Melinda J. Griffiths

LIPID PRODUCTIVITY AS A KEY CHARACTERISTIC FOR CHOOSING ALGAL SPECIES FOR BIODIESEL PRODUCTION

Microalgae are a promising alternative source of lipid for biodiesel production. One of the most important decisions is the choice of species to use. High lipid productivity is a key desirable characteristic of a species for biodiesel production. This paper reviews information available in the literature on microalgal growth rates, lipid content and lipid productivities for 55 species of microalgae, including 17 Chlorophyta, 11 Bacillariophyta and five Cyanobacteria as well as other taxa. The data available in the literature are far from complete and rigorous comparison across experiments carried out under different conditions is not possible. However, the collated information provides a framework for decision-making and a starting point for further investigation of species selection. Shortcomings in the current dataset are highlighted. The importance of lipid productivity as a selection parameter over lipid content and growth rate individually is demonstrated.

Selection of Direct Transesterification as the Preferred Method for Assay of Fatty Acid Content of Microalgae

Assays for total lipid content in microalgae are usually based on the Folch or the Bligh and Dyer methods of solvent extraction followed by quantification either gravimetrically or by chromatography. Direct transesterification (DT) is a method of converting saponifiable lipids in situ directly to fatty acid methyl esters which can be quantified by gas chromatography (GC). This eliminates the extraction step and results in a rapid, one-step procedure applicable to small samples. This study compared the effectiveness of DT in quantifying the total fatty acid content in three species of microalgae to extraction using the Folch, the Bligh and Dyer and the Smedes and Askland methods, followed by transesterification and GC. The use of two catalysts in sequence, as well as the effect of reaction water content on the efficiency of DT were investigated. The Folch method was the most effective of the extraction methods tested, but comparison with DT illustrated that all extraction methods were incomplete. Higher levels of fatty acid in the cells were obtained with DT in comparison with the extraction-transesterification methods. A combination of acidic and basic transesterification catalysts was more effective than each individually when the sample contained water. The two-catalyst reaction was insensitive to water up to 10% of total reaction volume. DT proved a convenient and more accurate method than the extraction techniques for quantifying total fatty acid content in microalgae.

Interference by pigment in the estimation of microalgal biomass concentration by optical density

Optical density is used as a convenient indirect measurement of biomass concentration in microbial cell suspensions. Absorbance of light by a suspension can be related directly to cell density using a suitable standard curve. However, inaccuracies can be introduced when the pigment content of the cells changes. Under the culture conditions used, pigment content of the microalga Chlorella vulgaris varied between 0.5 and 5.5% of dry weight with age and culture conditions. This led to significant errors in biomass quantification over the course of a growth cycle, due to the change in absorbance. Using a standard curve generated at a single time point in the growth cycle to calculate dry weight (dw) from optical density led to average relative errors across the growth cycle, relative to actual dw, of between 9 and 18% at 680 nm and 5 and 13% at 750 nm. When a standard curve generated under low pigment conditions was used to estimate biomass under normal pigment conditions, average relative errors in biomass estimation relative to actual dw across the growth cycle were 52% at 680 nm and 25% at 750 nm. Similar results were found with Scenedesmus, Spirulina and Nannochloropsis. Suggested strategies to minimise error include selection of a wavelength that minimises absorbance by the pigment, e.g. 750 nm where chlorophyll is the dominant pigment, and generation of a standard curve towards the middle, or across the entire, growth cycle.

