Micro Algae Biofuel Energetic

REVIEW
www.rsc.org/ees | Energy & Environmental Science
Microalgae as biodiesel & biomass feedstocks: Review & analysis of the biochemistry, energetics & economics
Peter J. le B. Williams* and Lieve M. L. Laurens
Received 26th November 2009, Accepted 18th February 2010 First published as an Advance Article on the web 22nd March 2010 DOI: 10.1039/b924978h Following scrutiny of present biofuels, algae are seriously considered as feedstocks for next-generation biofuels production. Their high productivity and the associated high lipid yields make them attractive options. In this review, we analyse a number aspects of large-scale lipid and overall algal biomass production from a biochemical and energetic standpoint. We illustrate that the maximum conversion efciency of total solar energy into primary photosynthetic organic products falls in the region of 10%. Biomass biochemical composition further conditions this yield: 30 and 50% of the primary product mass is lost on producing cell protein and lipid. Obtained yields are one third to one tenth of the theoretical ones. Wasted energy from captured photons is a major loss term and a major challenge in maximising mass algal production. Using irradiance data and kinetic parameters derived from reported eld studies, we produce a simple model of algal biomass production and its variation with latitude and lipid content. An economic analysis of algal biomass production considers a number of scenarios and the effect of changing individual parameters. Our main conclusions are that: (i) the biochemical composition of the biomass inuences the economics, in particular, increased lipid content reduces other valuable compounds in the biomass; (ii) the biofuel only option is unlikely to be economically viable; and (iii) among the hardest problems in assessing the economics are the cost of the CO2 supply and uncertain nature of downstream processing. We conclude by considering the pressing research and development needs.
1. Introduction and historical background

1.1 History of biofuels development Without question our society will need to move away from its strong dependence upon fossil fuels as sources of energy and to
School of Ocean Sciences, University of Bangor, Menai Bridge, Anglesey, UK LL595PP Electronic supplementary information (ESI) available: Appendices IIV. See DOI: 10.1039/b924978h Current address: National Bioenergy Center, National Renewable Energy Laboratory, 1617 Cole Blvd, Golden, CO 80401, USA
reduce the emissions of greenhouse gases. Although alternatives exist for land-based transport notably electrical power there is an understandable reluctance to abandon liquid hydrocarbons and internal combustion engines for many transport purposes. Further, in the case of aviation transport and shipping, there is no practical alternative in the foreseeable future. These are amongst a number of reasons for the drive for the industrial development of liquid biofuels. Systems to produce biodiesel and bioethanol from crop plants (so-called rstgeneration biofuels) have been developed and optimised over the past several decades and are currently run as protable businesses.
Broader context
There is a rapidly growing interest in the potential of microalgae as feedstocks for the next generation of biofuels. Working from fundamental biochemical principles, we consider the potential yields of organic production by photosynthesis. The maximum theoretical energy conversion of full spectrum sunlight to organic material lies in the region of 10%. The yields are constrained by thermodynamics and stoichiometry. The yields obtained with outdoor cultures are characteristically one third to one tenth of this theoretical yield; the losses are primarily due to the inability of the photosynthetic system to process the captured photons at the rate they are absorbed and so energy is lost. Overcoming this problem is a major challenge. The mass and, to a lesser extent, the energy yields are further reduced following the conversion of the primary photosynthetic products to the spectrum of biochemicals required by the cell. With a simple light-driven model, we explore the potential economics of a number of production scenarios of algae of varying lipid content (1550% lipid of the cells dry weight) at low (030 ), intermediate (3545 ) and high (4555 ) latitudes. The main conclusions were that (i) the fuel only option is not viable, markets need to be found for the other major and minor components of the cell; (ii) high lipid containing algae may not necessarily be the most favourable candidate organisms. We conclude that although the potential does appear to exist for economic production of algal biofuels, a major R & D programme would be called for to convert the concept to a reality.
554 | Energy Environ. Sci., 2010, 3, 554590 This journal is The Royal Society of Chemistry 2010
Although the production and development of biodiesel and bioethanol has increased rapidly since the start of biofuels development and production in the 1970s, at 3040% per annum, the total energy content in both biofuels is still less than 1% of the worlds energy production. The 2008 production gure for bioethanol was 2.5 1018 joules and that for biodiesel an order of magnitude less by comparison to a total global energy use of 5 1020 joules. Despite their relatively small contribution ($1%) to the overall production of liquid fuels, these rst-generation biofuels have come under considerable international scrutiny and criticism. The main criticisms are the encroachment of the biofuels feedstock production on valuable crop and virgin land, and the effect biofuels have on food commodity prices. These issues have raised major question marks surrounding their social benets, as summed up in the 2008 Gallagher report to UK House of Commons.129 The executive summary of that report noted that feedstock production must avoid agricultural land that would otherwise be used for food production and The introduction of biofuels should be signicantly slowed until adequate controls to address displacement effects are implemented and are demonstrated to be effective (the embolding is ours). This mood, which although not universal, is widespread and has given rise to searches for alternative, so-called second-generation biofuels and has re-stimulated interest in mass algal biomass production. 1.2. Algal biofuels potential
We have restricted the review to microalgae. Macroalgae (seaweeds) have long been grown commercially and may have a role as biofuels and although a number of the aspects of their physiology are shared with the microalgae, their mass production involves a substantially different set of considerations. The advantages of microalgae as a feedstock for biodiesel production, over terrestrial plants, are that there is no requirement for soil fertility and, if marine algae are used, there is no need to draw upon valuable and often scare supplies of
freshwater. Thus, their negative ecological impact is much reduced as compared with higher plants.1 The potential to produce the feedstock on waste or desert land and the reduced freshwater requirements addresses some of the concerns in the Gallagher report. The mass production of microalgae for lipid production has a long history. Prior to the present interest in producing biofuels to mitigate CO2 release, there had been an extended period of research interest motivated by security of oil supply. This was initially prompted by the 1973 oil crisis. In response to the recognition of the potential vulnerability of oil supplies, Exxon Research and Engineering Company, for example, funded work of lipid production by algae.2 At about the same time (1978 to be precise), the U. S. Department of Energy set up the Aquatic Species Program (ASP). This program reached peak funding in the mid-1980s; after which the funding dwindled, until the program was eventually closed down in 1996. All told, just over $25 million was invested in the program. The ndings, which were extensive and valuable, are collected in a major report.3 There was a concurrent Japanese programme, which purportedly cost in excess of $100 million, but which produced very little accessible science. The main conclusion of the ASP report was that algal biofuels are a potential valuable alternative to traditional biofuels, however, considering the oil prices of the early 1990s, the calculated economics turned out to not be protable. Nevertheless, considerable headway was made with regards to basic biology, strain selection, metabolic engineering and largescale growth cultures of selected strains. Algae have long been known to produce a great variety of lipids, hydrocarbons and other complex oils (reviewed recently in ref. 4,5). Cultured algae have been used as feeds for aquaculture applications because of their high content of nutritionally essential polyunsaturated fatty acids. A further motivation for algal culture has been the production of high value by-products such as the pigments astaxanthin (a food colorant and antioxidant from Haematococcus pluvialis) and b-carotene (a food additive produced from Dunaliella species). These sorts of
Peter Williams is professor emeritus at University of Bangor, UK. Originally trained as an industrial biochemist, he came to the view that oceanography offered more interesting challenges. He has held posts at the Woods Hole Oceanographic Institute, Southampton University, Bigelow Lab in Maine and the University of Gothenburg, returning to the UK to become Professor of Marine BiogeoPeter J: le B: Williams chemistry at Bangor Universitys School of Ocean Sciences. His long term research interest has been the dynamics of organic material in the oceans, but an opportunity provided by Shell, opened up a new interest in the science behind algal biomass and biofuel production.
This journal is The Royal Society of Chemistry 2010
Dr Lieve Laurens is a researcher at the National Renewable Energy Laboratory (NREL) in Golden, Colorado. Prior to joining NREL, she was a Research Associate at the Centre for Applied Marine Science in the School of ocean Sciences, at the University of Bangor, UK, where she started research on biodiesel from microalgae and the work leading to this review was initiated. Her Lieve M: L: Laurens recent work focuses on the quantication and analysis of lipid yield and composition in microalgae using high-throughput spectroscopic methods. She holds a PhD from the Department of Metabolic Biology at the John Innes Centre in Norwich, UK.
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production processes have been in place since the early 1950s, so many aspects of the technology of mass algal growth may be considered mature.6 Much historical research has focused on the lipid composition from either a taxonomic or nutritional standpoint. There is, however, a sharp difference between the growth of algae for nutritional and for fuel use. These nutritional products are high value: astaxanthin for example commands a price in the region of $3 million tonne1,7 compared with less than a $1000 tonne1 for crude oil. Thus the economics are profoundly different. As a result, the production of biofuels will require a fundamental change in the approaches to production compared with nutraceutical and aquaculture feed-grade products and without question fresh, major challenges. Algal biomass can serve as a feedstock for the production of a variety of different biofuels, e.g. biodiesel, hydrogen, methane and bioethanol. Furthermore, its production is also being seriously considered for the removal of carbon dioxide from the ue gases of fossil fuel power stations. In order to maintain focus, we limit our discussion to the use of algae as feedstock for biodiesel and biomass production. There have been claims in the literature over recent years hailing algae as the solution to the global energy crisis (e.g. ref. 8). In this review we aim to present the principles behind large-scale production of algal biomass and biodiesel production along with a critical and objective review of the literature to date. As others (e.g. ref. 9), we build our review on the biochemical fundamentals of photosynthesis and biomass production, and accordingly begin (Section 2) with a brief discussion of the biology and biochemical composition of microalgae, with particular regard to the major biochemical categories: proteins, carbohydrates and lipids. In Section 3, starting from rst principles, we establish the overall potential efciency of photosynthetic production of algae. A major challenge in algal biofuels production is maximising this stage, as economic models for biodiesel production from seed oils have acknowledged that the feedstock cost comprises a substantial portion of the overall biodiesel cost.10 Accordingly, in Section 4 we address the growth of algae, the controls on growth and the reported yields. These are then combined with the theoretical photosynthetic yields to give predictions of biomass yields for season and latitude. Section 5 contains a review of the harvest and processing of the biomass relevant to published processes. In Section 6, we bring together the ndings of the two preceding sections in the form of an economic analysis of various scenarios for low, mid and high latitudes. Finally, in Section 7, we explore the answers to two questions: (i) can the production of biofuels from algae be economically viable; and (ii) what R & D is needed to achieve a protable outcome.
the eukaryotes and bacteria. They are able to photosynthesise, and are found in a range of different habitats, from fresh to marine and hyper-saline environments.11 The large number of species are generally subdivided into 10 taxonomic groups which include the green algae (Chlorophyceae), diatoms (Bacillariophyceae), yellow-green (Xanthophyceae), golden algae (Chrysophyceae), red algae (Rhodophyceae), brown algae (Phaeophyceae), dinoagellates (Dinophyceae), Prasinophyceae and Eustigmatophyceae.12 The blue-green algae (Cyanophyceae) were originally grouped with the eukaryotic algae; however it was subsequently realised that they belong to the bacterial domain, hence their present common name cyanobacteriax. A major and signicant difference between the bacteria and the eukaryotes is that the former lack discrete internal, subcellular structures, organelles (chloroplasts, mitochondria, nuclei). Organelles are surrounded by lipid membranes whose bilayer structure requires strongly polar molecules. The triglycerides, the predominant lipid class in higher plant oils, lack the required polarity to form a sufciently stable bilayer. The more polar phospholipids and the glycolipids are the major components of cell membranes and as a result, in actively growing cells, these two groups are major components of algal lipids, with important consequences. The metabolism of organisms, particularly microorganisms, is strongly inuenced by their surface-to-volume ratio. Simple, single celled organisms can be approximated to spheres, thus their metabolism, and therefore growth rate, is inversely proportional to their cell diameters (see insert in Fig. 1). The growth rates of microorganisms can be very high; whilst some algae are able to divide once every 34 h, most divide every 12 days under favourable conditions (see Fig. 1). Accordingly their scope for growth is colossal and this, in major part, is the basis for the interest in their potential as biomass producers. In principle algal biomass crops may be harvested either daily or every few days (e.g. ref. 15, 16).
2.1.
Major biochemical groups, their presence and function
It is conventional to consider four principal biochemical classes of molecules: carbohydrates, proteins, nucleic acids and lipids. Table 1 gives the broad cell content of these major fractions, their elemental composition and energetic properties. (i) Carbohydrates. Microorganisms contain a wide variety of carbohydrates, both monomers and polymers. Carbohydrates serve both structural and metabolic functions and, as the early products of photosynthesis, they serve as the starting point for the synthesis of the other biochemicals. Different classes of algae produce specic types of polysaccharides. For example, green algae produce starch as an energy store, consisting of both amylose and amylopectin, similar to higher plants. The green alga
x Strictly the term alga should be limited to the eukaryotic phototrophs, since cyanobacteria (formerly known as blue-green algae) are a form of bacteria, with very different genetics and evolutionary history. It is however very cumbersome to qualify every reference to these two groups of single-celled phototrophs as algae and cyanobacteria, or as micro-photoautotrophs so, unless there is need to be specic, for convenience the two are referred in the text simply as algae.
2. Biology and biochemical composition of microalgae

