Introduction

The treatment and disposal of domestic sewage has become a serious issue in many parts of the globe including India. Among the various technologies used, waste stabilisation ponds or lagoons are adopted due to its low operation and maintenance (OM) costs and adequate land availability. These treatment systems work with an initial anaerobic stabilisation followed by the facultative and aerobic oxidation ponds (Mahapatra et al, 2011a) or high rate oxidation ponds (Park et al. 2011). In India >40 billion litres per day of wastewaters are generated mostly from the urban areas (CPCB, 2011) and these untreated or partially treated wastewaters find their way into receiving water bodies enriching the aquatic systems with nutrients (Mahapatra et al. 2011a,b). The suspended and attached growth bacterial systems are not popular in India due to the economic barrier apart from the complexities involved in the operation and management. This necessitates a simple, economic and sustainable wastewater treatment system at decentralised levels.

Algae based domestic wastewater treatment has been practiced for a long time for cost effective treatment and nutrient recovery (Oswald 1957; Pittman et al. 2011). These systems would offset OM costs through biomass use as bio-fertilisers, bio-chemicals as proteins, pigments etc., (de la Noue et al. 1992; de Bashan and Bashan 2004). Energy generation from algal biomass was advocated in 1960’s (Oswald and Golueke 1960) and gained momentum in recent times owing to the exhaustion of fossil based reserves resulting in increased fuel prices, and also due to increased green house gas (GHG) emissions (Ramachandra et al. 2009; Wang et al. 2010a,b; Pittman et al. 2011). Among the many species of algae that grow in wastewaters, oleaginous algae species act as a possible feedstock for biodiesel production as a viable alternative for fossil fuels (Chisti 2007). Oleaginous algal species have been used for municipal wastewater treatment due to their abilities to uptake nutrients and grows profusely in wastewater with varied Carbon (C), Nitrogen (N) and Phosphorus (P) concentrations (Mahapatra et al. 2011a,b; Noue et al. 1992; Pittman et al. 2011). However, there are constraints like light absorption due to dense algal growth, longer retention time, etc. (Savage 2011).

Earlier studies (Bhatnagar et al. 2010; Ruiz-Marin et al. 2010; Wang et al. 2010a) have reported high biomass productivity and complete removal of nutrients by Chlorella and Scenedesmus sp. Removal experiments include either batch or semi-continous laboratory or pilot-scale cultures (Ruiz-Marin et al. 2010; Wang et al. 2010b). Chlorella minutissima grown in wastewaters have  higher growth rates of 380 mg L^-1 under heterotrophic conditions compared to 73 mg L^-1 under phototrophic conditions in raw sewage (Bhatnagar et al. 2010). This suggests chlorophycean members such as Chlorella are better assimilators of nutrients. In addition to these, algal growth and nutrient removal have also been reported from artificial wastewaters (Voltolina et al. 1999; Li et al.  2010; Feng et al. 2011; Yujie et al. 2011).

Wastewaters provide a sustainable growth medium for algal-biofuel feedstock (Chanakya et al. 2012a,b) and the nature, type of lipids (saturates, unsaturates, polyunsaturates and glyco/phospho lipids or TAGS) and the quantity of lipids produced depends on the species and its growth conditions (Borowitzka 1992; Chisti 2007; Griffiths and Harrison 2009; Ramachandra et al. 2009). Reports reveal of high concentrations of lipids in algal cells (Griffiths and Harrison 2009) and low algal-biomass productivities (Dean et al. 2010), due to stress induction (N and P limitation). However, unlimited nutrient supply in wastewaters often results in the enhanced algal cell densities fixing larger amounts of lipid (Wang et al. 2010a). Nutrient deprivation studies have reported an increased lipid/protein (L/P) ratio and Carbohydrate/Amides (C/P) ratio (Stehfest et al. 2005; Dean et al 2010, 2012). Laboratory studies carried out in batch and semi-continuous cultures in bioreactors and ponds have reported lipid accumulation in the wastewater grown algae, yielding low (<15%), moderate (25-30%) lipid content and in many cases a very high lipid yield. Rapid ways of quantification for algal lipid accumulation and tracking lipids over the culture period include FTIR spectroscopy (Stehfast et al. 2005; Dean et al. 2010; 2012), which helps in the study of C transformations and allocation in algal cells (Dean et al. 2010, 2012). The role of mixotrophic algae like Chlorella sp. and Scenedesmus sp. has been widely reported in the wastewater treatment and lipid production (Wang et al. 2010a; Li et al. 2010). However there are no studies on euglenoides (Euglena spp.) for their abilities of nutrient removal and biofuel production. Euglenoides grow profusely under anoxic conditions in sewage with high organic loads. The current study investigates the role of mixotrophic indigenous algae Euglena sp. in the removal of nutrients from wastewater and lipid production. The objectives of the present study were

1. to evaluate the utility of Euglena sp. in wastewater treatment and assess the nutrient (C, N and P) removal capability;
2. to assess the cellular compositional changes in the algae with the culture time; and
3. to determine the biomass and lipid productivities together with fatty acid composition for assessing the suitability of wastewater grown Euglena sp. as a candidate for sustainable biofuel production