Optimization of cell disruption methods for efficient recovery of bioactive metabolites via NMR of three freshwater microalgae (chlorophyta) Nyuk Ling Ma a,⇑ , Kit Yinn Teh a , Su Shiung Lam c , Anne Marie Kaben b , Thye San Cha b a School of Fundamental Science, University Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia b Institute of Marine Biotechnology, University Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia c Eastern Corridor Renewable Energy Group (ECRE), School of Ocean Engineering, University Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia highlights  Homogenizing, cryogrinding, sonication, HCl extraction was tested on microalgae.  Chlorella variabilis, Scenedesmus regularis, Ankistrodesmus gracilis were tested.  HCl extraction shows the highest recovery of oil compounds (up to 90%).  Homogenized extracts shows the highest intensity of bioactive metabolites by NMR.  NMR as a tool to assess compound diversity and metabolites in microalgae extracts. article info Article history: Received 8 January 2015 Received in revised form 6 March 2015 Accepted 7 March 2015 Available online 13 March 2015 Keywords: Microalgae Disruption NMR Metabolite Bioactive compounds abstract This study demonstrates the use of NMR techniques coupled with chemometric analysis as a high throughput data mining method to identify and examine the efficiency of different disruption techniques tested on microalgae (Chlorella variabilis, Scenedesmus regularis and Ankistrodesmus gracilis). The yield and chemical diversity from the disruptions together with the effects of pre-oven and pre-freeze drying prior to disruption techniques were discussed. HCl extraction showed the highest recovery of oil compounds from the disrupted microalgae (up to 90%). In contrast, NMR analysis showed the highest intensity of bioactive metabolites obtained for homogenized extracts pre-treated with freeze-drying, indicating that homogenizing is a more favorable approach to recover bioactive substances from the disrupted microalgae. The results show the potential of NMR as a useful metabolic fingerprinting tool for assessing compound diversity in complex microalgae extracts. Ó 2015 Elsevier Ltd. All rights reserved. 1. Introduction Microalgae has been widely explored mainly on their potential to be transformed into biofuel feedstock (Brennan and Owende, 2010; Mercer and Armenta, 2011). However, the exploitation of microalgae for biofuel production is still in its infancy and has yet to achieve with the high maintainance and production cost. The cost of the use of current technology to produce biofuels from microalgae is still too high for commercialization; for example, it costs about US$ 200 to produce a barrel of oil but the oil could only be sold at US$ 1.13/kg. This creates a slow growing market with an estimated generation of US$ 100 billion annually as per 2014 and is expected to only reach US$ 130 billion by 2019 (Oilgae, 2011). On the other hand, secondary metabolites obtained from microalgae has higher economical value (Cardozo et al., 2007; Fleurence, 1999); the high value chemicals obtained from the first commercialized algae ranges from US$ 300 to 1500/kg. Microalgae being an ancient species have evolutionary surviving techniques to adapt to the abrupt changes in their living environment. These diversified defence mechanisms are potential and plausible sources for the development of health, cosmetics and natural prod- ucts (Ioannou et al., 2010). A wide range of biochemically active compound produced from microalgae with potential economic impact in food science, drug industry, and as a biofuel feedstock has been clearly reviewed (Cardozo et al., 2007, 2011). Over http://dx.doi.org/10.1016/j.biortech.2015.03.036 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +60 9 6683845; fax: +60 9 6683608. E-mail addresses: nyukling@umt.edu.my (N.L. Ma), t.kityinn@gmail.com (K.Y. Teh), lam@umt.edu.my (S.S. Lam), nyetebe@hotmail.com (A.M. Kaben), Cha_ts@umt.edu.my (T.S. Cha). Bioresource Technology 190 (2015) 536–542 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech