micro algae biofuel energetic

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Microalgae as biodiesel & biomass feedstocks: Review & analysis of thebiochemistry, 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

efficiency 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

field 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 andthe effect of changing individual parameters. Our main conclusions are that: (i) the biochemical

composition of the biomass influences 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

reduce the emissions of greenhouse gases. Although alternatives

exist for land-based transport notably electrical power there

is an understandable reluctance to abandon liquid hydrocar-

bons and internal combustion engines for many transport

purposes. Further, in the case of aviation transport and ship-

ping, 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 first-

generation biofuels) have been developed and optimised over

the past several decades and are currently run as profitable

businesses.

School of Ocean Sciences, University of Bangor, Menai Bridge, Anglesey,UK LL595PP

Electronic supplementary information (ESI) available: AppendicesIIV. See DOI: 10.1039/b924978h

Current address: National Bioenergy Center, National RenewableEnergy Laboratory, 1617 Cole Blvd, Golden, CO 80401, USA

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 maximumtheoretical 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

REVIEW www.rsc.org/ees | Energy & Environmental Science


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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 figure for bio-

ethanol 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 overallproduction of liquid fuels, these first-generation biofuels have

come under considerable international scrutiny and criticism.

The main criticisms are the encroachment of the biofuels feed-

stock 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 benefits, 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 significantly slowed until adequate controls to

address displacement effects are implemented and are demon-

strated to be effective (the embolding is ours). This mood, whichalthough 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 require-ment 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 wasinitially 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 findings, 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 wasthat algal biofuels are a potential valuable alternative to tradi-

tional biofuels, however, considering the oil prices of the early

1990s, the calculated economics turned out to not be profitable.

Nevertheless, considerable headway was made with regards to

basic biology, strain selection, metabolic engineering and large-

scale 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 antioxi-dant from Haematococcus pluvialis) and b-carotene (a food

additive produced from Dunaliella species). These sorts of

Peter J: le B: Williams

Peter Williams is professor

emeritus at University of Ban-

gor, UK. Originally trained as

an industrial biochemist, he

came to the view that oceanog-

raphy offered more interesting

challenges. He has held posts at

the Woods Hole OceanographicInstitute, Southampton Univer-

sity, Bigelow Lab in Maine and

the University of Gothenburg,

returning to the UK to become

Professor of Marine Biogeo-

chemistry at Bangor Uni-

versitys 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.

Lieve M: L: Laurens

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 oceanSciences, at the University of

Bangor, UK, where she started

research on biodiesel from

microalgae and the work leading

to this review was initiated. Her

recent work focuses on the

quantification 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.

This journal is The Royal Society of Chemistry 2010 Energy Environ. Sci., 2010, 3, 554590 | 555


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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 stand-

point. 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 tonne

1 for crude oil. Thus the economics areprofoundly 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 prod-

ucts 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 seri-

ously considered for the removal of carbon dioxide from the

flue 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 topresent 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 first principles, we

establish the overall potential efficiency 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 thefeedstock 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 findings 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 profitable outcome.

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

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 (Bacillar-

iophyceae), yellow-green (Xanthophyceae), golden algae

(Chrysophyceae), red algae (Rhodophyceae), brown algae

(Phaeophyceae), dinoflagellates (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 significant difference between the bacteria and

the eukaryotes is that the former lack discrete internal, subcel-

lular 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 sufficiently 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 importantconsequences.

The metabolism of organisms, particularly microorganisms, is

strongly influenced 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 prin-

ciple 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 forthe

synthesis of the other biochemicals. Different classes of algae

produce specific types of polysaccharides. For example, green

algae produce starch as an energy store, consisting of both

amylose andamylopectin, similar to higher plants. The green alga

x Strictly the term alga should be limited to the eukaryoticphototrophs, since cyanobacteria (formerly known as blue-green algae)are a form of bacteria, with very different genetics and evolutionaryhistory. It is however very cumbersome to qualify every reference tothese two groups of single-celled phototrophs as algae andcyanobacteria, or as micro-photoautotrophs so, unless there is needto be specific, for convenience the two are referred in the text simply asalgae.