In this section we introduce the aspects of algal biology and biochemistry that are relevant in the economical considerations (Section 6). The composition of the algal biomass with regards to lipids, carbohydrates and proteins will greatly determine its overall value. Microalgae are single cell organisms, found in either colonies or individual cells and are comprised of representatives of both
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Proteins have important commodity value as animal feed. Critical to this is their amino acid composition, as a number of amino acids are dietary essentials for mammals yet they are unable to synthesise them, for example. Fig. 2 shows the mean amino acid spectrum for a number of algae. It can be seen that the spectrum compares favourably with proteins of high nutritional quality. (iii) Nucleic acids. Nucleic acids (RNA and DNA), in conjunction with proteins and their monomers, provide the basis for algal division and growth. The nucleic acids comprise a small fraction of cellular biomass but the major part of the cells phosphate and the second most important site of nitrogen (see Table 2). (iv) Lipids. As with carbohydrates, lipids serve both as energy reserves and structural components (membranes) of the cell. The simple fatty acid triglycerides are important energy reserves. Membranes are mainly constructed from phospholipids and glycolipids, where the hydrophilic polar phosphate or sugar moieties and the level of saturation of the fatty acyl chains determine the uidity of the membranes. The microalgae have the facility for rapid adaptation to new environments (e.g. changes in temperature), through the de novo synthesis and recycling of fatty acids to maintain the membrane characteristics. A large fraction of the cells phosphate may be present in phospholipids. The membrane lipids associated with the thylakoids (the internal chloroplast membranes, also the sites for photosynthetic activity in eukaryotes, see Section 3.1(i)) contain a sulfur lipid (sulfoquinovosyldiacyl glycerol), which is the major site of the sulfur in algal lipids.11 Table 3 gives a summary of lipid class distribution derived from published analyses. A number of factors give rise to the variations seen in the table. Storage lipids, which are predominantly triglycerides, may gain a greater proportion of the overall lipid fraction as the metabolic rate slows down. As a consequence, shifts in lipid composition occur through the various phases of growth. Allowing for the scatter due to differences in analytical techniques, the frequency distribution suggests a minimum cell lipid content in the region of 15% (see Fig. 3). There appears to be a marked difference between the total lipid content of the
Fig. 1 Frequency analysis of algal growth rates (main pane), derived from data in ref. 13 and algal growth-size relationships from ref. 13, 14.
Tetraselmis suecica, accumulates 11 and 47% of its dry weight as starch in nutrient replete and deplete conditions respectively.19 Red algae synthesise a carbohydrate polymer known as oridean starch, consisting mostly of amylose.20 A commonly found polysaccharide in a large number of algal species is chrysolaminarin, a linear polymer of b(1 / 3) and b(1 / 6) linked glucose units.21 Chrysolaminarin often accumulates in pyrenoids, which are centres of high activity of carbon assimilation in the chloroplast. (ii) Proteins. As do carbohydrates, proteins also have both structural and metabolic functions. As enzymes, they are the prime catalysts for cell metabolism and so facilitate growth. Second, they serve a structural role. For example, they provide the scaffold upon which the chlorophyll molecules are assembled in the light harvesting complexes of the chloroplast (see Section 3.1(i)) and also are embedded in the lipid membranes, where they serve a similar structural role, as well as a metabolic role. Furthermore, it is known that the structural cell wall of Chlamydomonas reinhardtii consists primarily of cross-linked hydroxy-proline-rich glycoproteins.22
Table 1 Elemental composition of algal biochemical components. Data for the proteins and fatty acids were derived from means from the analyses shown in Fig. 3 and 5. The values for the lipid classes are derived from the mean fatty acid elemental composition and the composition of the other molecular structures in the overall molecule. That for the overall algal lipids is derived from the values for the individual lipid classes and the mean lipid class composition given in Table 3. The values for the nucleic acids and carbohydrates are taken from ref. 17. The caloric values (as kJ g1) of lipids and their derivatives have been calculated according to an equation for higher heating values (equivalent to the bomb caloric value) 35.17C + 116.25H 11.1O + 6.28N + 10.47S, where C, H, O, N, P and S are the mass fractions of the constituent elements.18 This equation gives a good approximation for these compounds. The hydrogen and oxygen atoms associated with phosphate and sulfate were not included in the calculation. The caloric values for proteins and nucleic acids are calculated from the caloric values of their component molecules, using 18 kJ per mol of water as the energy lost on polymerisation. The range given in the right hand column derives from the data compilations used to produce Fig. 5 (only the middle 90% of the data is used) Characteristic elemental composition C1H1.83O0.17N0.0031P0.006S0.0014 C1H1.83O0.096 C1H1.79O0.24S0.0035 C1H1.88O0.173N0.012P0.024 C1H1.91O0.12 C1H1.92O0.05 C1H1.56O0.3N0.26S0.006 C1H1.23O0.74N0.40P0.11 C1H1.67O0.83
Biochemical component Algal lipids Acylglycerides Glycolipids Phospholipids Algal fatty acid Methyl esters Protein Nucleic acid Polysaccharide
Calculated caloric value/kJ g1 36.3 40.2 33.4 35.3 39.6 43.0 23.9 14.8 17.3
Range of typical cell content (%) 1560 2060 35 1050
Fig. 2 Mean amino acid composition of algal protein derived from 28 species (9 classes) of eukaryotic algae.23 The upper bars are the standard deviations, the lower the standard errors. The low value for tryptophan will derive in part from loss of the molecule during acid hydrolysis of the protein. Characteristic values for high quality proteins are shown for comparison purposes.
eukaryotic and the prokaryotic algae (the cyanobacteria) the latter containing less than the former (see Fig. 3), likely to be due to the absence of internal membranes in the prokaryotes (see Section 2.1 (i)). So, although the cyanobacteria are easier organisms to manipulate genetically (as a result of their simpler DNA), their potential as lipid producers would not appear to be promising. The fatty acid composition of algal lipids (Fig. 4) is well documented, with a high occurrence of unsaturated (and polyunsaturated) fatty acids, with half of the fatty acids having a carbon number less than C18. The majority of the unsaturated fatty acids occur in the membrane lipids, where their main function is to maintain membrane uidity under different conditions. The high concentrations of unsaturated fatty acids in the extracted lipids, and ultimately in the resulting fuel, will be an important fuel quality determinant. The preponderance of the shorter chain fatty acids has signicance for their potential as diesel fuels (dened as alkyl chains of between 12 and 18 carbon atoms long). The level of unsaturation affects biodiesel properties.36 For example, fuels with a higher level of unsaturation of the acyl chains have a higher cloud point, which is desirable, but are also much more susceptible to oxidation. The highly unsaturated fatty acids found in algae may need to be hydrogenated to improve their potential fuel properties. Furthermore, a high level of unsaturated fatty acids in a fuel increases the danger of polymerisation in the engine oil and can cause problems with oxidative stability of the fuel. 2.2. Shifts in the biochemical composition with increasing lipid
proteins preferentially run down or do the carbohydrates and proteins decrease in relative proportions. These three trajectories are shown in the triangular plot in Fig. 5. In the case of rst of the three scenarios listed above, we might expect maintenance of growth rate with increases in lipid content up to 4050%, whereas in the others we would anticipate a reduction in growth rate as the lipid content increased (see discussion below in Section 2.3). The data we have been able to assemble in Fig. 5 does not give a clear sign of any systematic compositional trend and probably a dedicated study would be needed to elicit one, if it indeed exists. 2.3. Relationship between lipid content and growth and nutrient status Fig. 6 contains data from two papers2,42 and an analysis of the relationship between lipid content and growth rate. There is scatter in the data and the absolute rates differ, however there is a signicant inverse relationship between growth rate and lipid content (p value for ref. 2 data set is 0.0006 and that for ref. 42 data set is 0.0014). A similar inverse relationship is also reported in ref. 131. The product of the lipid content and growth rate will give a lipid-normalised production rate (in units of days1) and the relationship between this latter property and lipid content will take on a quadratic form, with an intermediate maximum. The rst derivative of the quadratic equation will give the lipid content for the maximum lipid production rate (mass of lipid produced per mass of lipid present in the culture per day). To demonstrate the outcome, and potential, of this procedure, we have processed the parameters from the two equations given in Fig. 6. The analysis gives a maximum lipid production rate at a lipid content of ca. 15% (from the analysis in ref. 42s data set) and ca. 30% (for ref. 2). From these values, an optimum growth rate for lipid metabolism may be calculated from the initial equation. Estimated maximum lipid production and growth rates are valuable for process design and management, e.g. to allow culture dilution rates to be optimised without deleterious effect on overall lipid yield. However, bespoke data sets are needed to establish to what extent the relationship seen in Fig. 6 is general. Overall production of lipid will be more complex than this simple analysis, as there are many other considerations, but the calculation brings home that high lipid content alone cannot regarded as the sole, perhaps not even the major, consideration when searching for suitable strains or optimum growth conditions.
As the lipid content increases, the percentage of the sum of the other components must go down. Fig. 5 gives a summary of the reported relative proportions of the three major biochemicals protein, lipid and carbohydrate. For two reasons the shift in protein content is critical. First these molecules set the level of the cells metabolism and in conjunction with the nucleic acids determine the growth rate potential. Second, from an economic point of view proteins are valuable bulk components of the cell (see Section 6). Thus, in designing a strategy to search and select for the economically most suitable algae, it is of prime importance to establish whether or not we can generalise over the change in protein content associated with changes in total lipid content. As the lipid content increases, the question arises, are the carbohydrates preferentially run down and the protein content held constant as long as possible. Alternatively, are the
Table 2 Calculated distribution of major elements in the four principal biochemical fractions for model low (15%), medium (25%) and high (50%) lipid-containing algae. The calculation is made using the elemental compositions and caloric values given in Table 1 and assumes that, as the lipid content varies, the nucleic acid content remains constant at 5% and protein : carbohydrate ratio remains constant at 3 : 2 (see legend to Fig. 5). These simplications mean that the data are mainly illustrative
Table 3 Mean ( standard error) lipid class content as a percent of total lipid of individual algal species, plus a composite from all reports for microalgae.2435 Simple lipids Diatoms Chaetoceros species Phaeodactylum tricornutum Range from Borowitzka (1988)130 Green algae Chlamydomonas species Dunaliella tertiolecta Dunalliella viridis Range from Borowitzka (1988)130 Blue green algae Range from Borowitzka (1988)130 Others Nannochloropsis oculata Isochrysis species Composite from all reports (n 46) 37 16 54 6 1460 48 10 71 13 1 2166 1168 22 1 36 3 35 3 Glycolipids 36 8 34 5 1344 44 13 67 1 44 3 662 1241 39 0 35 1 40 2 Phospholipids 25 8 11 1 1047 63 25 0 42 2 1753 1650 38 1 27 3 25 2
High
Medium
15 85 0 0 9 91 0 0 52 0 0 48 35 0 0 65 25 0 0 75 2 82 0 15 1 88 0 11 0 90 0 10
Low
High
Medium
Low
High
Medium
Low
8.7
7.8
5.4
0.7
0.81
1.1
0.5
0.4
36 64 0 0
Percentage major element in various biochemical fractions
It has been known since the 1940s that cell lipid content increases during nutrient limitation (see Fig. 7). As nutrient limitation also affects growth rate, this provides an explanation for the apparent inverse relationship between growth rate and lipid content (see Fig. 6). Ref. 43 drew the broad conclusion that when the nitrogen ran out, the organisms were forced to terminate the production of nitrogen containing material (proteins and nucleic acids) but continued to synthesise lipids and carbohydrates. Whereas it may explain the lipidnutrient relationship, it cannot explain observations where growth was controlled by light rather than inorganic nitrogen and where again a negative relationship was seen.42 This, and other work, suggests that the matter may be more complex than ref. 43 suggested. Regardless of the biochemical mechanism, the evidence for of a negative relationship between growth rate and lipid content (see Fig. 6) has an important bearing on the strategy adopted for biomass production, especially when products other than the lipid component are valuable (see e.g. Fig. 24).
High
Medium
33 24 36 7 14 32 48 6 8 34 52 6 63 22 13 2 36 39 23 2 23 47 28 3 59 24 13 3 33 41 23 3
Low
High
Medium
Low
High
Lipid level
Medium
55
60
7.2
7.6
8.6
30
29
24
2.4.
Algal lipids as fuels
Low
From their molecular composition, we can anticipate a number of properties relevant to the potential of algal lipids to serve as fuels. A summary of the fuel properties of biodiesel from a microalga (heterotrophically grown Chlorella protothecoides)44 is given in Table 4. Algal oils differ from higher plant oils in their high phospholipid and glycolipid concentrations. These lipid classes contain nitrogen, phosphorous and sulfur that may be problematic with regards to engine performance, if present in fuels. However, it is likely that the sulfur, phosphorus and nitrogen-containing compounds would end up in the watersoluble fraction following transesterication, so one would expect these elements to be low to non-existent in algal biodiesel. Since about 30% of the original lipid mass can be lost to the polar phase during esterication, the lipid class composition will also greatly affect the potential fuel yield by transesterication. Thus triglycerides have a > 99% biodiesel yield compared to a < 70%
High
Biochemical composition (as % total mass)
Medium
Lipids 15 25 Proteins 48 42 Polysaccharides 32 28 Nucleic acids 5 5 Caloric value/kJ g1 23.2 24.7 Element total (as % dry weight)
Lipid level
Low
50 27 18 5 28.5
20 49 27 3
53
Fig. 3 Frequency distribution of the lipid content (as % dry weight) of eukaryotic algae and Cyanobacteria. The values in parentheses are the number of data sets used for the analysis. Primary data and references in Appendix I (provided in the ESI).
Fig. 4 Upper histogram: mean fatty acid composition of various eukaryotic algal groups and Cyanobacteria. The gures in parenthesis are the numbers of analyses from which the means are derived. The upper error bars are standard deviations, the lower standard errors. Full data set and references in Appendix III (provided in the ESI). Lower histogram: data for comparison purposes for higher plant fatty acids (C. Price, Shell Global Solutions, personal communication).
yield for phospholipids.45 If the relative distribution of the lipid classes given in Table 3 is used, then the overall mass yield on producing biodiesel is calculated to be about 80%. This gure does not take into consideration the consumption of methanol during esterication; if the methanol used is considered as part of the original organic supply, as it could well be, then the overall organic yield is reduced to ca. 72%. The overall heating value of crude algal lipid is somewhat depressed to a value in the region of 36 kJ g1 (Table 1), due to the lower caloric value of the glyco- and phospholipids. However, as these fractions would be almost certainly separated off in the transesterication process, their low caloric value would have no effect on the properties of the nal biodiesel. From their calculated elemental composition (based on their reported fatty acid composition), we determine a heating value for the fatty acid methyl esters of 43 kJ g1, slightly higher than
the gure of 41 kJ g1 found in ref. 44. This derives from a slightly higher value in our case for the C : H ratio (see Table 4). The difference is small and, considering the two approaches used are so different, we do not place any great signicance on the variance between these two sets of values. The fuel characteristics will be greatly determined by the fatty acid composition and will thus vary with species and growth and nutrient conditions of the algal culture. As shown in Fig. 4, the chain length distribution of fatty acids occurring in microalgal species is more diverse compared with that of higher plants, making it possible that certain species will be cultured for selected fuel properties. For example, strains that primarily accumulate shorter (<C16) chain fatty acids may be more amenable for use in the production of jet fuels, compared to the application of very long chain fatty acids (>C20) in the lubricant market.
Fig. 5 A: triangular plot of the proportions of lipid, protein and carbohydrate content of algae, data taken from ref. 2, 27 and 3742. The red circle is the mean of the whole dataset for active growth (lipid 24.2%, protein 48.3%, carbohydrate 27.5%). The solid circles show the shift in composition from unlimited logarithmic growth (red) to N-limited growth (brown). The green line shows the trajectory on increasing lipid composition assuming the protein : carbohydrate ratio remains constant, the red line that if the carbohydrate content diminishes as the lipid content increases and the magenta line that if the protein content diminishes. Throughout this review we have used a rounded off value of 3 : 2 for the protein carbohydrate ratio.
Fig. 7 The effect of nitrogen limitation on the lipid content of eukaryotic algae. The values in parenthesis are number of observations from which the mean was derived. The upper error bar is the standard deviation, the lower the standard error. Full data set and references in Appendix II (provided in the ESI).
3. Principles and efciency of photosynthesis

