operation cost.

Removal of nutrients from undiluted anaerobically treated piggery wastewater by improved microalgae

This study aimed at improving the adaptability and biodegradability of tested microalgae in undiluted anaerobic fermentation slurry of piggery wastewater. For that, a two-stage method based on UV irradiation followed by gradual domestication was developed. The distinctness of this method was the elimination of a screening procedure and just needed the UV-irradiated cells with appropriate survival to be subjected to gradual domestication. The microalgae treated with the method not only grew well in undiluted slurry, but achieved outstanding removal efficiencies in total nitrogen (TN) and total phosphorus (TP). Large-scale application was conducted in an open raceway pond, and the concentrations of TN and TP after treatment were 43.80mg/L (removal rate of 89.5%) and 5.83mg/L (removal rate of 85.3%) respectively, which greatly excelled the Chinese discharge standards for livestock and poultry wastewater. The strategy is therefore a promising method for microalgae to purify piggery slurry containing high nutrient contents.

The near-ideal catalytic property of Candida antarctica lipase A to highly concentrate n-3 polyunsaturated fatty acids in monoacylglycerols via one-step ethanolysis of triacylglycerols.

Declining quantity/quality of available n-3 polyunsaturated fatty acids (n-3 PUFAs) resources demand innovative technology to concentrate n-3 PUFAs from low quality oils into value-added products/health-beneficial ingredients rich in n-3 PUFAs. This work proposed the catalytic property and specificity of an ideal enzyme required to tackle this task and identified Candida antarctica lipase A (CAL-A) is such a near-ideal enzyme in practice, which concentrates n-3 PUFAs from 25% to 27% in oils to a theoretically closer value 90% in monoacylglycerols (MAGs) via one-step enzymatic ethanolysis. Non-regiospecificity and high non-n-3 PUFAs preference of CAL-A are the catalytic feature to selectively cleave non-n-3 PUFAs in all 3 positions of triacylglycerols (TAGs); while high ethanol/TAGs ratio, low operation temperature and high tolerance to polar ethanol are essential conditions beyond biocatalyst itself. C-13 Nuclear magnetic resonance ((13)C NMR) analysis and competitive factor estimation verified the hypothesis and confirmed the plausible suggestion of catalytic mechanism of CAL-A.

Production of new human milk fat substitutes by enzymatic acidolysis of microalgae oils from Nannochloropsis oculata and Isochrysis galbana

Human milk fat substitutes (HMFs) with four kinds of n-3 fatty acid for infant formula were firstly synthesized using triacylglycerols (TAGs) from Nannochloropsis oculata rich in PA at the sn-2 position and free fatty acids (FFAs) from Isochrysis galbana rich in n-3 polyunsaturated fatty acids (n-3 PUFAs-ALA/SDA/DHA) via solvent-free acidolysis with Novozym 435, Lipozyme 435, TL-IM and RM-IM as biocatalysts. The results show that the resulting HMFs contain total n-3 PUFA of 13.92-17.12% and PA of 59.38-68.13% at the sn-2 position under the optimal conditions (mole ratio FFAs/TAG 3:1, 60°C (Novozym 435 and Lipozyme TL-IM) and 50°C (Lipozyme 435 and RM-IM), lipase loading 10%, reaction time 24h). Moreover, among the tested enzymes, Lipozyme 435, TL-IM, and RM-IM display the fatty acid selectivity towards SDA, LA and ALA, and OA, respectively. Overall, the examined lipases are promising biocatalysts for producing high-value microalgal HMFs in a cost-effective manner.

Rationale behind the near-ideal catalysis of Candida antarctica lipase A (CAL-A) for highly concentrating ω-3 polyunsaturated fatty acids into monoacylglycerols.

Dramatic decline in the quality and quantity of ω-3 PUFAs from marine resource demands new environmental-friendly technology to produce high quality ω-3 PUFAs concentrates in a better bioavailable form. Accordingly this work demonstrated an exceptionally highly efficient non-aqueous approach that non-regiospecific and non ω-3 PUFAs preferential Candida antarctica lipase A (CAL-A), functioning as a near-ideal biocatalyst, is capable to directly concentrate ω-3 PUFAs from 20% to 30% in oils to up to >90% in monoacylglycerols form through one step reaction. The rationale behind the experimental observation is justified and the catalytic property and specificity of an ideal enzyme tackling this task are defined. High selectivity and efficiency, excellent reusability of biocatalyst, general applicability for concentrating ω-3 PUFAs from both fish and microalgae oils, simple process for product recovery (e.g. by short path distillation), make this novel approach a highly industrially relevant and with potential application in food and drug industries.

Cost-effective biodiesel production from wet microalgal biomass by a novel two-step enzymatic process

In this study, a novel two-step enzymatic process was firstly established to produce microalgae biodiesel using wet Chlorella biomass. In the first hydrolysis step, to reduce energy consumption and effectively disrupt microalgal cell wall, among cellulase, hemicellulase, papain, lysozyme and pectinase, the highest hydrolysis efficiency (67.52%) was obtained by cellulase at pH 5.0 with enzyme dosage of 200 U/g dry biomass at 40 °C for 12 h. In the second transesterification step, compared with liquid CAL-A/B from Candida antarctica and PLA from Aspergillus oryzae, liquid lipase TL from Thermomyces lanuginosus achieved the highest biodiesel conversion at 81.15:1 (v/w) ethanol/g TFAs ratio in 78-83% water content with 100 PLU/g TFAs lipase loading at 25 °C for 48 h. Moreover, similar results were obtained with three Chlorella species by this process. Overall, this two-step enzymatic process was a green, low-energy and efficient method for cost-effective biodiesel production using wet microalgal biomass.

Isolation of an indigenous Chlorella vulgaris from swine wastewater and characterization of its nutrient removal ability in undiluted sewage

Bio-treatment of wastewater mediated by microalgae is considered as a promising solution. This work aimed to isolate an indigenous microalgal strain (named MBFJNU-1) from swine wastewater effluent and identify as Chlorella vulgaris. After 12days, the removal efficiencies of total nitrogen (TN) and total phosphorus (TP) in undiluted swine slurry were 90.51% and 91.54%, respectively. Stress tolerance in response to wastewater was verified by cultivating in artificial wastewater containing different levels of chemical oxygen demand (COD), TN and TP. MBFJNU-1 could grow well in undiluted swine slurry and artificial wastewater containing 30,000mg/L COD or 2000mg/L TN. Furthermore, global nuclear DNA methylation (5-mC) of MBFJNU-1 was employed to explore the possible mechanism in response to wastewater stress. The results showed that the level of 5-mC was inversely proportional to the growth of MBFJNU-1 in different diluted swine slurry, helping to understand 5-mC variation in response to stress environment.