Elsevier

Algal Research

Volume 2, Issue 2, March 2013, Pages 113-121
Algal Research

Strategic enhancement of algal biomass and lipid in Chlorococcum infusionum as bioenergy feedstock

https://doi.org/10.1016/j.algal.2013.01.005Get rights and content

Abstract

Algal biomass can serve as rich source of bioproducts including lipids for diverse commercial applications. Both biomass production and lipid accumulation are limited by several factors, of which nutrients play a vital role. In the present investigation, the nutritional requirement for the growth by a (an autotrophic) Chlorococcum infusionum was determined using a Plackett–Burman based statistical screening experiment. Five out of the fifteen factors of a reported production medium were found to be significantly affecting the biomass growth. The components NaNO3, K2HPO4, FeSO4.7H2O and KOH had direct proportional correlation with biomass production, while MgSO4 showed inverse proportional relationship in the selected experimental range. Nitrogen was the most influential factor with an effect contribution of 45.77% and a very low p-value of < 0.001. The most favorable nitrogen source was potassium nitrate which could replace both sodium nitrate and potassium hydroxide. More than two fold increase in biomass concentration was achieved by screening and standardizing the media components of Bold Basal Medium. Lipid accumulation under normal condition was 12–15% dry cell weight (dcw). Under nitrogen starvation condition, it was 30–35% dcw. However, a semi-starvation condition at 1.75 mM of sodium nitrate induced lipid production as high as 40 ± 2% dcw. FAME analysis in GC showed the presence of more saturated fatty acids. Results obtained in this work can further be applied to optimize production of algal biomass and lipid for applications like biofuel, fish or animal feed, fertilizer, etc. Also information obtained could be exploited for wastewater treatment processes.

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

References (36)

Cited by (83)

View all citing articles on Scopus
View full text