Since the basis of algal biomass production is directly proportional to the efciency with which the algal cells assimilate carbon from the atmosphere through photosynthesis, we discuss the basic principles and efciency of photosynthesis in this section. The aim of this section is to provide a background to the theoretical and practical yield calculations in the subsequent sections so that the limitations to mass algal production can be made clear. Photosynthesis is the predominant process maintaining a whole host of elements (notably carbon, nitrogen and sulfur) out of thermodynamic equilibrium, and thereby driving their global geochemical cycles. It is the basis of the food supply for most life on Earth, maintaining the biosphere; it is also the ultimate source of all fossil fuels. Photosynthesis, in turn, is driven by photons, which, when absorbed by chlorophyll molecules, give rise to a charge separation and the ejection of
electrons. These electrons are driving the dissociation of water, generating protons and oxygen. The protons and the associated electrons enable the reduction of carbon dioxide to organic material, whereby oxygen is essentially a waste product.
3.1.
Mechanism of photosynthesis
The simple stoichiometry of photosynthesis may be written as: H2O + CO2 / [CH2O]{ + O2 However, the above simple equation implies that at least 50% of the atoms of the oxygen produced must come from the carbon dioxide, whereas both derive from water, thus the more appropriate equation is: 2H2O + CO2 / [CH2O] + O2 + H2O This overall reaction can be separated into two phases: (i) a set of photochemical and redox reactions (conventionally called the light reaction) and ii) a sequence of enzymatic reactions, often referred to as dark reactions but better as light-independent reactions, as they occur both in the light and the dark. Light reaction: 2H2O / 4[H] + O2 + energy Light-independent reactions: 4[H] + CO2 / [CH2O] + H2O (The notation [H] refers to the combination of the reduced coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) and an electron.) These reactions + O2 + energy are intimately connected within the cell. The light reaction operates on very short time scales (from femtoseconds to milliseconds) whereas the
{ The notation [CH2O] is commonly used as shorthand for organic material in biology in the present context its use is restricted to organic material with the same elemental ratio as monosaccharides.
Fig. 6 The variation of growth rate with lipid content. The data set from ref. 2 derives from 15 strains of freshwater and 11 strains of marine eukaryotic algae; the data used were taken during active logarithmic growth. The data from ref. 42 comes from 8 eukaryotic marine algae commonly used in aquaculture. In this case growth rate was controlled by varying the irradiance and the cultures were sampled during active growth.
Table 4 Comparison of properties of biodiesel from microalgae and diesel fuel and the ASTM biodiesel standard. The data from ref. 44 comes from a heterotrophically grown Chlorella protothecoides culture. There are small differences between their observations and the values we calculate (given in square parentheses) from elemental composition and caloric values given in Table 1 Properties Density/kg dm3 Viscosity/mm2 s1@ 40 C Flash point/ C Solidifying point/ C Filter plugging point/ C Acid value/mg KOH g1 Heating value/kJ g1 H/C ratio Micro-algal 0.864 5.2 115 12 11 0.374 41 [43] 1.8 [1.9] Higher plant 0.8770.887 3.35.2 0.160.43 39.540.3 Diesel fuel 0.838 1.94.1 75 5010 3.0 (max. 6.7) Max. 0.5 4045 1.8 ASTM biodiesel standard 0.860.90 3.55.0 Min. 100 Summer max. 0 Winter max. < 15 Max. 0.5
light-independent reaction operates over timespans of seconds to hours. This profound mismatch of timescales gives rise to inefciencies in uctuating environmental circumstances, notably irradiance and temperature variations and is a major problem in maximising yields of mass algal culture. (i) Light, its absorption and the formation of reducing capacity. Not all incoming radiation is available for photosynthesis. The solar spectrum is described by Plancks radiation distribution equation; the spectrum of light arriving at the surface of the planet has been attenuated by some 30% by the gases in the atmosphere and losses due to light scattering and absorption by clouds. The irradiance spectrum is shown in Fig. 8. The primary pigment involved in photosynthesis is chlorophyll a, which has strong absorption bands in the regions 400450 and 650700 nm (see Fig. 9). This delimits the useful range of incoming radiation to 400700 nm so called photosynthetically active radiation (PAR). PAR amounts to 4550% of the total incoming radiation, the exact value mainly being determined by the moisture content of the atmosphere attenuating the infrared part of the spectrum.47 Since we use clear sky radiation as the
Fig. 9 The spectrum of incoming radiation (black line) and the absorption spectrum of chlorophyll and accessory photosynthetic pigments. Redrawn with permission from ref. 46.
Fig. 8 The distribution of energy (blue line) and photons ux (red line) in incoming radiation. The lower green line shows the residual energy after it has been transferred to a frequency equivalent to that of chlorophyll a at 680700 nm absorption bands.
basis for further discussion here, the 45% end of the spread is the more appropriate value to adopt. Because of its low absorption in the range 450650 nm, chlorophyll a itself only is able to capture some 3040% of PAR. Plants have overcome this by introducing additional light capturing pigments (e.g. b-carotene) that ll in much of the chlorophyll a window of the spectrum (see Fig. 9) so increasing the portion of the spectrum that can be used for photosynthesis. Algae can change the quantity of these accessory pigments (subject to phylogenetic constraints) to optimise light capture, but the adaptation process is slow compared with the time scales of the photochemical reactions. The photosynthetically active parts of the chlorophyll spectrum lie at 680 and 700 nm (see Fig. 9). The energy of photons, captured at shorter wavelengths, can be transferred to the 680700 nm region very efciently on a quantum basis. However, there is an inevitable loss of energy resulting from the transfer from high-energy, shorter wavelength to lower energy, longer wavelength, photons. Thus, although the resultant quantum efciency may be more or less constant throughout the PAR spectrum, there is a net loss of some 21% of the original energy; this is illustrated in Fig. 8.
Fig. 10 Illustration of the light reactions of photosynthesis (the so-called Z-scheme). The major functional units are represented as oval shapes; photosystem II (PSII), plastoquinone (PQ), plastocyanin (PC), cytochrome b6f complex (Cyt b6f), photosystem I (PSI), ferredoxin (Fd), ferredoxinNADP reductase (FNR) (in order of electron transport chain) and ATP synthase. P680 and P700, refer to the reaction centres of photosystem II (PSII) and I (PSI) respectively, the asterisk (*) indicates the excited state. The inset shows a schematic close-up of the light harvesting complex (LHC).
The broad mechanism of the conversion of the photochemical energy into metabolic energy and reducing capability is complex, although comparatively well understood. Briey, there are two functionally separate sites of photon absorption, coupled in tandem by a chain of redox carrier molecules. The photon absorption elicits a charge separation of at two reaction sites, at photosystem II (PSII; l 680 nm) there is a charge separation of 1.7 V and 1.6 V at photosystem I (PSI; l 700 nm) requiring respectively 164 and 154 kJ per einstein.k This is depicted in Fig. 10 as the so-called Z-scheme of photosynthesis. The electron ow away from the chlorophyll molecules draws electrons from water. Whereas the formation of electrons operates on a one-photon-per-electron basis, the dissociation of water requires an accumulation of four electrons to effect the production of one molecule of oxygen. A tetra-manganese complex acts as an accumulator, being oxidised stepwise at four stages, the fully oxidised form then draws the four electrons from two molecules of water in one step, producing a molecule of oxygen plus four protons. The functioning of the water splitting system is described in detail in ref. 48. This whole complex of photon capturing mechanisms, charge separation, generation of metabolic energy and reducing capability, and the water splitting system is embedded in the lipid membrane of attened sac-like structures present in the chloroplast, known as thylakoids.
The electrons pumped by the two reaction centres eventually give rise to the production of the reducing agent (NADPH) used in the process of carbon assimilation. Two molecules of NADPH are being produced per four electrons transported with a free energy gain of 220 kJ mol1 NADPH. At the same time protons are pumped across the membrane into the inner cavity of the thylakoid (the lumen). This sets up a charge gradient. On their return, the protons spin a molecular rotor,** which gives rise to the synthesis of adenosine triphosphate (ATP), the biological energy currency. In total, three ATP molecules are formed per 12 protons transported, with a free energy gain of ca. 50 kJ mol1 ATP; along with the two NADPH molecules; the total potential energy yield is 590 kJ per 4 moles electrons transported. Although in theory this could be supplied by four photons (two einsteins at 680 nm and two at 700 nm would yield 692 kJ), experimental observations suggest 810 photons are needed per four electrons transported. Some of the intermediates in the system have a limited lifespan, and the statistical probability of arrival of 8 photons at a single
k An einstein is dened as a mole of photons, of unprescribed wavelength.
** It has been held that Nature never evolved a wheel it however did a billion or more years ago and on the nanoscale! Only recently have we developed the skills to observe these molecular mechanisms. Depending upon the circumstances the DG for the hydrolysis of ATP varies from 45 to 55 kJ mol1. A mean gure of 50 kJ mol1 is used in the present account. There have been two schools of thought over the number of photons required per molecule of O2 split: either 36 photons or 810 photons (see ref. 56). Present consensus favours the latter.
capturing site within the required time frame is low. Nature has overcome this problem by linking together approximately 2000 chlorophyll molecules in a light harvesting complex (LHC) with a much smaller number of reaction centre chlorophyll molecules. On average there are about 300 chlorophyll molecules per active reaction centre. This is termed the photosynthetic unit (see inset in Fig. 10). The captured energy of photons very rapidly passes through this network of chlorophyll molecules, reaching the reaction centre within a timescale of ca. 1010 s. (ii) The light-independent reactions. The reducing power (NADPH + H+) and energy (ATP) produced by the light reaction is used in the enzymatic light-independent part of photosynthesis to enable the incorporation of CO2 into organic material and its subsequent reduction (see Fig. 11). The caloric value of the product is around 469 kJ mol1 C. The rst committed step is the carboxylation of the sugar ribulose 1 : 5 bisphosphate (Ru5BP) by the enzyme ribulose bisphosphate carboxylase oxygenase (RuBisCO). RuBisCO exists as large heteromultimeric protein complex (536 kDa) and is an outstandingly sluggish catalyst, xing 210 molecules of CO2 per active site per second. In addition, it has the curious property of catalysing both the carboxylation and oxidation of its substrate. Which reaction predominates depends upon the ratio of the partial pressures of the two gases; CO2 and O2. The oxidation reaction is wasteful of energy. Most, but not all, aquatic microalgae overcome the predominance of oxygen over carbon dioxide in oceans (ca. 30-fold) by actively pumping in CO2 and so increasing its concentration around RuBisCO. RuBisCO plays a central role in all plant photosynthesis, it accounts for a major fraction of all living protein. It has long
been a puzzle why an enzyme, of such crucial importance to life on Earth, has seemingly remained so inefcient since there have been a billion or more years for improvements to have evolved. It appears, however, that the enzyme is nearly perfectly optimised49 and that the perceived inefciency is a fallacy. The rst stable products of the reaction are 3-carbon organic acids. It is from these compounds that all major biochemicals (fats, fatty acids, sugars, proteins etc.) are eventually formed (see Fig. 11). Carboxylation of Ru5BP in the Calvin cycle leads to the production of two molecules of 3-phosphoglyceric acid (3-PGA), which is subsequently phosphorylated to 1,3-bisphosphoglycerate (1,3-BPGA) and reduced to glyceraldehyde-3-phosphate (G3P). This reduction step is where the reducing power generated during the photochemical reactions is used. These set of three carbon compounds are the building blocks for the synthesis of the basic biochemical fractions (see insert in Fig. 11). The biochemistry is complex50 and incurs further demands on energy and, where acetyl-CoA in an intermediate (lipids and some amino acids), loss of carbon and oxygen, and therefore biomass, as carbon dioxide. A simplied analysis is made in Box 1 for triglycerides and proteins based on hexoses as the starting point. Fig. 12 shows the biomass loss with increase in lipid production. Two matters are important to note here: rst high lipid content is achieved at a cost of biomass loss; second, whereas theoretical calculations of photosynthetic efciency are based on hexose production, the cells ultimately produce biomass which comprises lipids as well as proteins, this results in something in the region of a 3050% loss of the original photosynthetically produced organic material (see Fig. 12).
Fig. 11 Schematic simplied representation of the Calvin-Benson cycle in three parts, (i) CO2 xation, (ii) reduction and (iii) regeneration. The average cycling time of one round of CO2 assimilation is 100 to 500 ms. The necessary energy (ATP) and reductant (NADPH+ + H+) (not shown stoichiometrically) are originating from the photosynthetic light reactions. RuBisCO: ribulose bisphosphate carboxylase/oxygenase; RuBP: ribulose-1,5bisphosphate; 3-PGA: 3-phosphoglycerate; 1,3-BPGA: 1,3 bisphosphoglycerate; G3P: glyceraldehyde-3-phosphate; F6P: fructose-6-phosphate; G6P: glucose-6-phosphate; PEP: phosphoenolpyruvate.
Box 1: Biomass and energy losses incurred during the biosynthesis of lipids and proteins
Lipid synthesis: The biosynthesis of high caloric value compounds such as lipids from the primary products of photosynthesis hexoses must incur a metabolic penalty in the form of loss of mass. A simple minimum calculation may be made from the change in caloric value from 15.7 kJ g1 (a hexose) to 36 kJ g1 (a generic algal lipid). Thus, to conserve energy there must be a mass loss of about 2.5. This, however, is a minimum calculation, as the 2nd law of thermodynamics calls for some energy, and therefore additional mass, loss. This can be estimated from the biochemical stoichiometry of the various reactions during the transformation of hexoses into lipids. If there is a negative energy balance, then as well as the mass losses during the biochemical transformations the production of CO2 (see equation below), additional mass will be lost to provide the extra energy, which must come from respiration. The overall synthesis of the primary lipid product palmitic acid from glucose may be divided into two phases: (i) the formation of acetyl CoA and (ii) and the conversion of acetyl CoA to palmitic acid. 4C6H12O6 / 8 acetyl CoA + 8CO2 + 8ATP + 16 NAD(P)H 8 acetyl CoA + 7ATP + 14 NAD(P)H / CH3(CH2)14COOH Overall: 4C6H12O6 / CH3(CH2)14COOH + 8CO2 + ATP + 2NAD(P)H (3) (1) (2)
Thus, there is a small energy gain in the form of an ATP and two NAD(P)H; however there will be the loss of 3ATP molecules for the formation of the triglyceride and additional losses when palmitic acid is desaturated to form other fatty acids. These energy gains and losses are small (100200 kJ per palmitic acid molecule) as compared with the overall energy present in the molecule (ca. 10 000 kJ mol1) and can be largely ignored. There will be a small energy gain if the triose phosphates, rather than the hexose sugars are considered to be the starting point for lipid biosynthesis. Overall, the conversion of 4 molecules of a hexose to 1 mole of palmitic acid results in a drop in caloric value from 11 446 to 10 117 kJ. Thus, these calculations imply that, whereas there is a small loss of energy (ca. 10%), there is a substantial (2.9-fold) loss of mass. Algal lipid contains a substantial amount of phospholipid and glycolipid. The mass loss in making phospholipid may be estimated to be about 2.7 and making glycolipid 2.3 these values are not as certain as the triglyceride gure. If we take the mean gures for the proportions of these components of algal lipid given in Table 3, a weighted mean value of 2.6-fold loss of mass is obtained for algal lipid formation from hexose; this has been used for the calculation of the mass yield of cells with varying lipid content. Protein synthesis: The parallel calculation for the conversion of the photosynthetic products to proteins is more complex. The elemental composition of algal protein may be calculated from the amino acid analysis as C1H1.56O0.30N0.26S0.006 (see Table 1); the loss of mass on production protein from a hexose, assuming no respiration of carbon, is 1.36-fold, much less than the formation of lipid. The energy gain on a mass basis is much the same as the mass loss (1.33) on a carbon basis. However, there will also be metabolic work at two stages: (i) during the synthesis of the individual amino acids; and (ii) on their conversion to proteins (ref. 125 and personal communication) makes a very comprehensive analysis of the mass yield of synthesising the amino acids for protein synthesis and obtains a value of 1.65 g g1 protein. The energy for the second reaction, which incurs a considerable reduction in entropy, is estimated to be 120 kJ per peptide bond.46 The energy required per gram of protein synthesised can be estimated from the nitrogen content of the protein and is small fraction (1.4 kJ g1, i.e. 6%) of the overall caloric value of the protein, thus we may take a round gure of 1.7 g for the mass of hexose requires to synthesise 1 g of protein. Nucleic acid synthesis: The calculation for the nucleic acids is extremely complex and, in view of their relatively small contribution to the overall mass of the cell, the calculation has not been made. Calculating the mass yields: given the values calculated above, the general equation for the biomass yield is Y 1/(1.11C + 1.7P + 2.6L) (4)
where C is the carbohydrate content (assuming it to be a hexose-based polysaccharide), P is the protein content and L the lipid content, Y is the biomass yield in mass per mass hexose synthesised. Note: C + P + L 1. If it is assumed that the protein to carbohydrate ratio is 3 : 2 and that this ratio remains constant with increase in lipid content, then the equation simplies to: Y 1/(1.46 + 1.14L) (5)
Estimating mass loss from calculated the photosynthetic quotient (PQ): If there is little metabolic work involved in the conversion of photosynthetically produced hexose to another biochemical category, then the hexose mass required for its synthesis may be simply calculated as 30 PQ/FW (g g1), where FW is the formula weight (with carbon set as 1) and 30 is the formula weight of CH2O. For lipid, it underestimates the demand 9%, in the case of protein by about 15%, reecting the scales of the metabolic work needed. Note: NAD(P)H is used to refer to both NADH and NADPH.
compensation irradiance is seen as an offset on the photosynthesisirradiance curve (see Fig. 13A); reported values show a wide range from 03 to 12 mE m2 s1 PAR (equivalent to 0.01 to 0.46 MJ m2 d1 of incoming solar radiation). There are thought to be differences between taxonomic groupings of algae (see Fig. 13B) and ref. 55. The analysis in ref. 53 suggests that the major part of the variation can be attributed to individual cell biomass: Ec is approximately proportional to the square root of cell mass. The compensation irradiance takes on importance in dense algal cultures (see Section 4.4(ii) and Box 2). 3.3 Overall efciency of photosynthesis The energetics of the photochemical reactions in photosynthesis and the linked enzymatic reactions associated with carbon assimilation have been covered above. We now consider the relationship between the photon ux (the irradiance) and the resultant photosynthetic rate. Characteristically the relationship is nonlinear (see Fig. 14). In the near-linear initial part of the curve, there is excess enzymatic activity, with light limiting the photosynthetic rate, and maximum quantum yields are obtained. The attening off of the curve at higher irradiances is a consequence of the limitation of the rate of photosynthesis by
Fig. 12 Graph of the calculated yields of biomass (green line) from hexose for biomass with varying lipid contents. The protein : polysaccharide ratio is taken to be constant at 3 : 2, this assumption has a minor effect on the outcome of the calculations. The calculation is based on eqn (5), Box 1. Shown also is the hexose energy required to synthesise biomass of varying lipid content. The hexose is assumed to have a caloric value of 15.7 kJ g1.
3.2 Respiration At rst it may seem surprising that plants, that have the ability to create metabolic energy de novo, should need a supplementary energy generating process. There are a number of reasons, the two main ones being: (i) to sustain their metabolism over periods of darkness, so plants need some energy generating process independent of light; and (ii) the mismatch of time scales between the light-dependent and independent reactions in photosynthesis can give rise to trafc jams along the electron transport chain when the former reaction gets ahead of the latter. Respirationlike electron disposing reactions have evolved to alleviate this congestion. However, the penalty is wastage of the energy from the captured photons. This is a major limitation to achieving maximum photosynthetic efciency in other than ideal laboratory situations. Photosynthesis can in most part be treated as one of two alternative pathways. By contrast respiration is more complex. In addition to the widespread so-called mitochondrial respiration, ref. 51 list four other forms of respiration in plants. In the case of microalgae, overall respiration can be treated operationally as a composite of two components: (i) a biomassassociated component, which probably represents some basic maintenance energy; and (ii) a photosynthetically-associated component, which may result from the above decongestion process but also from repair or replacement of the components of the light gathering system which suffer photo-damage. A careful analysis of these two forms of respiration for the major algae groupings has been made.53 Normally, other than at very low rates of photosynthesis, the dominant loss term is the photosynthetically associated respiration and the fraction of photosynthesis lost to concurrent respiration lies in the range 812%. As respiration continues in the dark, these numbers are roughly twice as great over a 24 h diel cycle. By comparison, respirationassociated losses in terrestrial crop plants fall in the range of 30 60%.54 The point at which the respiration and photosynthesis curves intersect is the compensation point and the irradiance at this level is known as the compensation irradiance (Ec). Net growth only occurs at light levels above this value. The
Fig. 13 A: Schematic of the various components of respiration and their effect on net metabolism. B: Analysis of data obtained from cultures of algae in ref. 53, which illustrates the offset due to the compensation irradiance. Gonyaulax tamarensis, a large dinoagellate, would not be a candidate organism for mass algal culture but is included as the compensation irradiance is clearly shown with this organism.
Box 2: Estimating the light threshold for growth of dense algal cultures
In dense, well-mixed algal cultures, growth will only occur when the daily average irradiance (E) through the culture is greater than the compensation irradiance (Ec) i.e. (E)/z $ Ec (1)
where z is the depth of the culture. The minimum incoming irradiance level that will allow growth (Emin) may be calculated 0 according to Sverdrups critical depth theory.127 Assuming the distribution of light with depth complies with Beers law, then the above integral with depth has the following solution Pz
0E
(E0/k)(1ekz)
(2)
where k is the beam attenuation coefcient (extinction coefcient). In dense cultures ekz(1, thus the equation simplies to Pz
0E
E0/k
(3)
Combining eqn (1) and (3) gives the minimum irradiance for growth Emin Eckz 0 (4)
For cultures with a biomass of 0.25 kg dry weight m3, the light extinction coefcient will be in the region of 50 m1. This assumes chlorophyll, the main light absorbing pigment, to be 2% of the cell dry weight and to have a specic light attenuation of 10 m2 g1 (derived from data in ref. 128). Thus, for a raceway 15 cm deep, using the above numbers, eqn (4) reduces to: Emin 7.5Ec 0 (5)
For candidate algae, the 24 h compensation irradiance, based on total incoming radiation, has a wide scatter, generally falling in the region 0.03 to 0.3 MJ m2 d1 (see ref. 52, 53 and 55), with a geometric mean of 0.12 MJ m2 d1. This would give a range for Emin from 0.2 to 2 MJ m2 d1, with a median value in the vicinity of 1 MJ m2 d1. 0
enzymatic reactions. In this zone, the energy from the excess captured photons has to be disposed of in some manner, either by uorescence or by one of the respiratory decongesting mechanisms. It is in the context of this effect that the time scale mismatch of the photochemical and enzymatic parts of photosynthesis becomes signicant. If the high irradiances are sustained for an hour or so, then the algae will adapt to them; by either increasing their enzymatic capacity but more commonly by reducing the capturing efciency of photons. The latter is
Fig. 14 The photosynthesis versus irradiance (PvE) relationship. The dashed black line is a projection of the initial rate, and the hatched area between this and the blue curved line for photosynthesis is an indication of the photon wastage. The zone between the blue dashed line and the solid blue line for photosynthesis is that of unexpressed enzyme activity.
achieved by reducing the size of the light-collecting antenna, this lowers the slope of the initial part of the curve, such that the photosynthetic system saturates at higher irradiances. Thus, the algae can exist as two physiological types low light adapted (high chlorophyll content) and high light adapted (low chlorophyll content). It would appear that the low light adapted form is the default state. The phenomenon of light adaptation has a major inuence on the yields of optically dense cultures as will be discussed in a later section. It is important to stress that the following calculation of the efciency of photosynthesis restricts its attention to the ideal circumstance. Even under these circumstances it is not a straightforward calculation and all too often the complications are overlooked or bypassed. A rigorous analysis of the thermodynamics was made in ref. 56, and the energetics are concisely outlined in ref. 54. The present calculation is made for the production of one mol of organic carbon (of a formula CH2O), assumed to have a heat of combustion of 469 kJ mo11; a quantum yield of 8 moles of photons (8 einsteins) captured per mol of oxygen produced (and CO2 assimilated) is assumed. A quantum yield of 10 would give rise to a proportionately lower yield. A quantum yield of 8 (4 quanta at 680 nm and 4 at 700 nm) would have a total energy content of 1384 kJ per 8 einsteins. The consequential voltage jumps of 1.7 V (PSII) and 1.6 V (PSI) (equivalent to 1267 kJ in total) imply very efcient energy transfer at this stage. The reactions following these charge separations give rise to the formation of 3 moles of ATP and
Fig. 15 Energy losses during photosynthesis. A. The stepwise loss of energy during the production of 1 mole of organic carbon as glucose (caloric value taken to be 469 kJ mol1 C). B. The fractional energy losses at the major steps during photosynthesis. The hatched lines are stages at which the losses are variable.
2 moles of NADPH a combined yield of 590 kJ. These products of the light reaction in turn give rise to the formation of 1 mole of organic carbon with an elemental composition of CH2O. Taking this to be a hexose, with a caloric value of 469 kJ mol1 C, the overall efciency of the formation of 1 mole of organic carbon from the 8 absorbed moles of photons themselves would be 34%, with the major part of the loss occurring during the light reactions (see Fig. 15A and 15B) There are also inevitable energy losses preceding and following these two sets of reactions. Photons are gathered from across the whole 400700 nm range the balance will depend upon the photosynthetic pigments present in the cell. First, whereas the quantum yield can be remarkably constant across the PAR range, energy losses will be incurred transferring the energy to the lower frequencies of the longer wavelength photons (see Fig. 9). This loss is about 20%. This reduces the photosynthetic efciency to 27% (see Table 5). Second, the radiation available for photosynthesis is only some 45% of the incoming radiation. Thus, incorporating these losses, 3908 kJ of incoming radiation is required to produce one mol of organic carbon, reducing the overall yield to 12%. There are also energy losses associated with the metabolic conversion of the primary photosynthetic products (e.g. hexoses) to biomass i.e. lipids, proteins, polysaccharides, etc. This loss will be dependent upon two factors. The biochemical
composition of the cell will affect both biomass and energy yields. This is discussed in Box 1 and the effect of varying cell lipid content is illustrated in Fig. 12. A signicant change in yield can result from the state of oxidation of the nitrogen source whether the cells are using ammonia (or urea) or nitrate. If nitrate is used as a nitrogen source as opposed to reduced sources such as ammonia, then the reduction of nitrate calls for extra energy, characteristically increasing the energy demand by 2030%. A summary of photosynthetic yields calculated from the present analysis, along with the various yield terms are used in the study of the energetics of plant culture, is given in Table 5. The above efciencies may be regarded essentially as xed ceilings for photosynthetic efciency under natural conditions, as the losses are determined by the stoichiometry (metabolic losses) and the thermodynamics of the processes for which there is limited scope for variation. There are two further forms of loss, which are variable, where there may be possibilities for manipulation of the photosynthetic apparatus and/or cell metabolism: (i) concurrent respiration (Fig. 13); and (ii) photon wastage (Fig. 14). Taking a median estimate of the 24 h loss to respiration as 20% of photosynthesis, the overall yield of the incoming radiation is reduced to around 10%. A quantum requirement of 10, rather than 8, would reduce the overall maximum yields to 8% after respiration is considered.
Table 5 Calculation of maximum photosynthetic efciencies. Yield terminology and symbols adopted from ref. 57. CHO refers to organic material with a hexose-type elemental ratio, and a caloric value of 469 kJ mol1 C (15.6 kJ g1). CHON refers to the calculated carbon-normalised major element composition (C1H1.64O0.44N0.13) for a cell containing 42% protein, 28% carbohydrate, 25% lipid and 5% nucleic acid, with a caloric value of 541 kJ mol1 C (24.7 kJ g 1, data taken from Table 1). This gives photosynthetic quotients (DO2/DCO2) of 1.1 on ammonia as a N-source and 1.3 on nitrate. Values are calculated on PAR except those within brackets, which are rounded off values based on total solar radiation Type of yield Bioenergetic yield (J%) Energetic yield (JkJ) Quantum yield (JE) Photons/mol C xed Units % (kJ kJ1) g kJ1 g einstein1 einstein mol C1 26.7 17 103 3.8 8 CHO [12] [7.7 103] CHON (NH3) 17.7 7.2 103 2.1 10.2 [8] [3.2 103] CHON (NO3) 14.3 5.8 103 1.7 12.6 [6.4] [2.6 103]
Table 6 Observed and projected yields for crop plants and microalgae. Figures for annual insolation are calculated as for Fig. 18A; theoretical values are given for three latitude zones, the same as in Fig. 24. * allowing for respiration, ** assuming a caloric value of 20 kJ g1 dry weight for the crop as a whole, *** assuming a biomass caloric value of 24.7 kJ g1 dry weight (see Table 5) and a respiratory loss of 20% Maximum biomass yield/tonnes dry weight ha1 a1
Crop Higher plants Theoretical* (C3 plants) c% 4.6% Theoretical* (C4 plants) c% 6.0% Sugar cane Switchgrass Corn (grain) Poplar wood chips Soya Rape seed Oil palm Microalgae Theoretical c% 12% (Table 5) Projected raceway, algae unspecied Bioreactor raceway, algae unspecied Projected raceway, algae unspecied Best case raceway, algae unspecied Achieved bioreactor (Phaeodactylum) Achieved raceway (Pleurochrysis) over 10 months
Source
Low 210, mid 170, high 140** Low 270, mid 220, high 190** 7495 820 834 11 4.65.5 4.56 8.7 Low 410, mid 330, high 280*** 110220 175 127 120153 182 60
54 54 6768 19,67 66 69 66 66 66 Present account 16 4 4 9 70 71
Fig. 16 Open ponds (image courtesy of Nature Beta Technologies Ltd, Eilat, Israel, subsidiary of Nikken Sohonsha Co. Gifu, Japan).
4.1.
Algal growth and production
By comparison the estimated equivalent yield for higher plants54 (see Table 6) lies in the range 4.6 to 6%, the difference being due to the greater respiration losses in higher plants. If we take the irradiance information used to create Fig. 18A and the more cautious quantum requirement of 10, then, allowing for respiration, the maximum theoretical yields of a carbohydrate type molecule would be 410 tonnes dry weight ha1 a1 (112 g m2 d1) at low latitudes less than 30 , falling to 280 tonnes dry weight ha1 a1 (75 g m2 d1) at latitudes 4555 . Actual yields (see Table 6) fall short for a number of reasons; photon wastage (which can be up to 80%) being the major problem, when attempting to maximise the output from mass cultures of algae. Photon wastage is the principal reason why the actual yields fall short of the theoretical. This phenomenon continues to tax the ingenuity of researchers and engineers and is far from resolved. We return to this problem in the following section.
Initial work on mass culture of algae was carried out in ponds and tanks of various forms, frequently with some form of agitation. From these beginnings, two types of growth system have evolved: (i) raceways; (ii) photobioreactors (PBR). The former (see Fig. 16) are simple engineering developments of the pond/tank type system. At their simplest, they are oval in shape with depths 100300 mm and the water is kept in circulation with paddle wheels the most energy efcient system. Bioreactors (see Fig. 17) are enclosed systems of various geometries, characteristically, although not invariably, with a light capturing depth of less than 100 mm, often having internal dimensions as little as 30 mm. They may take the form of narrow tubes, horizontal or inclined tubes, vertical coils or columnar structures as well as at plate structures. Many of the nancial disasters in mass culture of algae have been associated with bioreactor-based systems (see ref. 58). The main problems with algal culture in bioreactors are the maintenance of the necessary turbulent ow in long lengths of narrow tubing and the possibility of inhibition of
4. Production rates: background and observations

