Strategic enhancement of algal biomass and lipid in Chlorococcum infusionum as bioenergy feedstock
Highlights
► Strategic enhancement of biomass production using statistical method. ► 5 out of 15 media components studied were significant for biomass production. ► Modified media yielded more than 2 fold enhanced biomass. ► Chlorococcum sp. produced as high as 40% lipid content under nutrient limiting conditions.
Introduction
Microalgal biomass can be used as feedstock for biofuel and other valuable products. Sustainable and eco-friendly feedstock is needed to overcome food security problem and address environmental concerns. Microalgae have been attracting much attention from the scientific community and have emerged as a potential sustainable biomass source due to their neutrality towards nature and versatility with respect to applications. Microalgae have the ability to convert inorganic matter into organically rich biomass by sequestering atmospheric CO2. Thus, the resulting microalgal biomass, which comprises important components like lipid, carbohydrate; pigments, proteins, etc., can be utilized in the field of therapeutics, cosmetics, nutraceuticals, animal feed, food, biofuel and wastewater treatment [1]. As a result of depletion of the world's fossil fuel reserves and increasing environmental concerns, there is a great demand for alternative renewable fuels and efficient environmentally friendly strategies for the treatment of industrial, municipal and domestic wastewaters. Advantages of microalgae include higher photosynthetic efficiency, enhanced biomass production, and faster growth-rates compared to other terrestrial crops without compromising landmass, making it an attractive option for biofuel and other commercially useful products [2].
Manipulations of the physicochemical parameters can have profound influence on biomass growth, fatty acid content and other bioactive metabolites [3]. The role of medium composition has been well recognized as very significant in influencing growth rate, product yield and biochemical composition of specific microalgae, since standard media generally result in low biomass productivities [4]. Among numerous macro and micro elements, some are rate limiting for algal growth, whereas others are important and involved in various enzymatic reactions for the biosynthesis of many compounds [5]. Conventional method for formulation of culture medium is a time-consuming and labor-intensive process. Statistical methods of media design have been successfully employed to deal with a large number of variables simultaneously in order to reduce the number of experimental runs that result in manifold improvement of the process performance [6], [7]. The Plackett–Burman experimental design is a useful tool to screen the most influential factors among numerous parameters with minimal number of experiments. It also determines the relative significance of various parameters and indicates the effects and concentration level of the various medium components [7], [8]. The disadvantages of this method are that it cannot determine the optimal concentration of each component and the interaction between different variables.
In any biomass cultivation system the presence of nutrients in the growth medium have influence on the nature, quantity and the composition of the product. In recent years microalgal cultivation has gained attention due to their ability to store high amount of lipids, which can further be extracted for the production of poly unsaturated fatty acid (PUFA) and biodiesel. It has been reported that the desired fatty-acid composition of lipid intended for a particular application can be achieved by screening critical media components and process parameters such as temperature, light intensity and concentration of various nutrients [9]. Among various approaches, exposing media to nitrogen limiting conditions, has been proved to be effective in accumulation of neutral lipid by algae for biodiesel application [9].
As lipid is considered to be a secondary metabolite, two step strategies are generally adopted to produce algal biomass [10]. Accordingly in the first step optimal conditions best suited for high biomass production are provided, followed by necessitating medium with nutrient limiting conditions to induce lipid biosynthesis. To the best of our knowledge there is scanty information in the literature reporting systematic studies on screening of critical medium components to enhance biomass and lipid yields by Chlorococcum sp. So in the current study, with a view to developing a modified medium, the statistical Plackett–Burman design was used to screen the most influential medium components and to determine the optimal ranges of concentration of nutrients. Further, lipid accumulation studies were carried out under nutrient starved conditions to obtain high lipid content for potential biodiesel and healthcare applications.
Section snippets
Microalga maintenance and culture conditions
The microalgal strain C. infusionum was obtained from the Botany Department, Calcutta University, Kolkata. It was isolated from brackish water, grown and maintained in bold basal medium (BBM), whose composition is given in Table 1 [11], [12], [13]. Algal strain was grown at 25 ± 2 °C and at pH 6.8–7 under 14:10 light–dark cycle with light intensity of 1500 lx. All the media chemicals used in this study were obtained from Hi-media and Merck, India.
Inoculum preparation and experiments
Seed culture was prepared by inoculating C. infusionum
Statistical analysis of experiments based on Plackett–Burman design
The biochemistry of algae, that significantly resembles the photosynthetic plant systems, depends on a wide range of inorganic salts called macro- and micro-nutrients. Most of the inorganic media components are micronutrients that are trace metal salts playing important roles in optimal health in terms of overall viability and physiology and are integral to many metabolic pathways and processes [18]. These trace metals are mainly used as cofactors for various enzymes in overall photosynthetic
Conclusion
This investigation helped identify the optimal nutritional requirement for C. infusionum growth using the 20-trial Plackett–Burman experimental design involving 15 major medium components. The modified medium (MM-1), which contains a judicious combination of trace metals like iron, magnesium, manganese, copper and zinc in high or low concentrations as dictated by the results of the P-B experiments, stimulated higher biomass and lipid production for potential application as a feedstock for
Acknowledgments
AK acknowledges Department of Biotechnology (DBT), New Delhi, India for the research fellowship. AK is grateful to Dr. Soumen Mukherjee for his valuable inputs. This study was undertaken as a part of CSIR-NMITLI project (Grant no.: 5/258/64/2009 - NMITLI; Date: 19-04-2010) on “Biofuel from marine microalgae” co-funded by Ministry of Earth Sciences (MoES). RS and RP thus thankfully acknowledge CSIR and MoES, Government of India for financial support. We are also grateful to Dr. P K Ghosh
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