Advantages and Challenges of Microalgae as a Source of Oil for Biodiesel

Microalgal oil is currently being considered as a promising alternative feedstock for biodiesel. The present demand for oil for biofuel production greatly exceeds the supply, hence alternative sources of biomass are required. Microalgae have several advantages over land-based crops in terms of oil production. Their simple unicellular structure and high photosynthetic efficiency allow for a potentially higher oil yield per area than that of the best oilseed crops. Algae can be grown on marginal land using brackish or salt water and hence do not compete for resources with conventional agriculture. They do not require herbicides or pesticides and their cultivation could be coupled with the uptake of CO2 from industrial waste streams, and the removal of excess nutrients from wastewater (Hodaifa et al., 2008; An et al., 2003). In addition to oil production, potentially valuable co-products such as pigments, antioxidants, nutraceuticals, fertilizer or feeds could be produced (Mata et al., 2010; Rodolfi et al., 2009). Despite these advantages, algal fuel is not currently in widespread use, largely due to its high cost of production (Chisti, 2007; Miao & Wu, 2006). Despite strong interest from the commercial and scientific sectors, there are currently no industrial facilities producing biodiesel from algae (Lardon et al., 2009). One of the major economic and technological bottlenecks in the process is biomass and lipid production by the algae (Borowitzka, 1992; Sheehan et al. 1998; Tsukahara & Sawayama, 2005). Productive strains and optimized culture conditions able to produce cells with a simultaneously high growth rate and lipid content are required. The high cost and energy demand of harvesting unicellular algae also remains a major challenge. The small cell size (often < 10 m in diameter) and dilute biomass produced requires innovative solutions to minimize the consumption of water and energy as well as processing costs (Rodolfi et al., 2009). This chapter provides an overview of microalgae as a source of oil for biodiesel, focusing on:  A description of algae and their properties with regards to oil production  Requirements and key factors in microalgal cultivation  Methods and challenges in harvesting and processing of algal biomass  Economic and environmental feasibility of microalgal biodiesel  Mechanisms to enhance lipid productivity of microalgae and future research directions.

Major Commercial Products from Micro- and Macroalgae

Macro- and microalgae are used in a variety of commercial products with many more in development. This chapter outlines the major products, species used, methods of production, extraction, and processing as well as market sizes and trends. Foods, nutraceuticals, and feeds are the major commercial products from algae. Well-known culinary products include Nori, Wakame, Kombu and Dulse, from whole macroalgal biomass. The microalgae Spirulina and Chlorella have been widely marketed as nutritional supplements for both humans and animals. Several microalgae with a high nutritional value and energy content are grown commercially as aquaculture feed. The major processed products from macroalgae are the hydrocolloids, including carrageenan, agars, and alginates, used as gelling agents in a variety of foods and healthcare products. Pigments extracted from algae include β-carotene, astaxanthin, and phycobiliproteins. These are generally used as food colorants, as additives in animal feed or as nutraceuticals for their antioxidant properties (Radmer in Bioscience 46:263–270, 1996; Pulz and Gross in Applied Microbiology and Biotechnology 65:635–648, 2004). Polyunsaturated fatty acids (PUFAs) are another high value product derived from microalgae. Other potential products include fertilizers, fuels, cosmetics and chemicals. Algae also have application in bioremediation and CO2 sequestration, as well as producing many interesting bioactive compounds. Algae have great potential to produce a wide range of valuable compounds, beyond their current exploitation. To date, commercialization of new products has been slow (Milledge in Reviews in Environmental Science and Biotechnology 10:31–41, 2011; Wijffels in Trends in Biotechnology 26:26–31, 2007; Radmer in Bioscience 46:263–270, 1996; Pulz and Gross in Applied Microbiology and Biotechnology 65:635–648, 2004; Spolaore et al. in Journal of Bioscience and Bioengineering 101(2):87–96, 2006), however, microalgal biotechnology is a relatively new industry, and therefore, it is unsurprising that significant challenges remain to be solved. The advantages associated with algal production are likely to ensure that efforts continue.

Techno-economics of Algal Biodiesel

Algal biodiesel production presents a possible carbon-neutral source of transportation fuel. Whilst algal biodiesel circumvents some of the issues arising from the use of crop- and waste–biomass-based fuels, the lack of commercial success raises questions regarding the feasibility of the process. Numerous economic and environmental impact assessments have produced highly variable results, predicting costs from as little as 0.42–72 USD L−1. A meta-analysis of these assessments reveals that areal productivity and provision of nutrients, as well as energy and water usage, are the key challenges to algal biodiesel production. A consideration of maximum achievable photosynthetic activity indicates that some scope exists for increasing areal productivity; hence, the factors influencing productivity are discussed in detail. Carbon dioxide supply may represent the single most important challenge to algal biodiesel, while recycling of other nutrients (specifically nitrogen and phosphate) is essential. Finally, a careful balance must be struck between energy and water consumption; this balance is primarily influenced by bioreactor design. It is unlikely that algal biodiesel will supply a substantial portion of the world’s transportation energy demand, but it may fill niche markets such as aviation fuel. Process economics are enhanced by integrating biodiesel production into a biorefinery, producing a suite of products.