Building on the background information on photosynthesis, we consider the theoretical limitations to biomass production, based on the incoming radiation and efciency of energy transfer from photons to biomass. We discuss two types of algal biomass production systems, closed bioreactors and open ponds.
Fig. 17 Photobioreactors (Greenfuels image courtesey of http:// www.ickr.com/photos/jurvetson/58591531/).
photosynthesis due to the accumulation of oxygen in the closed system. Active pumping with air-lift pumps induces turbulence and also helps to drive off the accumulating oxygen. On the positive side, bioreactors, being closed, are much less prone to contamination than open raceways. A coupling of the above systems has been used and found to be very reliable and successful. In these coupled systems, large diameter (ca. 300 mm) bioreactors act as a nursery stage, where a pure culture is maintained, after which the cultures are periodically ooded into large area raceways, which serve as the grow-on phase. Such systems have been successfully used for growth of the astaxanthin-producing algae Haematococcus pluvialis.7,16 Microbial production (or yield) may be regarded as a product of biomass (crop) and net growth rate i.e.: Production (M L2 T1 or M L3 T1) biomass (M L2 or M L3) growth rate (T1) Biomass may be expressed as moles or mass of organic carbon or, more commonly in algal mass culture, as ash-free dry weight (per unit area or volume). Growth rate is subject to a number of modifying physical, chemical and biological factors. Maximising the output of these systems, with the number of interacting factors is complex, as without exception, the relationships are non-linear. Further, with probably only a single exception (the apparent Arrhenius constant in the temperature response curve), the values for the parameters cannot be anticipated from theory with any degree of certainty.
activation energy of ca. 50 000 kJ mol1. Biologists customarily use a simple logarithmic alternative to the Arrhenius equation the Q10 dened as the change in rate over 10 C the value is characteristically close to 2. Temperature is different to nutrients and light, in that it is treated as a determinant of mmax, rather than a moderator. Relevant to outdoor mass algae culture (and in common with light), as well as the immediate response, algae also have some capability to adapt to the ambient temperature. There are also physiological interactions between the responses to temperature and light, as the photochemical system is essentially temperature insensitive, whereas the enzymatic system is temperature sensitive. Photoinhibition, and associated losses, is high at low temperatures and high irradiances.60 4.3. Yields
4.2.
Controls on algal growth
Growth of microorganisms is usually modelled as a speciesspecic maximum growth rate (mmax), reduced by one or a combination of growth limiting factors the two key ones in the case of the algae are light and inorganic nutrients (e.g. nitrogen, carbon or phosphorus). Characteristically the effect is non-linear, with the initially controlling factor reaching a point where it no longer affects the growth rate. This is modelled by the biological equivalent of the Langmuir isotherm (MichaelisMenten equation), giving broad nutrient-limited and nutrient-unlimited zones. In the case of light, there is a further zone of light inhibition (see Fig. 14). A number of equations have been devised to model this latter curve (see ref. 57 and 59). It is debated whether growth is controlled by the most severe limiting factor or a product of all factors at one instance in time. Two additional factors affect the growth rate of algae; both are of considerable importance in modelling and designing growth conditions. The rst is respiration (R), which essentially subtracts from growth (m), i.e.: mnet m R The second factor is temperature. The overall temperature response has three cardinal points a temperature minimum, a temperature maximum and a temperature optimum. The zone from the temperature minimum to approaching the temperature optimum closely follows the Arrhenius equation, giving effective
(i) Theoretical yields. From the information derived in Section 3 (summarised in Table 5) and the calculated incoming radiation, it is possible to calculate maximum theoretical yields of biomass per unit area for various latitudes and times of the year. One must stress that, for a number of reasons, this particular calculation is intended to provide nothing more than a high ceiling value. The calculation of incoming radiation is as follows: with a clear sky, the incident solar radiation with the sun vertically overhead may be taken as 1000 W m2, of which some 50% (500 J m2 s1or 2300 mE m2 s1) lies within the PAR region. Given this, the irradiance may be calculated for each hour angle of the day and for the cycle of the solar declination angle through the year. Finally a correction is made for reective losses from the surface of the water using Fresnels equation although this correction is very small. Details and the overall calculation may be found in ref. 47 and 61. The calculated daily irradiances for various latitudes are given in Fig. 18A. For comparison purposes, eld data of total incoming radiation, derived from satellite observations, are given in Fig. 19. The point made by this latter gure is that local atmospheric conditions notably cloud cover give rise to a much more complex distribution in space and time of incident radiation than the simple calculation from which Fig. 18A is derived. Once the irradiances have been obtained, then a simple linear calculation of organic production may be made from the product of the irradiance and the energetic yield (see Table 5). The curves given in Fig. 18B are for a ceiling value of bioenergetic yield of 10% this is obtained by correcting the 12% yield for a hexose (Table 5) for a 20% loss due to respiration. To transform the energy yields to mass yields (i.e. kJ to g dry weight) an energy/ mass conversion factor is required. Commonly (see ref. 56) this is calculated theoretically from elemental composition or established empirically by bomb calorimetry. These approaches do not take account of the metabolic work on transformation from hexoses (taken as the primary photosynthetic product) to other biochemicals.xx A more correct value may be derived from the
xx Bomb calorimetry involves the conversion the nitrogen in the molecule to dinitrogen, whereas the starting and end nitrogen compound in microalgal growth is commonly ammonia. Strictly a correction should be made to the reported bomb calorimitry values for the heat of formation of ammonia from dinitrogen (46 kJ mol1) otherwise the DG of the reaction will be overestimated. The error is small, about 0.4 kJ g1 in the case of protein and 0.2 or less in the case of algal biomass, and is customarily ignored.
Fig. 18 A. Monthly average daily clear sky total isolation for various latitudes. The calculation takes into account the loss from the surface due to reection. Calculation made following ref. 47 and 61. B. Estimated monthly averaged theoretical daily photosynthesis (as biomass dry weight, composition as in Table 5, assumed caloric value 24.7 kJ g1) through the year at various latitudes; the calculation (see text) is based on a 10% bioenergetic yield (see Table 5); no biological losses, respiration or organic excretion are taken into account. C. Variation of calculated productivity with latitude, assuming 3 and 10% bioenergetic yields (again no biological losses are taken into account) and also using the tanh relationship shown in Fig. 22. D. Monthly averaged theoretical daily productivity (as dry weight) through the year at various latitudes again using the same tanh relationship as insert C.
to control by other factors such as temperature or inorganic nutrients so in this respect the rates obtained perhaps are best regarded as maximal ones, unless major improvements can be made to the light capturing system. (ii) Observed yields. Fig. 20A summarises an extensive collation of reports of area yields of algal biomass derived from various culture systems. The reported rates have been plotted against time, as there have been improvements over the years, and separated into ponds, bioreactor systems and raceways. They have been further separated into average (or sustained values) and maximum values. Two exceptionally high rates (130 and 174 g m2 d1) have been omitted, as they appear close to or beyond accepted theoretical yields. Generally values above 40 g m2 d1 come from narrow path length (<50 mm) bioreactors. The clusters of low rates (>20 g m2 d1) for raceways in the post 2000 period come from observations during the solar winter at latitudes greater than 30 . This is not anticipated from the radiation calculations and may be due to temperature limitation arising from evaporative cooling effect on the raceways (see the following section). Furthermore, some of the data come from high lipid algae, which will result in a depression of the yield (see Box 1) and Fig. 12. Ignoring the low cluster (see above) there would appear a general upward drift due to improvements with time to a gure in the region of 3540 g m2d1 very much the prediction in ref. 65. The means of the maximum and average/sustained rates are given in Fig. 20B (a geometric mean has been used to avoid biases due to the wide scatter). Where the appropriate information is available, the bioenergetic yield (j%) has been calculated (see Fig. 20C). The data set however is very small; some 19 sets of observations in total and predominantly from high performance
Fig. 19 Global distribution of total incoming radiation through the rst half of the year.62
mass of hexose required to produce a given mass of biochemical and the caloric value of hexose itself (taken as 15.7 kJ g1, ref. 63, 64). The former can be estimated from either the protein/ polysaccharide/lipid content (see Box 1, eqn (4)) or from the lipid content, assuming a xed protein/carbohydrate ratio of 3 : 2 (Box 1, eqn (5)). The 3 : 2 ratio for the protein carbohydrate proportions comes from the analysis of data in Fig. 5. Fig. 18D shows the results of a calculation using a non-linear relationship between photosynthesis and radiation the tanh relationship between photosynthesis and irradiance (see discussion in Section 4.4 (i) and Fig. 21). In Fig. 18C the data has been integrated through the year giving the distribution of calculated annual yields with latitude, the calculations are made for a 10% bioenergetic yield, a 3% yield (an average obtained value, see Fig. 20) and the above tanh relationship. The calculation assumes that only light limits growth no consideration is given
due to the fact that, in the case of the higher plants, the reported crop (e.g. grain) may only be part of the above-ground plant yield, and the below-ground yield (roots) is almost inevitably omitted. There are major uncertainties with the estimations for algal biomass yields in that there are very few reports of long time series (i.e. data for a full year). When better sets of data become available, we may nd some downward revision of the numbers. However, this may be offset by improvements in strains and culturing techniques. We simply have to wait and see. 4.4 Controls on yields Broadly, the yields commonly achieved in outdoor mass culture systems lie between one tenth and one third of the theoretical value of 10% of the energy in the total incoming radiation (cf. Table 5 and Fig. 20C). A number of factors give rise to this. In Section 4.1, light, temperature and oxygen were identied as factors that would affect photosynthesis; further controls may be set by carbon dioxide concentrations, turbulence, inhibitors and contamination. (i) Light. Given the above efciencies, a large fraction (60 90%) of the incoming radiation is not used for photosynthesis. Some 50% of the total incoming radiation is outside the range useful for photosynthesis and so cannot be used. But this is not the full story. Losses may result from light not being collected by the culture or that the photons collected by the algae are in large part wasted and their energy is lost as heat or uorescence. The former loss can be essentially disregarded with cultures with light paths of 100 mm and biomasses of 1 kg m3, as the fraction of the light emerging from the culture is a very small proportion (<104) of that entering. The main loss is associated with the wastage of photons after absorption by the algae, which results in the culture as a whole using light very inefciently: the algae in the rst 520 mm of the light path being light-saturated or light inhibited, whereas those in remaining 9095% of the culture are severely light limited. The resolution of this problem has been, and continues to be, the major challenge to maximising the productivity of mass cultures (see Section 4.5(i)). There is a paucity of reported data on the seasonality of production rates for PBRs and raceways, which allows the overall relationship between daily irradiance and production rates to be explored. Fig. 21 and 22 show the sets of data we have been able to locate.70,72,73 The relationship between light and areal production rates was analysed using the tanh model for the PvE relationship (P Pmax*tanh(a*E/Pmax), where Pmax is the maximum photosynthetic rate, a is the initial slope, E the irradiance; see ref. 47. The tting to the data was undertaken using a least squares analysis. We found it necessary to allow for an offset on the irradiance axis. The tanh model was chosen as it provides a direct parameterisation of the initial slope (a), from which the photosynthetic efciency (j%, see Table 5) may be calculated. In order to calculate the photosynthetic efciency, a caloric value has to be prescribed for the organism, this was calculated from the reported lipid content for the organism and the caloric values reported in Table 1. The data have been separated into PBRs and raceways. At rst sight there would appear to be a signicant difference between the rates in raceways and PBRs (as seen also in Fig. 20B and C) but in ref. 72 the
Fig. 20 A. Analysis of published production rates of outdoor cultures, grouped according to type of growth system. Colours designate the type of system, crosses maximum rates, solid circles sustained rates, PBR: photobioreactors, RW: raceways. B. Average (geometric means) production rates of ponds, bioreactors and raceways. Upper line is the standard deviation, lower the standard error. C. Reported or calculated bioenergetic yield (j%, see Section 3.3 and Table 5), based on photosynthetically available radiation (PAR; n 15) and total solar radiation (TSR; n 4). TSR is the TSR efciencies, plus the PAR values corrected to TSR values by dividing by 2 (see Section 3.1(i)). The theoretical maximum is taken from Table 5 allowing for 20% respiration. Data and sources given in Appendix IV (provided in the ESI).
photobioreactors, where the mean bioenergetic yield (based on PAR) is close to 10%; the mean for raceway observations is much lower at 2%, with these values being around 30% and 10% respectively of the theoretical yield (see Table 5). The generally lower values for raceways, compared with bioreactors, may in some part be due to design limitations, but more likely that the bioreactors are far less susceptible to contamination and the populations in raceways may be growing sub-optimally. Table 6 sets out yield data for crop plants along with those available for microalgal cultures. In round gures, the higher plant annual yields fall generally in the range of 530 tonnes crop dry weight per hectare (sugar cane being the striking exception), the algal crops 50150 tonnes. The basis for the difference, although striking, is not wholly clear. In part, it may be that the longer growing period in the case of higher plants results in respiration taking a higher toll on the yields. It is also likely to be
Table 7 Parameters derived from the analysis of data shown in Fig. 21 and 22. Sources: ref. 70, organism cultured: Phaeodactylum tricornutum, ref. 72, organism cultured: Tetraselmis suecica, ref. 71, organism cultured: Pleurochrysis carterae Growth system Dimensions/mm diameter or depth Reference Pmax/g m2 d1 a/g MJ1 Offset/MJ m2 d1 Lipid content (% dry weight) Calculated caloric value/kJ g1 Observed photosynthetic efciency (J%) PBR 30 70 1400 1.7 4.2 16 23.3 3.9 PBR 60 70 86 3.5 0.5 16 23.3 8.2 PBR 35 72 20 2.9 4.6 26.5 25.0 7.1 RW 150 72 20 1.9 3.3 26.5 25.0 4.6 RW 150 71 41 2.3 9.7 22.5 24.3 5.5
Fig. 21 Analysis of three data sets70,72 of the seasonal changes in biomass yield in outdoor photobioreactors. The study in ref. 70 was undertaken in southern Spain at a latitude 36 480 N; the culture organism was Phaeodactylum tricornutum. The study in ref. 72 study was made in central Italy at 43 80 N; the culture organism was Tetraselmis suecica. Using a least squares analysis, the data were tted to the tanh expression P Pmax*tanh(a*(E Emin)/Pmax), where P is the observed rate of photosynthesis, Pmax is the maximum photosynthetic rate, a is the initial slope, E the irradiance.47 The lines were not forced through zero, but an offset on the irradiance axis (Emin) allowed. The derived parameters are given in Table 7.
same organism was grown in parallel in PBRs and raceways and the rates are essentially identical, so the apparent differences may be more due to the organism or the environmental or operating circumstances. The derived parameters from the analyses and the photosynthetic efciency and its calculation are summarised in Table 7 and a number of features are worth noting. Most important is low spread of values for the initial slope (a); the mean value is 2.4 0.75 g MJ1 gives a calculated photosynthetic efciency of 5.9 1.8%; allowing for a 20% loss incurred as respiration, a value for the primary photosynthetic efciency of 7.4% is obtained, very close to the theoretical value of 8% for the production of biomass (see Table 5). The second important outcome is the apparent irradiance offsets (3.3 to 9.7 MJ m2 d1)
seen in the data of ref. 72 and 73. This offset is not as evident, or perhaps absent, in the data from ref. 70, but that data set lacks observations at the low irradiances needed to establish whether or not there is an intercept, furthermore, the plot for the 30 mm PBR certainly looks anomalous. Thresholds, equivalent to 59 MJ m2 d1, were reported for mass cultures in ref. 74 (see Fig. 14.4 and Table 14.5 in ref. 74). The simple explanation for the threshold would be that it represents the compensation irradiance (see Section 3.2) but an analysis for dense cultures (see Box 2) suggests this would give rise to a maximum value of 2 MJ m2 d1, distinctly below the observed range of 39 MJ m2 d1; so it cannot be the sole explanation. Water temperature also varies with irradiance and will have an effect upon photosynthetic, and respiration rates, thus the observed offset may be a combined effect of temperature and irradiance. A temperatureparameterised model would be needed to resolve this. The data in Table 7 have been used as the basis of parameterisation for the production model used elsewhere is the review. (ii) Temperature. The incoming radiation has a marked diurnal effect upon the temperature of the systems. ref. 71, 75, 76 observed 1025 C increases in temperature during the day due to solar heating. In the case of shallow open systems there is a counter effect on temperature due to evaporative cooling. Evaporative losses at middle to low latitudes fall in the region of 2 m per annum. On a daily basis this results in a daily cooling effect equivalent to some 12 MJ m2 (ca. 300 calories cm2). With 150 mm deep raceways this generates a potential cooling of 20 C per day. Given a Q10 of 2, this would give rise close to a 4-fold reduction in metabolic rate and possibly a shift in the photosynthesis/respiration ratio. The matter is clearly more complex than this, as there will be some moderating feedback between cooling and evaporation but as evaporation rates are proportional to degrees Kelvin, the feedback will be weak. Temperature is very likely a major factor determining the productivity of outdoor growth systems open to the atmosphere and may limit the latitudinal extent to which they can be successfully used. Clearly supplementary heating is impractical, as the energy required would far exceed the energy gain from photosynthesis. It does not seem practical to use solar panels as heat collectors to offset the heat loss due to cooling, as the incoming radiation (1025 MJ m2 d1, see Fig. 18A) is, as it should be, not too dissimilar from that associated with the evaporative associated losses (12 MJ m2 d1), so the heat
Fig. 22 Analysis of two data sets71,72 of the seasonal changes in biomass yield in outdoor raceways. The study in ref. 71 was undertaken in Perth in Western Australia (latitude 31 570 S), the organism cultured: was Pleurochrysis carterae. The irradiance data were obtained from the Australian Bureau of Metrology database. Other details as Fig. 21.
collectors would need to be broadly of the same area as the growth systems and would almost certainly not be economic. (iii) Oxygen. Oxygen is a by-product of photosynthesis and it has been long known that high oxygen concentrations inhibit photosynthesis (the so called Warburg effect). Part of this derives from the competing oxygenase activity of RuBisCO (see section 3.1(ii)). Oxygen concentrations in the region of 400% saturation (ca. 2 mM) essentially switch off photosynthesis. Commonly encountered photosynthetic rates in mass cultures (ca. 700 g dry weight m3 d1) result in net oxygen production rates of 30 mol m3 d1 or a rise in oxygen concentrations of 30 mM over the daylight period; well in excess of the 2 mM inhibitory level. The exchange of gases across the air/water interface results in some 20 cm depth of water being re-aerated per hour. Thus with open raceway systems, provided the system is turbulent (ow rates in excess of 0.05 m s1 ensure this, see the following section), accumulation of oxygen should not occur. In the case of enclosed bioreactors, oxygen may be calculated to accumulate at rate of about 4 mol m3 h1 (ref. 60 gives a gure of 6 mol m3 h1 for Spirulina), thus reaching inhibitory concentrations in 2030 min. Bioreactor systems characteristically use airlift pumps to circulate the water, which should strip off the accumulating oxygen, however, these rapid rates of oxygen production impose constraints on the maximum length of run and the minimum ow rates within the bioreactors. There is apparently a combined effect of temperature and oxygen. Ref. 60 reported that a fall in temperature from 35 to 25 C, accompanied with a rise in oxygen concentrations from 0.69 mM to 1.9 mM, gave rise to a substantial (60%) fall in photosynthetic rates. (iv) Carbon dioxide. Many of the details for oxygen are shared with carbon dioxide, although the implications are profoundly different. Consider the example of seawater, where there is a broad chemical predictability. Seawater total inorganic carbon dioxide concentrations are approximately 2 moles m3. At daily production rates of 30 moles m3 (see above), the carbon dioxide species in seawater will be exhausted within an hour of so. The rate of CO2 replacement is about one tenth that of oxygen, as the driving force of the reaction is associated with the dissolved CO2, which is only a minor component of the seawater carbonate system. The consequence is that re-carbonation of the water by simple exchange with the atmosphere will not sustain the rates of removal, thus the CO2 needs to be replaced continuously by the addition of gaseous or liquid CO2. The supply of CO2 could well be a major constraint on the siting and consequential scale of algal biomass production facilities. (v) Turbulence. It had been demonstrated early on in the study of the mechanism of photosynthesis that the yield of oxygen per photon was greater in intermittent light.77 The basis for this is that a single 400 chlorophyll photon collector can process some 2000 excitations per second, whereas downstream enzymatic carbon assimilation can process at best 100200 electrons per second.78 The dark period, between the ashes, allows the slower enzymatic reactions to catch up with the photochemical reaction. The optimum dark period is temperature-dependent but generally falls in the region of 50 ms. Ref. 79 extended the early studies in ref. 77 for growing cultures and
showed that, to have a signicant effect, the dark period needed to be 10 times greater than the light, consistent with the above generalisation on relative processing rates of the photochemical and enzymatic reactions. The gains in efciency are lost at ash periods shorter than 10 ms. In dense cultures, where perhaps 90% of the culture is in effective darkness, turbulence cycles the cells through a dark/light cycle. There has been extensive theoretical and experimental work on this effect. Ref. 80 undertook a very detailed study with dense algal cultures (36 kg m3) and found the ashing light effect to cut out at about 10 Hz. Ref. 81 concluded that the response in outdoor tubular bioreactors saturated at frequencies greater than 1 Hz. Ref. 74 concluded that power consumption considerations would limit ows to 0.150.2 ms1. At these ow rates, in raceways 150 to 200 mm deep, the cross transfer frequency due to turbulent diffusion would be of the order of 0.1 Hz,{{ thus there is potential for improvement of production rates by increasing the turbulence in raceways. Ref. 82 used simple hydrofoil spoilers to induce turbulence and with a raceway system was able to achieve a mixing frequency of 0.5 to 1 Hz, which gave rise to a two-fold or more gain in areal production. In a subsequent paper, using the same arrangement, ref. 83 report sustained production rates of 40 g m2 d1, with short term, maximum rates in the region of 6070 g m2 d1. With the exception of a couple of questionably values, these rates are the highest achieved with raceway cultures, and are equivalent to bioenergetic efciencies (based on total incoming radiation) of 5.5%, with a high value of 9.5%. Whether such systems can be made economic remains an open question.
4.5 Maximising yields (i) Maximising biomass yields. A full discussion of maximising the overall output and economy of algae biomass/biofuel production, as implied before, is complex and beyond the scope of the present review. We restrict our focus on maximising the utilization of photons, i.e. maximising the photosynthetic efciency. This has long been seen as crucial to the success of algal biomass production.15,78 The problem is peculiar to mass algal culture. Near theoretical yields can be achieved in short light path lengths of dilute cultures at low irradiances. Mass algal production involves optically dense cultures (biomasses $1 kg m3, giving light attenuation coefcients of $200 m1) and supraoptimal irradiances. The consequence is that 90% of the photons are absorbed in the rst 10 mm by algae suffering severe light inhibition and so use photons very inefciently wasting perhaps some 8090%. Thus the remaining 90% or more of the culture is using photons very efciently, but exists in virtual darkness. Mixing by inducing turbulence alleviates the problem to some extent but would not appear to solve it fully. In essence, the root of the problem is that, in mass cultures, the algae are presented with a very unnatural situation and they are not equipped physiologically to deal with it. Photosynthesis occurs as three events with badly matching time responses:84 photon capture rates have timescales of 106 s or less, whereas enzymatic CO2 xation and reduction have
{{ This is calculated as the transverse timescale h3/(0.06kz) h3/(0.06u*xh) h2/(0.06 0.05 u) z 30h2/u; where h is the depth and u is the velocity, all in SI units.
timescales $1 s and the adaptation to changes to elevated light levels even longer timescales $103 s. Thus, the adaptive response is far too slow to resolve the problem of super-optimal irradiances. It is possible to mix the culture on time scales of fractions of a second, but not on the required microsecond time scale, so although mixing will alleviate the congestion in the electron chain to some extent with a consequential increase yield, it does not solve the fundamental problem. The solution lies in either speeding up the enzymatic reactions or slowing down the photochemical ones. The slow working, apparently inefcient enzyme RuBisCO has been an obvious target based on the supposition being that one could improve on evolution. However, the analysis made in ref. 49 would appear to show this assumption to be false. Even if one were able to engineer improvements in the reaction rate of RuBisCO, or modify the cell to produce more, the probability is that another enzyme would then become limiting and the problem would end up processing through the complex cascade of enzymes of the dark reaction. Solving the other end of the problem, by reducing the photon capturing capacity, has long been seen as the more likely solution (e.g. ref. 15, 78). When exposed to sustained high irradiances, algae adapt by reducing the size of the light harvesting antennae and so reduce the rate of collection of photons. The problem is that the time-scale of the adaptation is long as compared with the other timescales in photosynthesis and relies upon the algae being held for periods of tens of minutes to an hour or so at high irradiances. In mass culture, the algae exist in a rapidly uctuating light regime making it impossible for them to adapt to the high irradiances. Furthermore, unless the high irradiances are sustained, the algae appear to revert back to the low light/high chlorophyll state.85 The solution is to manipulate the physiological control mechanism and to switch the organism permanently into the high-light adapted state. This problem has attracted research interest and headway has been made.8591 The chlorophyll antennae are assembled on a scaffold of so called light-harvesting proteins and it is these proteins that set the size of the chlorophyll antennae. It has been possible to down regulate these proteins and so reduce the photon capturing ability of the algal photosynthetic system (see e.g. ref. 86). The photosynthetic unit associated with photosynthetic system II can be reduced some 3-fold from some 300 chlorophyll molecules down to 95, but this is not achieved in the case of photosynthetic system I.88 The reduction in the size of the light collecting antenna gives rise to 2-fold or more enhancement in photosynthesis and growth at high irradiances,86,88 importantly without apparently compromising enzymatic capacity and there appears to be no penalty due to rise in respiration rate. The results from a simple spreadsheet model show that if the chlorophyll content is reduced, a greater number of photons are delivered to the deeper parts of the culture, resulting in a more efcient photon usage and greater areal productivity. The effect is most pronounced at intermediate irradiances and as a consequence higher latitudes. This simple model suggests that 2-fold gains in photosynthesis can be expected, consistent with the supposition in ref. 15. However, it is unlikely that one will be able wholly eliminate the problem. Improving productivity of the culture in this way probably negates some of the gains that can be achieved by mixing, so a priori the two approaches to improving the utilization of
photons cannot be expected to be complementary. It is not possible without a sophisticated model to determine the combined effect of these two approaches. The cautious conclusion is that given these developments one could reasonably expect to approach 50% of the theoretical efciency, giving a maximum potential bioenergetic yield in the region of 5%. (ii) Maximising lipid yields. At rst sight a logical strategy to improve the economy of algal biodiesel production would seem to be to obtain and grow algae of high lipid content. This was a major research thrust of the US Aquatic Species Program. Broadly, two lines of research were followed, the rst was to isolate and screen algae for high lipid producers. The second approach aimed to increase the extent of lipid production by identifying the rate limiting enzyme and modify the cells genetics to increase its expression and so increase yield. The enzyme acetyl-CoA carboxylase, which catalyses the carboxylation of acetyl-CoA to malonyl-CoA, is the rst committed step in lipid synthesis and was regarded as the limiting reaction. It had been possible to increase the level of expression of the enzyme two- to three-fold; however, no associated increase in lipid levels was found (see ref. 3, p. 138). Unfortunately this work was in its infancy when the ASP was terminated, so the feasibility of raising lipid yields by such means has not been fully explored. An alternative approach to producing lipid-rich cells is to drive the cells into nutrient deciency, when lipid accumulates (see Section 2.3, Fig. 7). There are, however, penalties in producing cultures with high lipid content. First, as the fraction of lipid is increased some other cell component (e.g. protein, nucleic acid or carbohydrate) must decrease. The proteins and nucleic acids are major determinants of maximum growth rates, and it is far from clear how these biochemical groups change as lipid content changes (see Fig. 5). The coefcient of relationships shown in Fig. 6 implies that an increase in lipid content from 15 to 30% would give rise to a 50% or greater reduction in growth rate. The reduced growth rate is accompanied with a proportionate increase in the period the organisms would need to be held in the growth system and a consequential increase in costs. Increasing their lipid content, by driving the algae into nutrient deciency, ultimately gives rise to the same problem; it extends culturing time and increases the space and capital requirements of the production plant. At the present it would appear that there is limited potential to increase the lipid content of the algae and for a number of reasons 35% lipid content appears to be a realistic maximum target. An apparently unrecognised penalty of growing algae of high lipid content is the signicant overall biomass loss that necessarily occurs when lipids are synthesised from the primary products (hexoses) of photosynthesis. This is analysed in Box 1. There is a 50% loss in mass resulting from removal of oxygen during triglyceride synthesis; a further third is lost as CO2. In combination, and allowing for the different component lipids and their proportions in algae, we estimate there to be around a 2.6-fold reduction in biomass during the synthesis of microalgal cell lipid from a hexose this effect can be seen in the yield calculations for cells of varying lipid contents in Fig. 23. If the economic analysis focuses exclusively on biodiesel and lipid production, then this may not be a problem. However, if the components of the cell that are diminished at the expense of lipid
Fig. 23 Estimates of algal areal biomass yields (both daily and annual) for a range of latitudes. The calculations are based on calculated clear sky total insolation (see Fig. 18A) coupled to a tanh model of the relationship between photosynthesis and irradiance, described in Fig. 21. The parameters used were derived from the analysis of the data in Fig. 21 and 22, summarised in Table 7. The value for the initial slope (a) was the average of the full data set, the irradiance offset (Emin) was derived from ref. 70, 72; the dataset from70 lacked the observations at the low irradiances necessary to adequately constrain the intercept. Pmax was taken from ref. 71 the maximum rates in ref. 72 are low when compared with the data shown in Fig. 20A and accordingly were not thought to be appropriate. The initial calculation is of primary photosynthetic production of a hexose, so the individual values for a and Pmax have to be scaled up to take account of the fact that the observations were of biomass, which has incurred a loss of mass on converting hexose to proteins, polysaccharides and lipid (see Box 1). The nal values used were a 3.55 (dimensionless), Emin 5.86 MJ m2 d1, Pmax 76.9 g m2 d1; the calculation also requires the caloric value for the primary photosynthetic product, which was taken as 15.7 kJ g1 (ref. 63, 64). The yields for various lipid concentrations are derived from primary hexose production and the lipid content using the eqn (5), Box 1. This assumes a protein to carbohydrate ratio of 3 : 2, the mean value found in Fig. 5. The eld observation of yields (vertical bars) placed at latitude 20 come from ref. 16; the values at placed at latitude 35 come from ref. 71, 72.
increase are an important part of the economic equation, then this loss on conversion could well be a problem, especially if they were to command a price comparable to or greater than biolipid (see Section 6). Fig. 12, which derives from eqn (5), Box 1, shows the effect of the cell lipid content on the total biomass and lipid yields from the primary hexose photosynthetic products. (iii) Predicting maximum yields. We have attempted to incorporate the ndings of this section into a model of algal biomass production (see Fig. 23). In essence we have used the calculated clear sky irradiance values (Section 4.3(i) and Fig. 18A) and parameters derived in major part from an analysis of the data reported in ref. 71 to produce biomass production rates. We chose the Moheimani data set as it comes from raceways, which it is consistent with our economic analysis (see Section 6). The initial calculation we make is the rate production of hexose. In order to do this we need to back calculate the rates of hexose formation from the biomass-derived rates. This is done by reversing the calculation of the mass loss during the biomass. Ref. 71 observed a mean lipid content of 36.5% of dry weight. This (from eqn (5), Box 1) gives a gure of 1.88 for the biomass to conversion hexose and scales up the Pmax value from 42 g m2 d1 (Table 7) to 77 g m2 d1 and the initial slope (a) from 2.43 to 3.55 g MJ1. The data sets of ref. 71, 72 show a mean offset on the irradiance axis of 5.86 MJ m2 d1, this mean has been used for the calculation. The calculated photosynthetic hexose production rates are then converted to production for biomasses of varying lipid contents (15, 35 and 50% of cell dry weight) using eqn (5) in Box 1, assuming a protein : carbohydrate ratio of 3 : 2. Given the above parameterisations, we can calculate the yields per unit area, and their variation with latitude from the insolation data derived in Fig. 18A and the results are shown in Fig. 23. Shown for comparison purposes are results from three reports of seasonal productivity abstracted from published sources. It can be seen in Fig. 23 that there is broad agreement between the
observed integrated rates and the predicted ones for algae with realistic biochemical compositions. The calculation is made from clear sky radiation data, which will overestimate the incoming radiation (cf. Fig. 18A and 19) so overestimate growth rates. There is little to be gained at this stage by making a detailed calculation based on observed insolation rates. As a counter to this, the calculation makes no attempt to anticipate improvements of the light capturing apparatus of the algae which we may expect to occur (see Section 4.5(i)). There is some steepening of the poleward gradient between the latitudes 3555 . This may limit year-round outdoor culture systems to latitudes less than 35 (see also Fig. 19). This appears to be the case with commercial production facilities. The longstanding, and successful, Earthrise Farms in southern California (latitude 33 080 N) only operates its raceways for 9 months of the year, whereas the systems at Kanahoe Point on the Big Island of Hawaii (latitude 19 470 N) are operated successfully on a yearround basis, with no evident seasonal reduction of productivity.16,92 The experimental facility at Roswell (33 N), which was part of the ASP programme, ran into problems of snow and ice during the winter period. In the model, the trend with latitude derives mainly from the reduction in annually integrated insolation; the effect will very likely be much greater in reality as one may expect additional strong temperature effects on growth. This afrms the need for temperature-parameterised models of photosynthesis and respiration. Fig. 23 shows the very marked negative effect of the lipid content on the yields, and this questions the search for high lipid species for, unless they possess uniquely high intrinsic photosynthetic rates, they will give low biomass yields. There may be an additional effect due to the reduction in growth rate at high lipid content, resulting in the algae failing to achieve the rates that the irradiance would allow. Thus, there would appear to be diminishing returns with increased lipid content (see also Fig. 12); this is seen when the economics are considered (see Fig. 24).
5. Harvesting and processing of microalgal biomass

Before considering the economics of the process (Section 6), we want to highlight the challenges associated with the harvest and processing of microalgal biomass. The methods that are most used for harvesting of algal biomass are discussed in this section, with an emphasis on the use of occulation and centrifugation. With regards to biomass processing, we briey discuss the principles behind the methods relevant to the economical process we discuss in section 6. Out of all steps considered in an algal biomass production process, harvesting and processing of the biomass contains the largest uncertainty surrounding cost and effectiveness. The separation step in producing algal biomass as feedstock for biodiesel is difcult and inherently more expensive by comparison with oilseed crops. For algal biomass, there are three main phases in the preparation process prior to biodiesel production: (i) harvesting of the algae; (ii) disrupting the cells; and (ii) extraction of the lipids from the biomass. The efciency and methodology used in these three steps can have a major impact on the economics of commercial algal biofuel production. These processes have been largely avoided in past reviews, despite being identied as a high priority for the future R&D.9395
5.1 Biomass harvest Cell harvest we take as the initial 50200-fold concentration step of the algal cultures yielding a biomass product that can be used for further processing. The choice of harvesting methodology, likely to be a combination of individual methods, depends highly on the biomass type and requirements of the downstream processing (Sections 5.2 through 5.4). Settling rate (vs) of microalgae is a functon of two forces (drag, Fd and gravity, Fg) and the diameter (rc) and shape of the cells. The relationship at low Reynolds numbers for a sphere is described by Stokes law: vs Fg/Fd 2/9grc2 (rcell ruid)/h The settling rate of cells in suspension follows a square relationship with the radius of the cells and a linear relationship with the difference in density (r) between the cell and the uid and the kinematic viscosity of the uid (h). Centrifugation is the most common method for harvesting algae from large volume cultures and is widely applicable to a variety of algal species.15,96,97 Although this process is effective, it is also expensive and energy intensive. The operational variables, such as centrifugal force, ow rate, biomass settling rate and settling distance will determine the efciency of centrifugation. There are three different types of in-line industrial centrifuges: multi-chamber, nozzle and solid bowl. All three types are continuous in that there is a continuous ow of culture through the centrifuges. A multi-chamber centrifuge consists of a number of tubular bowls arranged coaxially, causing each chamber to collect particles of a specic size due to the distinct centrifugal forces exerted in each chamber. A solids content of 20% can be achieved, however the solids need to be removed from the bowl
manually. An alternative design is the solid bowl centrifuge in which the bowl contains a disc stack, causing the biomass to collect at the outer part of the bowl. However this set-up also requires manual intervention in removing the biomass from the bowl. A third type of centrifuge is the nozzle-type centrifuge, similarly containing internally stacked discs, and in which the shape of the bowl is modied so that the conical section of the bowl has evenly spaced orices (nozzles). The biomass is collected in a conical space on the side of the bowl where it is automatically discharged. This last type is a true continuous centrifugation system with minimal manual intervention98 and could be considered the most appropriate type to harvest the large algae cultures. These three types of centrifuges vary in the volume processed per hour, with a maximum of 50 to 100 m3 h1 (Table 8; ref. 15 and centrifuge industry representatives, personal communication). An alternative to settling is otation of biomass, where dispersed air bubbles in the culture adhere to ocs formed during occulation and cause the cell aggregates to oat. This process can operate more efciently and rapidly than sedimentation or settling and can also achieve a higher solids fraction.15,97 This process is potentially scalable to large-scale algal ponds.15 However, it is probable that an extra concentration step (possibly centrifugation) may be necessary because of the relatively low nal product concentration. Flocculation, in combination with centrifugation or dissolved air otation, is also widely method used for harvesting microalgal cells. Flocculation refers to the aggregation of particles in suspension to form clumps that can overcome the Stokes drag force and increase the settling rate. This reduces or avoids the need of energy intensive separation mechanisms like centrifugation. It has been suggested that this process could be adapted to be the lowest cost harvesting process.15 However, occulation rate and efciency are highly dependent on cellular characteristics, such as cell volume and cell wall properties, or culture characteristics, such as cell number, culture age and growth phase.99,100 Two mechanisms that give rise to occulation are: (i) charge neutralisation, i.e. the attraction between the negatively charged algal cell surface and the positively charged occulentl; and (ii) bridging, when larger occulent molecules span a distance large enough to adsorb to multiple particles and hence bridge the particles.101,102 Several different types of occulants can be used: polyvalent metal salts, synthetic cationic polymers, high pH and bioocculants. The low cost of metal salt occulants make these the more popular occulants. However, the potential toxicity of the wastewater fraction and the undesirable presence of metals in the biomass to be processed are concerns that arise and may inhibit their widespread applicability in the microalgal biomass production process. This would be particularly the case if the spent medium were to be recycled. The main advantage of occulation as a harvesting method is the relatively cheap nature of the process and high volume processing. However, disadvantages include the species and growth stage-dependent nature of occulation efciency. Until now, no clear correlation has been demonstrated between occulation efciency, dose and algal taxonomic group. Another disadvantage is the low solids content (i.e. < 10%) of the biomass harvested by occulation, which requires further concentration
Final product concentration refers to the algal biomass dry weight in the harvested product (e.g. 10% 10 g dry biomass from 100 g of centrifuged paste). b Capital costs presented, expressed on a 1 m3 h1 basis, are at best approximate since these were obtained from earlier reports and simply adjusted for ination, not taking improved engineering into account. c Values given in brackets indicate the harvesting costs, for the sake of comparison the calculation uses a 1 kg m3 cell concentration upon harvesting, assuming a 0.4 and 0.079 g dw L1 cell concentration for ref. 37 and 103 respectively. d Values presented are averaged costs of biomass from cultures grown at unregulated pH, pH 9.6 or pH 9. e The cost calculations have been adjusted for ination and converted into todays US dollars (conversion factor 1 AUS $ 0.81 USD). f The cost of harvesting using otation was reconsidered in ref. 15, where according to equipment and information available in 1996 the cost of harvesting 1 m3 h1 by otation was 20% of the cost of centrifugation. These gures did not include the cost of occulant. Assuming a relative cost of 0.31 for occulation, we could derive a relative harvesting gure of 0.51. g Current centrifugation engineering allows processing of up to 100 m3 of culture per hour; na not applicable.
Table 8 Summary of comparisons between relative harvesting throughput, applications, capital and operating costs. Settling refers to autoocculation, changes in pH in a stagnant culture will induce settling of algal biomass, otation refers to the separation process based on the adhesion of air bubbles to ocs of suspended algae after which these oat to the surface and separation can occur. Centrifugation is subdivided into multi-chamber, nozzle and solid bowl centrifugation. The relative harvesting costs include reference to operating, maintenance and capital costs and are based on gures from ref. 97, 15 and 94. The capital costs presented are adjusted for ination and converted into US dollars (USD, $)
Volume processed/m3 h1
na na 1591900 1520g 2.55 20 0.53
using other methods. As a result, occulation is often combined in a process with centrifugation to increase the solids content of the harvested biomass. Another method for harvesting the biomass is ltration. The vacuum drum lters and chamber lter press units are most commonly employed for harvesting fairly large microalgae. However, because ltration efciency is highly dependent on the size range of the species to be harvested, certain types of ltration are ineffective for species in the few micrometre size range, such as Scenedesmus, Dunaliella and Chlorella due to the rapid clogging of the lters.97 This species-dependent nature of the ltration process makes this less attractive compared to centrifugation and occulation and will not be considered further. An exact economic calculation and comparison of methods for harvesting is complex and practically impossible without information on the species chosen, production system and growth stage for harvesting. We have summarised the costs published in the literature to date in Table 8. This table contains the bestcharacterised processes and we have compared these on the basis of processing volume, species application, capital and operating costs. The purpose of the overview table is to provide relative costs estimates, not absolute comparisons. The cited literature lacked detailed information on, e.g. the type and energy requirements of the centrifuges used. Furthermore, species and developmental information is largely lacking in the cited reports, and together with large uncertainties on capital an operating costs it makes exact cost comparisons difcult (if not impossible). The harvesting costs are taken into consideration further in Section 6, where the economic analysis of the complete algal production process is considered. In summary, most of the literature on the economic considerations of harvesting processes has focused on the need for highly puried products for the food and feed industry and waste water purication. The application to harvesting biomass for biofuels will not require the stringent product purity that is the case for the nutraceutrical industry, which will make the process cheaper. It has been mentioned before97 that there is not a universally valid (one-size-ts-all) optimum separation process. However, the harvesting process is a major consideration in the set-up of the production process. The biomass concentration and form will affect (and most likely be dictated by) the downstream treatment processes. 5.2 Biomass pre-treatment and extraction of the lipid fraction The harvested biomass (consisting of about 520% solids, depending on the harvesting method used) has to be processed further to produce the feedstocks (i.e. lipid fraction) for biofuels production. In most economic analyses the exact harvest, extraction and process details are not discussed. This is largely due to the uncertainty surrounding the effectiveness, extraction yield and the costs associated with the catalytic upgrading of the lipid fraction. Most of the economic analyses published extrapolate the oil extraction procedures from a seed oil extraction process to algal biomass. Seeds require very little pretreatment or processing and the oils (mainly triglycerides) are traditionally extracted either by pressing the oil out of the biomass or using an apolar solvent to selectively extract the triglycerides from the biomass. Because of
Moheimani (2004)103 d,e Molina Grima (2004)94 Benemann (1996)15 Mohn (1988)97 Molina Grima (2004)94 Benemann (1996)15 Mohn (1988)97 Final product concentration (% solids)a Continuous process? Becker (1994)37
Cost per kg/$ c
Relative costs/m3
Capital cost/1000 $ m3 b
Settling/autoocculation Flocculation Flotation (dissolved air otation) Centrifugation 3-chamber centrifuge nozzle centrifuge solid bowl or decanter
N N Y Y N Y N
13 7 8 1227 20 <15 >20
33 15 12 4
19105 55
9 11
0.31 0.91f 1 0.82 0.26
0.051 0.60.8 0.40.6 1
0.61 [0.24] 1.39 [0.56] 1.71 [0.68]
1.1 [0.08] 5.2 [0.41] 4.6 [0.36] 3.8 [0.29] 4.1 [0.32]
large differences between algal and seed biomass properties and oil composition, specic procedures will have to be developed, applied and costed for an algal production process. Pretreatment, such as drying or cell disruption, of algal biomass has been shown to aid lipid extraction.94,104,105 One advantage of processing dried biomass is better percolation of the solvents in the biomass, leading to an increase in lipid extraction.104,106,107 However drying is not considered an economical option for biomass pretreatment for a biofuels production process because of the high-energy requirements of the drying process. Alternative pretreatment methods include cell rupture of the harvested biomass, which could aid in the lipid extraction process by avoiding or reducing the use of solvents. A range of cell rupture techniques, mechanical, chemical and enzymatic, has been described in the literature as applied to oilseeds and algae.104,108112 However, no such method has been adopted for algae on a large-scale. This is, most likely, because the effectiveness of the cell rupture methodology depends heavily on the physical properties of the algal cell wall and thus will change or have to be adapted with particular species used in the culture. The composition and strength of the cell wall varies considerably across species and throughout the algal growth cycle. For example, Chlorella sp. and Scenedesmus sp. both have robust cell walls with high cellulose content, rendering the cells difcult to digest or rupture.113 Alternatively, species like Spirulina (a cyanobacterium) lack a rigid cell wall and are thus easier to disrupt,114 however, cyanobacteria are characteristically low in lipid content. 5.3. Production of biofuels
The lipids obtained by the above processes can then be catalytically converted to biodiesel (methyl esters of the fatty acid portion of lipids) or green diesel (deoxygenated fuel derived from fatty acids). Conventional biodiesel production occurs via the transesterication of pre-extracted lipids in the presence of an alcohol and a catalyst, which can be a strong acid or base to produce fatty acid methyl esters. Green diesel refers to the production of alkanes through for example catalytic decarboxylation of fatty acids. A detailed discussion of the methodology and efciency of these processes falls outside of the scope of this review and can be found elsewhere.115 It is worth mentioning that the factors determining the yield and conversion efciency, such as temperature, time of incubation and catalyst concentration, are highly dependent on the nature and composition of the feedstocks. No systematic study has been undertaken to optimise, develop and price the cost of a biodiesel production process specically for algal lipids or biomass. A detailed costing scheme was developed for biodiesel production from soybean oil.10 However, there are too many unknowns in the algal biodiesel process to be able to provide an estimate for the cost of this process. Furthermore, it is anticipated that this process will be species, growth- and environmental conditions-dependent. 5.4. Processing of the residual biomass and co-products
more, valuable than the biodiesel itself. The process that is utilized in the cost model discussed in section 6 includes anaerobic digestion as an option for residual biomass utilization. Anaerobic digestion is a long established technology in wastewater management for the disposal of organic waste and the recouping of energy. In essence, in the absence of oxygen, bacteria turn to a variety of alternative proton acceptors to dispose of the protons the organisms abstract from their organic substrates. If available, bacteria will use nitrate, sulfate and carbon dioxide in that order of preference. If nitrate and sulfate are absent, or at low concentration, carbon dioxide is the main proton acceptor and the products are methane and CO2. The yield of methane can be high and the energy recovery likewise high, a gure of 70% may be calculated from the data in ref. 15. If decomposition can be driven to near completion then the process has the potential to recycle the inorganic nitrogen (as ammonia) and phosphate, thus saving costs and the carbon footprint associated with the HaberBosch production of ammonia. The recycling of nitrogen, of course, could only occur if the protein and nucleic acid constituents of algal biomass were put to digestion. If the protein were sold for animal feed, then there would be very limited recycling of nitrogen, as 8090% or so of the nitrogen resides with these molecules (see Table 2). The remainder of the nitrogen is principally associated with nucleic acids. This class of compounds have limited nutritional value and could usefully be put to anaerobic digestion, were it economic it would also result in recycling a major part of the cells phosphorus (see Table 2). Despite its obvious benets, the economics of anaerobic digestion are not promising; ref. 15 (p. 141) Thus, power generation (from anaerobic digestion) is not a major prot centre. Our own analysis of recycling the protein and carbohydrate fractions via anaerobic digestion likewise suggests that after allowing for the offsetting of the cost of electricity from the energy derived from the methane, there is no net revenue from anaerobic digestion (see Fig. 24). 5.5 Summary From the foregoing discussion it can be seen that there are a number of routes available for the separation and processing of the algal culture. In many cases their effectiveness is dependent upon specic features of the algae, which may vary between species and with their physiological and growth status. Thus, no general strategy can be specied, although some may be eliminated based on the current state of technology (e.g. ltration). Uncertainties surrounding the optimum strategy for harvest and biomass processing would not be a major concern if the separation stage made an insignicant contribution to the overall budget. However, this does not appear to be the case in the economic analysis undertaken in the present study (Section 6), harvest and extraction accounted for 15% or so of the operating budget (50% of the labour costs has been attributed to these processes). Ref. 15 gives a lower gure for operating costs but attribute a higher contribution ($12%) to capital costs. Presently, the estimates for the cost of harvesting and separating should be seen as best guesses, and without doubt they are major sources of uncertainty in any economic analysis. Until target organisms have been identied this may have to remain to be the
Critical to the economic success of algal biofuel production is the use to which the cell co-products are put, as these can be as, if not
case. Furthermore, advances in engineering and improvements in centrifuge technology could also alter the costing. Similar uncertainties surround the fate of the residual biomass. The high protein content of the residual biomass could provide an excellent source of feed. However, if the lipid extraction process is solvent-based, prior to considering animal feed as an option, toxicology tests will have to be carried out. The carbohydrates in the residual biomass could be used for ethanol fermentation, however, not enough information is present in the literature to date regarding the composition of individual sugars and concentration in the residual biomass as well as large unknowns about the recalcitrance of microalgal structural carbohydrates. The potential of the different scenarios on the use of the residual biomass will be discussed in the following section.
6. Economics of biomass and biofuel production

Widely varying estimates for the cost of algal biodiesel may be found in the recent literature (see Table 10). This, in large part, reects the immaturity of the subject. The aim of this section is not to produce a denitive costing presently there are too many uncertainties to allow this but give a basis to examine the pattern of the economics for various management scenarios. To this end a set of objective costings is needed. There is only a limited amount of information to draw upon to construct a costing algorithm. The DOE Aquatic Species Program commissioned a report (ref. 116) to produce costings; ref. 15 incorporated the essence of this in the 1996 Report to the Pittsburgh Energy Technology Centre. A detailed costing was prepared for a pond/raceway type system.117 Although the capital costs were specied, it is difcult to extract the operational costs with any certainty. Ref. 94 gives a costing for a tubular bioreactor and ref. 103 costed a raceway system. Some of the numbers in the report in ref. 103 are recycled from ref. 15 and 116, so it cannot be taken as a wholly independent analysis this is also the case for a number of other reports and to some small extent the present one. 6.1 Parameterising the economic model We have based our analysis upon a recent set of costings prepared for the US Department of Energys Ofce of Energy Efciency and Renewable Energy118 for a combined photobioreactor (PBR)/raceway system; all costings were made de novo. This has been updated with published costings and from discussions with colleagues in the eld. From this a spreadsheet model has been generated. All the items we have been able to cost are built into the spreadsheet. However, there are a number of processes we have been unable to cost with any certainty: the processing of the cell debris to separate the carbohydrate and protein fractions and the subsequent isolation and processing of the protein fraction to animal feed. There are additional uncertainties surrounding the harvesting step. Estimating the cost of the necessary CO2 supply is problematic as it will be very much dependent upon the nature of the overall facility, i.e. whether the algal growth system is an adjunct to a CO2 source (i.e. a power station) or an isolated facility. Our solution to this problem is, rather than to put in a costing that is circumstance-specic, to leave it out of the costing algorithm but to return to it in our
discussion of the analysis of scenarios. A hybrid system, such as that described in ref. 16, comprising a front end PBR nursery stage and grow-on stage in raceways is used as the physical arrangement as it appears to be the most cost effective option. An outline of the costings in the model is given in Table 9. Without doubt it can be added to and improved upon. Recently a comprehensive and thorough review of the economics of algal biomass production report has been prepared for the British Columbian Innovation Council.119 These authors worked largely, although not entirely, from fresh estimates. Their analysis was not available when our spreadsheet was generated. We are encouraged to nd that their capital costs ($209 500 ha1, converted from Canadian dollars using a Can/US exchange rate of 0.8) are almost identical to our estimate ($209 000 ha1). Their estimate for operating costs, with the capital repayment costs removed, was $20 425 per year; our estimate was $26 375; however, the similarity in these values is almost certainly fortuitous, since there is considerable variance in the estimates for several major individual items between the two budgets. Their energy costs are about 15% of ours; (deriving in part from the low cost of hydropower in British Columbia) whilst their labour costs are around four times greater (indicating that ours may need revisiting). There is, however, no signicant change to the economic pattern seen in Fig. 24 if their labour costs are substituted into our economic analysis. In the economic analysis we consider three lipid levels (15, 35 and 50% lipid as dry weight) and three latitudes: low (030 ), mid (3545 ) and high (4555 ) latitude bands using the production rates shown in Fig. 23. We have explored three scenarios surrounding the exploitation of the non-lipid fraction: (i) the carbohydrate and protein part of the cell debris is sold as animal feed ($60% protein, taken from the 3 : 2 protein to carbohydrate ratio used throughout this work, see legend to Fig. 5); (ii) all of the non-lipid cell debris (plus the glycerol and other water soluble products from the transesterication process) is put to anaerobic digestion, with the production of methane (used to generate energy in the process the nitrogen and phosphate are recycled); (iii) the carbohydrate from the algae and the glycerol and other water soluble products are digested anaerobically when all the phosphate will be recycled, but only the small fraction of the nitrogen in nucleic acids and phospholipids, about 1015% of the total nitrogen (see Table 2). The protein is sold as high grade protein-rich material, which could have a market as supplements for low protein animal feeds such as corn meal (15% protein). Assigning cash values to these various products (algal lipid, protein and carbohydrate plus protein mix) has to be a matter of speculation as no market exits for them as yet. Animal feeding trials on algal preparations in general have been underway for some time.120 However, they will need to be extended to look at, amongst other things, the possible effects of materials used during harvesting and processing and it will be some time before a product value will be established. We have approached the problem by setting a starting gure from the expected chemical property of the product. The Food and Agriculture Organization (FAO) statistics132 for the prices of animal feed show a broad correlation with protein content, thus given a protein content we can derive an objective estimate of price. For feed with 60% protein content a value of $750 tonne1 is obtained. An alternative calculation may be made pro rata from the price of soya
meal (protein content 45%; median commodity value ca. $400 tonne1); this gives a lower gure of $533 tonne1. We have conservatively used a rounded down value of the lower gure $500 tonne1. For the protein-only material the protein/price analysis is not regarded to be reliable as it exceeds the available data, a gure of $890 may be calculated pro rata from soya, this has been rounded off to $900 for the purpose of calculation. Its important to note that these values are only used as starting values for scenario analysis, so their exact values are not critical. Somewhat more critical is the price we assign to algal biodiesel. We have drawn a mean value of $916 73 tonne1 from recent analyses by market specialistskk and have rounded this down to $900 tonne1 this approximates to $125/barrel. Whether algal biodiesel will be more valuable than traditional biodiesel remains to be determined and will depend on the market when its fuel properties are better established, as well as social attitudes towards higher plant biodiesel (see highlighted comment in the Gallagher report, Section 1). As a starting point we have used the above value, plus two additional values: $550 tonne1 and $700 tonne1, these are rounded off values for $75/barrel and $100/ barrel. The required nutrients, nitrogen and phosphorus, are assumed to be supplied as a mixture of di-ammonium phosphate and ammonia (costed at $325 tonne1 and $300 tonne1, respectively) matched to the calculated nitrogen and phosphorous content for cells of low (15%), medium (35%) and high (50%) lipid content (see Table 2). When all the non-lipid fraction is put to anaerobic digestion, the nutrient cost is set to zero (this assumes 100% recovery). In the scenario where the protein fraction is not digested but sold, the phosphate addition is set to zero and ammonia alone is added to satisfy the nitrogen requirement. The model assumes that the cells protein : carbohydrate ratio is 3 : 2 and does not vary with lipid content. This assumption needs verication as it has a marked effect on some aspects of the costing. Furthermore, the separation of the protein and carbohydrate fraction will have inuence on the costing, however, no data on this separation step was found in the literature. There are three losses that need to be taken into consideration: (i) the production of extracellular dissolved organic material during growth; (ii) an allowance for operational downtime; and (iii) losses during harvesting. There is a substantial literature surrounding the losses due to the release of organic material (a gure of 5% is used) and downtime is assumed to be 1 week per annum (a 2% loss). Losses on harvesting are presently not established. A round gure of 10% is taken for the combined loss from these three sources, although we suspect that this may be an underestimate. The spreadsheet (see Table 9) scales the costings, where appropriate, to the tonnage cell material produced, the tonnage of lipid processed and the tonnage put to anaerobic digestion. The capital and operating costs of anaerobic digestion were supplied by a UK manufacturer. The gures are not too dissimilar from costings (ination adjusted) used by ref. 15 and 67. Anaerobic digestion is assumed to lose 25% of the caloric value of the feedstock during the production of methane, the latter is then converted to electricity assuming 35% efciency,
kk Values for Europe, Asia and USA from ICIS pricing and for Europe from Kingsman (http://www.kingsman.com).
Table 9 Basic structure of the spreadsheet costing model, shown for one example: a low latitude situation, with a 28 g m2 d1 (103 tonnes ha1 a1) productivity and a lipid content of 35% of dry weight. In this example, the protein is separated from the carbohydrate and sold at $900 tonne1 as high-grade animal feed, the carbohydrate and other water soluble products are processed by anaerobic digestion. The biodiesel, as methyl esters, is sold at $900 tonne1. The costings are intended to be for a scale where they are no longer dependent of the size of the plant (i.e. there is no further economy of scale), but determined, where appropriate, by the size of the culture area and the scale of processing. To convert biodiesel from mass to volume units, it is assumed to have a specic gravity of 0.88 and a barrel is taken as 159 litres. All large numbers have been rounded off to 3 signicant digits; dw dry weight ASPECT OF PRODUCTION FACILITY UNITS
Operating Parameters & Assumptions Areal production 28 g dw m2 d1 Annual tonnage produced 103 tonnes dw ha1 a1 Lipid content 35% dw Protein : carbohydrate ratio 3 : 2 mass Lipid production 36 tonnes dw ha1 a1 Bidiesel production (at 70% biolipid 25.2 tonnes dw ha1 a1 production) Carbohydrate used for anaerobic 100% digestion Protein used for anaerobic digestion 0% Annual tonnage used for anaerobic 35 tonnes dw ha1 a1 digestion Electricity yield from anaerobic digestion 139 000 MJ ha1 a1 Growth area, as % site area 88% DAP costs per tonne biomass 0 $ tonne1 NH3 costs per tonne biomass 21 $ tonne1 Capital costs 41 800 $ ha1 a1 Site prepa Culture systema 148 000 $ ha1 a1 Engineeringa 22 000 $ ha1 a1 Harvestb 3570 $ ha1 a1 Extractionc 4560 $ ha1 a1 Anaerobic digester @ $225 tonne1 annum1 8730 $ ha1 a1 throughputd TOTAL CAPITAL COSTS 228 000 $ ha1 a1 Running costs 4430 $ ha1 a1 Laboura Growth power requirement electricity 7020 $ ha1 a1 @ $0.07/kWha Harvest power requirement electricity 2660 $ ha1 a1 @ $0.07/kWha Dewatering power requirement electricity 105 $ ha1 a1 @ $0.07/kWha Diammonium phosphate (DAP)b 0 $ ha1 a1 Ammoniab 2210 $ ha1 a1 Transesterication methanolc 1650 $ ha1 a1 Transesterication power requirement 166 $ ha1 a1 natural gasc Waste energy value @ $0.07 kWh1 d 1120 $ ha1 a1 Anaerobic digester operating costsd 2720 $ ha1 a1 Captial costs @10% per annum 22 800 $ ha1 a1 TOTAL COSTS 42 700 $ ha1 a1 Product costs & revenues Biomass cost 0.41 $ kg1 Biodiesel cost 1.69 $ kg1 Biodiesel cost 237 $ bbl1 Revenue from biodiesel (@ $900 tonne1 22 700 $ ha1 a1 Revenue from pure protein @ $900 36 200 $ ha1 a1 tonne1 Total revenue 58 900 $ ha1 a1 Total expenditure 42 700 $ ha1 a1 NET REVENUE 16 200 $ ha1 a1
a Scaling of parameters: scaled to culture area. b Scaled to biomass production. c Scaled to lipid processed. d Scaled to tonnage digested.
Fig. 24 Results of a spreadsheet model of costings and economics for the production of biodiesel and associated product streams. The calculation assumes biodiesel to have a commodity value of $900 tonne1 (ca. $125 bbl1). The details of the model are given in Table 9 and the text. A. Net revenue. Lipid levels: blue 15%, green 35%, red 50%. B. The value the separate products, biodiesel at $900 tonne1, animal feed at either $500 tonne1 (as low grade protein dark blue bars) or at $900 tonne1 (as high grade protein pale blue bars). The net revenue calculation for anaerobic digestion involves the revenue gain from electricity production and the recycling of nitrogen (where it occurs) and phosphate minus the operating and capital costs of the anaerobic digestion plant. C. The cost of biomass ($ tonne1) and biodiesel ($ bbl1) assuming them to be the sole marketed product.
Fig. 25 A matrix of the outcome of analyses of economic Scenarios 1 and 3 for the net revenue resulting from various commodity prices for the algal products.
waste heat from the generators is not put to any specic use in our analysis, although it certainly would be in the plant design. The digester is assumed to enable 100% recycling of the nitrogen and phosphate in the feedstock for use in the culture media. All of these assumptions need conrmation. 6.2 Analysis of scenarios An illustration of the three end-product management scenarios is given in Fig. 24A. A general trend of reducing revenue with increasing lipid content is apparent. Furthermore, only at a lipid content of 50% of the cells dry weight does the revenue of biodiesel exceed that from selling the algal proteins. However at 50% lipid levels, the assumption in the productivity model that sufciently high growth rates can be sustained and only light limits growth, becomes uncertain (see Fig. 6). A summary of a matrix analysis for Scenarios 1 and 3 is shown in Fig. 25. Given a value of $125 bbl1 for biodiesel, positive economic solutions can be found if the value of the protein + carbohydrate product (Scenario 1) is above $350 tonne1, and for the protein-only scenario (Scenario 3) above values of $600 tonne1. The analyses are not particularly sensitive to lowering of oil prices an analysis for $350 tonne1 (nominally $50 bbl1) is much the same as the analysis for $550 tonne1. However, a rise of the same scale to from $100 to $125 bbl1 would make all the scenarios in Fig. 25 protable on the costings used. Anaerobic digestion (Scenario 2), even allowing for savings arising from the recycling of energy and nutrients, is not a net source of revenue (Fig. 24B). This was also the earlier conclusion in ref. 15. The costs of anaerobic digestion come from temperate latitudes, where a considerable proportion of the energy yield has to be used to maintain the temperature of the digester; one may expect these circumstances to be very different at low latitudes. This may make the anaerobic digestion of protein, along with carbohydrate, a more attractive proposition. This would be essential if the intention were for biodiesel to be the sole
commodity. Whether the alternative of fermenting the carbohydrate and glycerol to ethanol offers better economic rewards would be worth exploring. The analysis makes the point that the co-products, notably protein, have the potential to be very valuable. Perhaps the most important nding is that the simple aim to increase lipid yields using high lipid containing algae may not necessarily be a sound economic strategy if the biochemical and other losses associated with lipid and biodiesel production are taken into consideration, gains from high lipid production only appear at biodiesel prices above $125 bbl1. It is appropriate at this point to recognise that no costing has been made for CO2 supply. At liquid CO2 prices of $10 tonne1 the pattern in Fig. 24 remains much the same. At $50 tonne1, the price determined from a recent study in ref. 121 for carbon capture and sequestration, protable scenarios were only found at the higher price levels, at $100 tonne1 protable scenarios appear only with biodiesel prices of $125 and above. There is no doubt that our analysis can be improved upon and that there are uncertainties associated with many of the numbers used in the calculations. Large uncertainties surround the costing of the plant notably the harvesting and subsequent treatment of the biomass and the eventual market values of the various biomass components. Uncertainties over the estimated areal production rates will condition the reliability of our economic analysis. There is active ongoing research and this is one area where one can anticipate substantial advances in the not too distant future. The present model assumes that light alone limits production, the algae are presumed not to enter nutrient limitation, it this respect it may be providing optimistic estimates. On the other side of the coin, the sense exists that there is potential to improve upon present yields. Potential gains in productivity could arise from modications of the cells light capturing apparatus (see Section 4.5). We have chosen not to make allowance for these potential improvements. By far the majority of algal farms to date have focused on high value products, so
Table 10 A comparison of published costings for algal biomass and biodiesel production. We have only taken the median value from ref. 122. To convert biodiesel from mass to volume units, it is assumed to have a specic gravity of 0.88 and a barrel is taken as 159 litres. The estimates have been adjusted to 2007 prices, the currency conversions used are: $ Aus/$ US 0.81, $ Can/$ US 0.8 and V/$ US 1.28 Biomass costs Assumed productivity $ tonne1 g dw/m2 d1 % dw 50 50 Not given Not given Not given 30 30 30 40 Not given 25 15 15 25 50 1550 5900 470 370 600 200015 000 2140 6100 47006900 30 400 110360 360650 241 148 1600 [224] 1 6800 35070 000 (1 4600 8200) PBR RW Hybrid PBR 20 3.230 (14.4 8.2) 30 60 Not given 20 1620 27 72 35 1960 2.714 15.3 9.4 10 25 50 1837 Assumed lipid content $ tonne1 [$ bbl1] RW 490 [69] 300 [42] 1200 [168] 2050 [287] 7500 [1050] 2250 [315] 750 [105] Hybrid 280910 [39127] 9003500 [126490] Biodiesel costs
Source
Organism
Borowitzka (1992)117
Not specied Various Mean (+StDev)
Benemann & Oswald (1996)15
Not specied Not specied
Molina Grima et al. (2004)94
Phaeodactylum
Moheimani (2005)103
Dunaliella Pleurochrysis
van Harmelen & Oonk (2006)122
Not specied
Chisti (2007)4
Huntley & Redalje (2007)16
Not specied
Carlsson et al. (2007)123
Not specied
Alabi et al. (2009)119
Pienkos & Darzins (2009)95
Not specied Not specied Not specied
Present account
Not specied
the incentive to undertake expensive R & D to increase yields has been low. This investment will need to be made to increase sustained algal biomass production rates if we are to use algae for relatively low value products such as biodiesel and animal feed. 6.3 Comparison with other economic analyses We have compared the costings published in the literature. Table 10 gives a collation of published costings. At rst sight they seem disturbingly disparate: estimates for biodiesel costs range for $280$7500 tonne1 ($39$1050 bbl1) and biomass from $110 $30 400 tonne1. Ref. 117 contains his own estimate and summarises earlier work the average of these estimates in the region $15 000 tonne1. Part of the reason for the high costs in the latter study are the conservative assumed yield rates (15 to 20 g dry weight m2 d1) used in the analysis, but also in Borowitzkas study an apparently high operational cost. The estimate in ref. 103, which again is high, likewise contains high values for manpower and harvesting, which seems out of line with other costings. Further, the numbers must also be considered in context the high estimate reported in ref. 94 derives from an advanced PBR system, intended for a high value product. The costings in ref. 123 are based on an extremely cautious assumption of growth yields (2.714 g dry weight m2 d1). We nd it difcult to see how systems can be run in prot with biomass yields below 20 g dry weight m2 d1. The careful analysis in ref. 119 also stands out as high-cost estimates; however, they made their analysis for the latitude of British Columbia ($50 N), where biomass yields would around half those from latitudes of 030 . If we consider the remaining analyses and disregard the high cost, non-viable, versions of the present report and the worst case scenario of the analysis in ref. 95, the spread is still large but reduced to $110$600 tonne1 for biomass and $280$2050 tonne1 ($39$315 bbl1) for biodiesel. Despite the uncertainties, the broad patterns in the economics are probably correct, although it would be unrealistic to expect the exact values to be. Thus, given the most favourable form of culture a photobioreactor/raceway hybrid we can envisage a number of scenarios that would appear to have the potential to yield net prot.
7 Prospects and research needs

In summing up the review we explore the answers to two questions that arose through the course of the review: (1) Can the production of algal biomass (or biolipid) be made protable? (2) What R & D is needed to achieve these aims? 7.1 Can the production of algal biomass (or biolipid) be made protable? The evidence from our analysis is a conditioned yes. First, in our analysis in Section 6 we found that, with prices for biodiesel up to $1400 tonne1 ($200/barrel), we are only able to nd protable outcomes if all the major biochemical products are processed and marketed. We are unable to nd economic solutions for the marketing of biodiesel alone and the associated recycling of the nutrients and energy in the non-lipid fractions by anaerobic
digestion. Our nding is in agreement with ref. 122, in which it was concluded that the fuel only option was not economically plausible other revenue sources were needed to bring the system into prot. If the rst option is adopted, i.e. to market other biomass products, notably protein as animal feed, in addition to biolipid, the market for protein will control the overall scale of production. This has implications, potentially setting a limit to the scale of production. There are broadly four areas of uncertainty in the economic analysis. First, we have not been able to obtain costings for anaerobic digestion at low latitudes. This could extend the economic viability. A second major uncertainty relates to the cost of harvesting. This is probably the most complex of the problems, as the properties of the alga used, its nutritional and physiological status, will determine the strategy that can be used for harvesting. Harvesting costs will have a major, if not overriding, effect on strain selection. Estimates of harvesting costs (capital and operating costs), as a percentage of total costs, vary considerably in different economic analyses. The analysis in ref. 103 gave a range of 4050% for the harvest and processing costs for the calcium carbonate-containing coccolithophorid Pleurochrysis carterae and 63% for Dunaliella salina. The analysis in ref. 15 gave range of 2025%, and the analysis in ref. 117 analysis implies a similar gure: 1525% of the capital costs and 20% of the operating costs were associated with harvesting. We derive a gure in the region of 10% in the present analysis, similar to the estimate of 12% in ref. 119. Much of this variability derives not from the uncertainty in the costings themselves, over which there is reasonable consistency, but in the uncertainty over what procedures will be used. In particular, whether or not and to what extent centrifugation, which has high capital and operating costs, will be used; i.e. whether it has to be used for primary separation (which seems unlikely) or nal concentration. Doubling the harvesting costs in our analysis gives rise to a 1015% fall in revenue of the more protable scenarios at low latitudes, but a greater (1530%) loss at high latitudes. A third area of uncertainty deals with the future market prices for the various commodities notably the value and acceptability of algal protein as an animal feed and, of course, the market price of biodiesel. If legislation for minimum additions of biodiesel to fuels persists and the ecological concerns over higher plant fuels continues or rises, then this may inate the value and so price of algal biodiesel. The fourth major uncertainty lies in the prediction of biomass yields. As discussed in Section 6, yields have a pronounced effect upon the economics. If, for example, the yields are reduced to two-thirds from about 3040 g dry weight m2 d1 (100140 tonnes ha1 a1) to 2025 g dry weight m2 d1 (6793 tonnes ha1 a1) then we nd it difcult to produce economic scenarios. Although predicting yields is a complex matter, we would see in many respects that producing models of algal production is a more tractable problem than many of the others above and the potential exists to produce robust models in the not too distant future. 7.2 What R & D is needed to achieve these aims? A number of reports have considered the R & D required to produce algal biomass economically, and on an industrial
scale.3,5,15 The list of R & D needs is very long and there is no need to detail these at length. The aim here is to give emphasis to the R & D requirements that this review has indicated to be of greatest importance and urgency: optimising yields, advancing our understanding of harvesting and processing strategies and resolving the problems of scaling up. (i) Optimising yields. This may be subdivided into two broad problems increasing production rates and maximising the economics of product yields. In Section 3 we developed the broad basis for the theoretical photosynthetic yields a gure in the region of 10% of the energy in incident irradiation incorporated into organic material is a widely accepted value. This maximum yield, we point out, is constrained by stoichiometry and thermodynamics and, for all practical purposes, may be considered to be immovable. Actual yields fall short of this, often being in the region of one third to one tenth of the theoretical maximum (see Section 4.3(ii)). As far as we can determine, there is no fundamental barrier to achieving the theoretical yields, however nding solutions that are economically viable present severe practical challenges. The losses are principally due to wastage of the energy of the collected photons at high photon ux rates. This derives mainly from a mismatch between the processing rates of the photochemical and enzymatic reactions. The problem was discussed at length in Section 4.5(i) and its probable resolution lies not in increasing the rates of the dark reaction, there seems limited capability to do this, but by slowing down the photochemical reaction by reducing the photon capturing capability of the cell. There has been work done on this and progress made. This will require modifying the control of the cells light capturing apparatus, by some intrusive procedure essentially locking the cell into the high light adapted state. It is very unlikely to come from screening of natural species but rather through genetic engineering. In ref. 15 it was noted that the type of strain needed would be as as common as hens teeth in Nature, as they would have little to no competitive capability. A major issue is whether this could be achieved by classical plant breeding techniques induced mutations (so called accelerated evolution techniques) or by direct manipulation of the genome. Some major players in the eld, sensitive to the negative public reaction towards genetically modied organisms (GMOs), have set their face against going down the latter route. This, as other issues that surround the use of GMOs, will need to be debated. In a growing microalgal culture there is a complex of interactions between the organism and the physical and chemical environments, involving a number of positive and negative feedbacks (see Section 4.4). Commonly used, simple rst order empirical models based on energetics, such as that employed in Section 4.5(iii), cannot incorporate these feedbacks in any explicit way and thus can only be considered as rst order solutions. Fundamental physiological models of algal growth exist,124 but do not appear to be extensively used, as yet, in the eld of mass production of algae. They will be needed to optimise production rates further they have the potential to serve as an exploratory tool to determine the environmental criteria necessary for economic production. One matter that needs urgent consideration is the apparent irradiance threshold (see Fig. 22, and text in Section 4.4(ii)). We conclude that this cannot
be solely attributable to the compensation irradiance, but there may also be a secondary effect of light mediated through temperature. It may set an upper latitude boundary for yearround production. In ref. 122 a latitude boundary of 35 was suggested, they used a mean annual temperature of 15 C as a criterion. Such latitudinal boundaries would exclude yearround production in the EU, Russia and Canada, and geographically limit it in the US and China. Most of Africa, the Middle East, Central and South America, India, South-East Asia and Australia would however be well placed to develop such technologies. There is a pressing need to establish whether or not this apparent irradiance threshold is a general phenomenon and, if it is we need to gain a better understanding of its physiological basis. Many of the previous analyses of R & D needs have pointed to the requirement to isolate or develop high lipid producing algae. In relation to this, a pressing matter is to determine whether the apparent negative relationship between lipid content and growth rate exists (see Fig. 7 and the associated discussion in Section 2.3). This brings to the fore the need to distinguish between high lipid containing algae and high lipid yielding algae. Lipid content by itself would seem of limited value, if it has to be achieved at the expense of growth rate. This is particularly the case when products other than lipids are part of the overall economic equation (see Section 6 and Fig. 24). Following on from the above, as soon as the economics are extended beyond the production of lipids and biodiesel, the value and the dynamics of the quantitative distribution of the other major biochemicals within the cell become a major issue. A collation of the reported proportions of the major biochemical fractions (lipids, carbohydrates and proteins) was analysed in Fig. 3 and no pattern was evident. This may well be the case, but the compilation came from very disparate studies and in many cases required data conversions and any pattern may have been lost or concealed within the resultant noise. Network models for the ows of material during metabolism have been developed for higher plants (see ref. 50 and 125). These could be adapted to microalgae. However, these models will need dedicated studies on microalgae in order to calibrate them. Once they are available, one could use these models as powerful predictive tools. (ii) Advancing our understanding of strategies for harvesting and processing of algal biomass. Reviews and reports invariably point to processing, and particularly harvesting, as a major area of uncertainty that has major economic implications (as discussed in section 5.1). The present review reafrms this. The point has been made on a number of occasions that centrifugation, although conceptually and operationally simple, may not be an option as a primary form of separation on cost grounds. However, advances in centrifugation engineering may improve the economics. Similarly, the current status of ltration and micro-straining technology does not allow for a viable option for harvesting algae on a large scale. Again, technological advances in membrane and systems engineering could change this supposition. Furthermore, the strong species-dependent nature of the ltration process makes it difcult to draw conclusions about the general applicability of this process. One is left with some form of occulation, agglutination or co-precipitation in combination with a centrifugation step to harvest cells. To provide some
strategic basis we need a much more detailed understanding of the surface properties of the cell and how this varies with species, physiology and culture conditions. As we start from a position of very little existing knowledge in the case of microalgae, it will call for a major research effort, however without some basic understanding we will be committed to continue to fumble our way through the problem by trial and error. Once a concentrated slurry of cells has been produced we have a number of processing options. Three scenarios, which involved overall various ways of handling the major biochemicals, were considered in Section 6 (see Fig. 24). These raise major R&D questions; the cost, practicality and utility of separating the protein, lipid and carbohydrate fractions from one another, and the further separation of valuable, more specialised biochemical, nutraceutical and potential pharmaceutical products. In the analysis in Section 6, carbohydrate was relegated to a low value commodity; research is needed to determine whether the bulk carbohydrate fraction or a sub-fraction can be put to better use than simply recouping a fraction of its energy by anaerobic digestion. Compositional analysis of the algal biomass will determine whether the carbohydrate fraction can be used to generate a second biofuels stream; e.g. bioethanol after fermentation of the structural sugars. However, current published carbohydrate information does not allow predictions of their potential yield. Major questions surround the application of anaerobic digestion to the processing of waste algal debris. Whereas we have extensive knowledge of the application of anaerobic digestion to human and animal waste and a growing knowledge of the treatment of domestic waste, by comparison we have a very limited knowledge of the digestion of microalgal debris. We are, however, not without experience of applying anaerobic digestion to algal material, as this was undertaken as part of the high oxidation rate ponds used in some of the US west coast sewage treatment plants. How easy it would be to transfer this knowledge to the residual cell material from mass algal culture needs to be explored. We need insight on a number of matters surrounding anaerobic digestion: (i) the speed and completeness with which nitrogen (presumably as ammonia) and particularly phosphate are released and therefore recycled; (ii) the energetic budget and whether or not the net energy yield improves with the increasing ambient temperature at lower latitudes; and (iii) the fraction that remains undigested over some economic time scale and whether it has value as a fertiliser or soil conditioner. There remains the question whether, rather than separating and processing the individual biochemical fractions, there is value in subjecting algal biomass to some form of thermal upgrading for producing biofuels. Work on this was recently reviewed in ref. 115; however, a great deal more will be required before we can establish the practicalities and their relative economics. (iii) Resolving the problems of scaling up. There are two levels of scaling up: (i) from the laboratory to the pilot plant/modest production plant scale, (ii) from the pilot plant scale to a scale that would make a signicant and sustainable contribution to global liquid hydrocarbon fuel production. The rst is essentially scaling up from gram to tonnage production and is associated with the R&D needs outlined above. The second brings along
a number of special considerations. To make a signicant contribution to global fuel production one needs to consider the question of sustainable production approaching the near gigatonne scale. This, not surprisingly, brings along a host of problems: political, social and economic as well as scientic. There are a number of potential show stoppers to sustained production on this scale, three of them merit early consideration and research. Algal mass culture, in addition to land and sunlight, requires, at the minimum, signicant quantities of water and nutrients. A rst possible show stopper is the availability of the nutrients, nitrogen and phosphorus in particular, required for algal mass culture. Of the three major nutrients, nitrogen might be argued not to be a problem as, given energy, it can be generated from atmospheric nitrogen. However even if the energy were derived from within the algal production facility, and thereby carbon neutral, nitrous oxide, a powerful greenhouse gas, is a by-product of the HaberBosch process (used for ammonia synthesis) and its release into the atmosphere will need to be incorporated into any life cycle analysis. In contrast to nitrogen, supplies of phosphate would need to come from very nite reserves. At present rates of exploitation the global reserves are estimated to have a lifespan no more than 50100 years.126 One-off use of phosphate on a gigatonne scale of algal lipid production would add signicantly to the present demand and shorten the lifespan of the global phosphorus reserves. This returns us again to the problem of the recycling of phosphate within the overall scheme of production and the successful, or otherwise, use of anaerobic digestion to achieve this. A second possible show stopper is the availability and delivery of signicant quantities of CO2. As discussed in Section 4.4(iv), in order to achieve rapidly growing dense cultures, carbon dioxide has to be added to the culture in some way, as the natural exchange across the atmosphere is too slow. It may be possible to achieve this by active aeration relying upon the ambient concentration in air, although this would be costly or it may be necessary to actively separate the CO2 from the atmosphere by liquefaction and add pure CO2 to the culture. Neither is likely to be a low cost solution. The third potential show stopper, not surprisingly, is water. As soon as one anticipates production plants of considerable scales, local water supplies are unlikely to be adequate and so water (for a number of reasons most probably seawater) will need to be pumped onto the culturing site and after use disposed of in some manner. The engineering and energy costs for pumping could be considerable and almost certainly one would need to resort to recycling water to some extent. Recycling of the spent culture medium is embedded in the conceptual scheme in ref. 95. Recycling of used water brings along a number of problems, the spent culture medium will contain soluble organic products released during algal growth; these will accumulate and may have a positive or negative effect upon the growth rate of subsequent cultures. Similarly any residual additives, for example material added to induce occulation will be present or will have to be removed from the spent medium. Biological contaminants, e.g. competitors and predators, and, perhaps more problematic, pathogens such as viruses if not removed will be recycled. This may call for further treatment e.g. sterilisation of the recycled water with inevitable cost implications. There is experience of these matters from the existing production plants,
whether this is sufcient to plan large-scale production facilities or whether further systematic R & D is needed will require careful consideration. In conclusion, the production of biodiesel and other algal biomass components on an industrial scale does appear to have the potential to be a sound economic proposition. It would, however, be unwise to underestimate the scale of R & D required to make this a reality. The diversity of knowledge required is such that it is unlikely that a single institution or corporation would have the resources to undertake the work exclusively in house. The review National Algal Biofuels Technology Roadmap prepared as part of the US Department of Energy Biomass Program makes a strong play for openness in the R & D and the value of collaborative work and exchange of information between industry and academic centres. This steer should be noted. It is widely accepted that it will need considerable political will and social change if we are to nd solutions to the problem of greenhouse gas emissions. The same will and changes in attitude and approach will be needed if we are to contemplate putting algal biomass and biofuel production on a scale where it is a signicant part of the overall equation. James Lovelock once observed that it characteristically takes at least 25 years for new major technologies to come on line, this broadly matches the timescale of many of the major political aspirations and promises surrounding the tackling the problem of CO2 emissions. Thus, that the task may seem daunting should not deter us.
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Acknowledgements
We have valued the discussions with many of our colleagues, in particular P. W. has appreciated inputs and guidance from Ian Archibald (Shell Global Solutions), John Beardall (Monash University), Ian Joint (Plymouth Marine Laboratory), Phillip Williamson (University of East Anglia), Dylan Evans (Anglesey Sea Zoo) and David Bowers (School of Ocean Sciences, University of Bangor) and L. L. has appreciated input from Marti~a Ferreira-Novio (Universidade de Santiago de Comn postela, Spain) in Section 5.1. Al Darzins and Andy Aden (NREL) are gratefully acknowledged for their comments on the manuscript. We thank Jasvir Atwal, Account Manager of ICISICIS Pricing & ICIS News for providing a copy of their publication Market. P. W. also thanks Jrg Schwender of the Biology o Department of the Brookhaven National Laboratory for help with the calculations of the conversion efciency of protein given in Box 1. We thank Carolyn Walburton for help with preparing the images and Alice Bowers for digitising the Pedroni et al. data used in Fig. 20 and 21. Finally, much of the data present in the Appendices and P. W.s understanding of the subject are an outcome of contracts from Shell and the company is thanked for their support and for their willingness to allow this material into the public domain.
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www.rsc.org/ees | Energy & Environmental Science

1. Introduction and historical background

Energy Environ. Sci., 2010, 3, 554590 | 555

Major biochemical groups, their presence and function

2. Biology and biochemical composition of microalgae

This journal is The Royal Society of Chemistry 2010

Range of typical cell content (%) 1560 2060 35 1050

This journal is The Royal Society of Chemistry 2010

Energy Environ. Sci., 2010, 3, 554590 | 557

Percentage major element in various biochemical fractions

Algal lipids as fuels

Biochemical composition (as % total mass)

This journal is The Royal Society of Chemistry 2010

3. Principles and efciency of photosynthesis

This journal is The Royal Society of Chemistry 2010

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562 | Energy Environ. Sci., 2010, 3, 554590

k An einstein is dened as a mole of photons, of unprescribed wavelength.

This journal is The Royal Society of Chemistry 2010

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564 | Energy Environ. Sci., 2010, 3, 554590

This journal is The Royal Society of Chemistry 2010

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This journal is The Royal Society of Chemistry 2010

This journal is The Royal Society of Chemistry 2010

568 | Energy Environ. Sci., 2010, 3, 554590

This journal is The Royal Society of Chemistry 2010

54 54 6768 19,67 66 69 66 66 66 Present account 16 4 4 9 70 71

Algal growth and production

4. Production rates: background and observations

Fig. 17 Photobioreactors (Greenfuels image courtesey of http:// www.ickr.com/photos/jurvetson/58591531/).

Energy Environ. Sci., 2010, 3, 554590 | 569

Controls on algal growth

This journal is The Royal Society of Chemistry 2010

This journal is The Royal Society of Chemistry 2010

This journal is The Royal Society of Chemistry 2010

5. Harvesting and processing of microalgal biomass

na na 1591900 1520g 2.55 20 0.53

Cost per kg/$ c

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0.61 [0.24] 1.39 [0.56] 1.71 [0.68]

578 | Energy Environ. Sci., 2010, 3, 554590

6. Economics of biomass and biofuel production

This journal is The Royal Society of Chemistry 2010

Energy Environ. Sci., 2010, 3, 554590 | 581

582 | Energy Environ. Sci., 2010, 3, 554590

This journal is The Royal Society of Chemistry 2010

584 | Energy Environ. Sci., 2010, 3, 554590

Not specied Various Mean (+StDev)

Benemann & Oswald (1996)15

Not specied Not specied

Molina Grima et al. (2004)94

van Harmelen & Oonk (2006)122

Not specied Not specied

Huntley & Redalje (2007)16

Carlsson et al. (2007)123

Alabi et al. (2009)119

Not specied Not specied

Pienkos & Darzins (2009)95

Not specied Not specied Not specied