Omega-3 fatty acid production from enzyme saccharified hemp hydrolysate using a novel marine thraustochytrid strain
Introduction
The microbial biosynthesis of sustainable biochemicals or biofuels from lignocellulose biomass obtained from agricultural feedstock, industrial and urban bio-residue or woody biomass, has attracted significant attention (Wu et al., 2014). Lignocellulose biomass (LCB) can serve as an inexpensive carbon source for growing microbes including oleaginous microorganisms thus reducing the total production cost of biofuels, value added metabolites and co-products. LCB consists mainly of lignin, cellulose and hemicellulose that requires pretreatment for the opening of the compact structure by breaking cross-linked bonds (Puri et al., 2012, Cao et al., 2013). Pretreatment increases the hydrolysability of complex materials and maximizes enzyme penetration by increasing the cellulose surface area, thus producing sugar hydrolysate (SH) that can be used for producing ethanol and lipids (Pakarinen et al., 2012, Abraham et al., 2014). Reducing sugars such as glucose, xylose, mannose, arabinose, glucan, rhamnose, and cellobiose are the main constituents in the sugar hydrolysate and their concentration vary based on the biomass composition (Toquero and Bolado, 2014).
In addition to biofuel production, LCB can be utilized for the production of nutraceuticals or other chemicals of industrial value. A recent report has described the potential of LCB for producing fatty acids and their derivatives (Liu et al., 2014). Utilization of inexpensive and abundant lignocellulose feedstocks such as rice straw, sugarcane bagasse, corn stover, and corncob for the production of microbial lipids using oleaginous yeast has been investigated in the previous studies (Gao et al., 2014). Metabolic engineering has also been applied to produce engineered microbes capable of metabolising multiple sugars from LCB to produce biofuels and other important metabolites (Yao and Shimizu, 2013).
Thraustochytrids are oleaginous marine protists found in mangrove and estuarine environments with the ability to accumulate higher amounts of polyunsaturated fatty acids (PUFAs) (Gupta et al., 2012). These microbes originating from marine environments are promising sources of industrially important long chain fatty acids. The utilization of various carbon sources by thraustochytrids for fatty acid production has been reported previously, including the use of conventional hexose sugars and complex cellulosic matter (Hong et al., 2012).
In the present study, a novel isolate named Schizochytrium sp. DT3 was employed to utilize the SH obtained after the enzymatic saccharification of pretreated hemp biomass (Cannabis sativa), for the production of omega-3 fatty acids. Hemp is an annual dicotyledonous angiosperm plant under Cannabaceae family and includes bast, xylem and marrow. It is mainly grown for the outer covering or bast fiber and the applications extend across many industries. Hemp stalks are used for industrial purpose due to the higher volume of stalk (two times higher) than bast fiber. Interest in the use of hemp biomass is due to its high cellulose content (cellulose ∼55%, hemi-cellulose ∼16%, pectin ∼18% and lignin ∼4%) (Jaldon et al., 1998), easier availability, high biomass yield, low need of fertilization and insensitivity to frost (Pakarinen et al., 2012, Abraham et al., 2013). Hemp has been used as a potential biomass for the production of bioenergy such as ethanol, biogas, electricity and methane (Barta et al., 2013). Thus production of PUFAs was investigated using SH from hemp biomass as the sole carbon source, in comparison to glucose.
Section snippets
Chemicals and culture maintenance
All chemicals used in this study were of analytical grade obtained from Sigma–Aldrich, (St. Louis, MO, USA) and Merck Chemicals (Frankfurter Strabe, Darmstadt, Germany). Instant ocean sea salts, was obtained from Aquarium Systems Inc. (Blacksburg, VA, USA). The biomass for this study was obtained from Commins stainless manufacturing, Whitton, NSW (Australia). A new thraustochytrid strain, Schizochytrium sp. DT3 isolated in our lab was used for this study (Genbank accession number KF682125). It
Isolation and identification of Schizochytrium sp. DT3
Our earlier optimized “pine-pollen baiting method” was used for the isolation of thraustochytrids from the local Australian marine environment (Barwon waters, Victoria). We found that pine pollen acted as specific substrate where thraustochytrids were found to readily attach (Gupta et al., 2013b). During the isolation, cells were visualized under 40× magnification using a differential interference contrast microscope to observe their morphology. Cell size of the isolate ranged between 20 and 25
Conclusion
Schizochytrium sp. DT3 was found to be a potential lipid producer with the production of DHA when grown on hemp SH. This strain utilized low concentrations of carbon sources (glucose and SH) present in the hemp derived SH, but the presence of inhibitors decrease biomass and DHA productivity at higher SH levels in the medium. Nonetheless, the fatty acid profile for this strain when grown on hemp derived SH shows competitive percentages of DHA as % of TFA at 30–38%, and so this strain may have
Acknowledgement
The authors are thankful to the Strategic Research Centre for Chemistry and Biotechnology, Deakin University, Australia, for providing funds to support bioprocessing research.
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Equal contributions, joint-first authors.