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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 florideanstarch, consisting mostly of amylose.20 A commonly found poly-

saccharide in a large number of algal species is chrysolaminarin,

a linear polymer ofb(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 theyserve a similar structural role, as well as a metabolic role.

Furthermore, it is known that the structural cell wall of Chla-

mydomonas reinhardtii consists primarily of cross-linked

hydroxy-proline-rich glycoproteins.22

Proteins have important commodity value as animal feed.

Critical to this is their amino acid composition, as a number of

amino acids aredietary essentials formammals yettheyare unable

to synthesise them, forexample. Fig.2 shows themean 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 basisfor algal division and growth. The nucleic acids comprise a small

fraction of cellular biomass but the major part of the cells phos-

phate andthe secondmostimportant 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 fluidity of the membranes. The microalgae have

the facility for rapid adaptation to new environments

(e.g. changes in temperature), through the de novo synthesis andrecycling 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 thyla-

koids (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 tech-

niques, 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.

Table 1 Elemental composition of algal biochemical components. Data for the proteins and fatty acids were derived from means from the analysesshown 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 othermolecular structures in the overall molecule. That for the overall algal lipids is derived from the values for the individual lipid classes and the mean lipidclass composition given in Table 3. The values for the nucleic acids and carbohydrates are taken from ref. 17. The calorific values (as kJ g1) of lipids andtheir derivatives have been calculated according to an equation for higher heating values (equivalent to the bomb calorific value) 35.17C+ 116.25H11.1O + 6.28N+ 10.47S, where C, H, O, N, Pand Sare the mass fractions of the constituent elements.18 This equation gives a good approximation forthese compounds. The hydrogen and oxygen atoms associated with phosphate and sulfate were not included in the calculation. The calorific values forproteins and nucleic acids are calculated from the calorific values of their component molecules, using 18 kJ per mol of water as the energy lost onpolymerisation. 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)

Biochemical componentCharacteristic elementalcomposition Calculated calorific value/kJ g1 Range of typical cell content (%)

Algal lipids C1H1.83O0.17N0.0031P0.006S0.0014 36.3 1560Acylglycerides C1H1.83O0.096 40.2 Glycolipids C1H1.79O0.24S0.0035 33.4 Phospholipids C1H1.88O0.173N0.012P0.024 35.3 Algal fatty acid C1H1.91O0.12 39.6 Methyl esters C1H1.92O0.05 43.0 Protein C1H1.56O0.3N0.26S0.006 23.9 2060Nucleic acid C1H1.23O0.74N0.40P0.11 14.8 35Polysaccharide C1H1.67O0.83 17.3 1050



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eukaryotic and the prokaryotic algae (the cyanobacteria) the

lattercontainingless than theformer (see Fig. 3),likely tobe dueto

theabsence of internal membranes in theprokaryotes (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 docu-

mented, with a high occurrence of unsaturated (and poly-

unsaturated) 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 func-

tion is to maintain membrane fluidity under different conditions.

The highconcentrations of unsaturated fattyacids in the extracted

lipids, andultimately in theresulting fuel, willbe an importantfuel

quality determinant. The preponderance of the shorter chain fatty

acids has significance for their potential as diesel fuels (defined as

alkyl chains of between 12 and18 carbonatomslong). The level of

unsaturation affects biodiesel properties.36 For example, fuels

witha higher level of unsaturationof the acyl chains have a higher

cloud point,whichis desirable, butare 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

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 inprotein 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 impor-

tance 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

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 first 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 significant inverse relationship between growth rate and lipid

content (p value for ref. 2 data set is 0.0006 and that for ref. 42data 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

first derivative of the quadratic equation will give thelipid 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

ofca. 15% (from the analysis in ref. 42s data set) and ca. 30% (for

ref. 2). From these values, an optimum growth rate for lipidmetabolism may be calculated from the initial equation. Esti-

mated 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 produc-

tion 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.

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.



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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 termi-

nate the production of nitrogen containing material (proteins

and nucleic acids) but continued to synthesise lipids and carbo-

hydrates. 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).

2.4. Algal lipids as fuels

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 theirhigh 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 water-

soluble fraction following transesterification, 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 esterification, the lipid class composition will also

greatly affect the potential fuel yield by transesterification. Thus

triglycerides have a > 99% biodiesel yield compared to a < 70%Table2

Calculateddistributionofmajorelementsinthefourprincipalbiochemicalfractionsformodellow(15%),medium(25%)andhigh(50%)lipid-containingalgae.Th

ecalculationismadeusing

theelementalcompositionsandcalorificvaluesgiveninTable1andassumesthat,a

sthelipidcontentvaries,thenucleicacidco

ntentremainsconstantat5%andprotein:carbohydrateratioremains

constantat3:2(seelegendtoFig.5).Thesesimplificationsmeanthatthedataaremainlyillustrative

Biochemical

composition(as%

totalmass)

Percentagemajorelementinvario

usbiochemicalfractions

C

H

O

N

P

S

Lipidlevel

Lipidlevel

Low

Medium

High

Low

Medium

High

LowM

edium

High

Low

Medium

HighLo

w

Medium

High

Low

Medium

High

Low

Medium

High

Lipids

15

25

50

20

33

59

23

36

63

8

14

33

0

1

2

25

35

52

9

15

36

Proteins

48

42

27

49

41

24

47

39

22

34

32

24

90

88

82

0

0

0

91

85

64

Polysaccharides

32

28

18

27

23

13

28

23

13

52

48

36

0

0

0

0

0

0

0

0

0

Nucleicacids

5

5

5

3

3

3

3

2

2

6

6

7

10

11

15

75

65

48

0

0

0

Calorificvalue/kJg1

23.2

24.7

28.5

Elementtotal(as%dryweight)

53

55

60

7.2

7.6

8.6

30

29

24

8.7

7.8

5.4

0.7

0.81

1.1

0.5

0

0.4

Table 3 Mean ( standard error) lipid class content as a percent of totallipid of individual algal species, plus a composite from all reports formicroalgae.2435

Simplelipids

Glyco-lipids

Phospho-lipids

DiatomsChaetoceros species 37 16 36 8 25 8Phaeodactylum tricornutum 54 6 34 5 11 1

Range from Borowitzka (1988)130 1460 1344 1047Green algaeChlamydomonas species 48 10 44 13 6 3Dunaliella tertiolecta 7 1 67 1 25 0Dunalliella viridis 13 1 44 3 42 2Range from Borowitzka (1988)130 2166 662 1753

Blue green algaeRange from Borowitzka (1988)130 1168 1241 1650

OthersNannochloropsis oculata 22 1 39 0 38 1Isochrysis species 36 3 35 1 27 3

Composite from all reports (n 46) 35 3 40 2 25 2



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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 figure

does not take into consideration the consumption of methanol

during esterification; if the methanol used is considered as part ofthe 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 calorific value of the glyco- and phospholipids.

However, as these fractions would be almost certainly separated

off in the transesterification process, their low calorific value

would have no effect on the properties of the final 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 figure 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 significance on the vari-

ance 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 (C20) in the lubricant

market.

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 figures 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).



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3. Principles and efficiency of photosynthesis

Since the basis of algal biomass production is directly propor-

tional to the efficiency with which the algal cells assimilate carbon

from the atmosphere through photosynthesis, we discuss the basic

principles andefficiency of photosynthesis in this section. Theaim

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 mole-

cules, 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 appro-

priate 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

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. 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

varyingthe irradiance andthe culturesweresampled during active growth.

Fig. 7 The effect of nitrogen limitation on the lipid content of eukary-

otic algae. The values in parenthesis are number of observations from

which the mean was derived. The upper error bar is the standard devi-

ation, the lower the standard error. Full data set and references in

Appendix II (provided in the ESI).

{ The notation [CH2O] is commonly used as shorthand for organicmaterial in biology in the present context its use is restricted toorganic material with the same elemental ratio as monosaccharides.



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light-independent reaction operates over timespans of seconds to

hours. This profound mismatch of timescales gives rise to inef-

ficiencies in fluctuating 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 photosyn-thesis. 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

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, chlo-

rophyll 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 fill 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 efficiently 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 efficiency 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.

Table 4 Comparison of properties of biodiesel from microalgae and diesel fuel and the ASTM biodiesel standard. The data from ref. 44 comes froma heterotrophically grown Chlorella protothecoides culture. There are small differences between their observations and the values we calculate (given insquare parentheses) from elemental composition and calorific values given in Table 1

Properties Micro-algal Higher plant Diesel fuel ASTM biodiesel standard

Density/kg dm3 0.864 0.8770.887 0.838 0.860.90Viscosity/mm2 s1@ 40 C 5.2 3.35.2 1.94.1 3.55.0Flash point/C 115 75 Min. 100Solidifying point/C 12 5010

Filter plugging point/C 11 3.0 (max. 6.7) Summer max. 0Winter max. < 15

Acid value/mg KOH g1 0.374 0.160.43 Max. 0.5 Max. 0.5Heating value/kJ g1 41 [43] 39.540.3 4045 H/C ratio 1.8 [1.9] 1.8

Fig. 8 The distribution of energy (blue line) and photons flux (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 chlo-

rophyll a at 680700 nm absorption bands.

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.



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The broad mechanism of the conversion of the photochemical

energy into metabolic energy and reducing capability is complex,

although comparatively well understood. Briefly, there are two

functionally separate sites of photon absorption, coupled intandem 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 flow away from the chlorophyll molecules draws elec-

trons 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 produc-

tion 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 twomolecules 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 meta-

bolic energy and reducing capability, and the water splitting

system is embedded in the lipid membrane of flattened 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 freeenergy 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 ofca. 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

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), ferredoxin-

NADP 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).

k An einstein is defined as a mole of photons, of unprescribedwavelength.

** It has been held that Nature never evolved a wheel it however dida billion or more years ago and on the nanoscale! Only recently havewe developed the skills to observe these molecular mechanisms.

Depending upon the circumstances theDGfor the hydrolysis of ATPvaries from 45 to 55 kJ mol1. A mean figure of50 kJ mol1 is usedin the present account.

There have been two schools of thought over the number of photonsrequired per molecule of O2 split: either 36 photons or 810 photons (seeref. 56). Present consensus favours the latter.



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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. 10

10 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 photosyn-

thesis to enable the incorporation of CO2 into organic material

and its subsequent reduction (see Fig. 11). The calorific value of

the product is around 469 kJ mol1 C. The first 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 hetero-

multimeric protein complex (536 kDa) and is an outstandingly

sluggish catalyst, fixing 210 molecules of CO2 per active site per

second. In addition, it has the curious property of catalysing boththe 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 inefficient 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 inefficiency is

a fallacy.

The first 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 glyceral-

dehyde-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 inter-

mediate (lipids and some amino acids), loss of carbon and

oxygen, and therefore biomass, as carbon dioxide. A simplified

analysis is made in Box 1 for triglycerides and proteins based onhexoses as the starting point. Fig. 12 shows the biomass loss

with increase in lipid production. Two matters are important to

note here: first high lipid content is achieved at a cost of

biomass loss; second, whereas theoretical calculations of

photosynthetic efficiency 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 mate-

rial (see Fig. 12).

Fig. 11 Schematic simplified representation of the Calvin-Benson cycle in three parts, (i) CO2 fixation, (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 stoichio-

metrically) are originating from the photosynthetic light reactions. RuBisCO: ribulose bisphosphate carboxylase/oxygenase; RuBP: ribulose-1,5-

bisphosphate; 3-PGA: 3-phosphoglycerate; 1,3-BPGA: 1,3 bisphosphoglycerate; G3P: glyceraldehyde-3-phosphate; F6P: fructose-6-phosphate; G6P:

glucose-6-phosphate; PEP: phosphoenolpyruvate.



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Box 1: Biomass and energy losses incurred during the biosynthesis of lipids and proteins

Lipid synthesis: The biosynthesis of high calorific 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 calorific 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 trans-

formation of hexoses into lipids. If there is a negative energy balance, then as well as the mass losses during the biochemicaltransformations 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 (1)

8 acetyl CoA + 7ATP + 14 NAD(P)H / CH3(CH2)14COOH (2)

Overall:

4C6H12O6/ CH3(CH2)14COOH + 8CO2 + ATP + 2NAD(P)H (3)

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 energygains 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, theconversionof 4 moleculesof a hexoseto 1 mole ofpalmitic acid results in a drop in calorificvaluefrom11 446to 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 amountof phospholipidand glycolipid. Themass loss in makingphospholipidmay be estimated to

be about 2.7 and making glycolipid 2.3 these values are not as certain as the triglyceride figure. If we take the mean figures 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 bemetabolic 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 g 1, i.e. 6%) of the overall calorific value of the

protein, thus we may take a round figure 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 contri-

bution 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)

whereC

is the carbohydrate content (assuming it to be a hexose-based polysaccharide),P

is the protein content andL

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 simplifies 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%, reflecting the scales of the metabolic work

needed.

Note: NAD(P)H is used to refer to both NADH and NADPH.



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3.2 Respiration

At first 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 traffic jams along the electron transport chain

when the former reaction gets ahead of the latter. Respiration-

like 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 achievingmaximum photosynthetic efficiency in other than ideal labora-

tory 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 biomass-

associated 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 algaegroupings has been made.53 Normally, other than at very low

rates of photosynthesis, the dominant loss term is the photo-

synthetically associated respiration and the fraction of photo-

synthesis 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, respiration-

associated 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

compensation irradiance is seen as an offset on the photosyn-

thesisirradiance 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 indense algal cultures (see Section 4.4(ii) and Box 2).

3.3 Overall efficiency 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 flux (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 flattening off of the curve at higher irradiances is a conse-quence 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 pro-

tein : 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 calorific value of 15.7 kJ g1.

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 dinoflagellate, would not be

a candidate organism for mass algal culture but is included as the

compensation irradiance is clearly shown with this organism.



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enzymatic reactions. In this zone, the energy from the excess

captured photons has to be disposed of in some manner, either by

fluorescence or by one of the respiratory decongesting mecha-nisms. It is in the context of this effect that the time scale

mismatch of the photochemical and enzymatic parts of photo-

synthesis becomes significant. If the high irradiances are sus-

tained 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 efficiency of photons. The latter is

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, thealgae can exist as two physiological types low light adapted

(high chlorophyll content) and high light adapted (low chloro-

phyll content). It would appear that the low light adapted form is

the default state. The phenomenon of light adaptation has

a major influence 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

efficiency 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 thermo-

dynamics was made in ref. 56, and the energetics are concisely

outlined in ref. 54. The present calculation is made for theproduction 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 efficient energy

transfer at this stage. The reactions following these charge

separations give rise to the formation of 3 moles of ATP and

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 (Emin0 ) may be calculated

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 coefficient (extinction coefficient). In dense cultures ekz(1, thus the equation simplifies to

Pz

0E E0/k (3)

Combining eqn (1) and (3) gives the minimum irradiance for growth

Emin0 Eckz (4)

For cultures with a biomass of 0.25 kg dry weight m3, the light extinction coefficient 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 specific 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:

Emin0 7.5Ec (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

Emin0 from 0.2 to 2 MJ m2 d1, with a median value in the vicinity of 1 MJ m2 d1.

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.



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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 CH 2O. Taking

this to be a hexose, with a calorific value of 469 kJ mol1 C, the

overall efficiency 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 reac-

tions (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, whereasthe 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 efficiency

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 significant 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 efficiencies may be regarded essentially as fixed

ceilings for photosynthetic efficiency 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.

Fig. 15 Energy losses during photosynthesis. A. The stepwise loss of energy during the production of 1 mole of organic carbon as glucose (calorific

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.

Table5 Calculation of maximum photosynthetic efficiencies. Yield terminology and symbols adopted from ref. 57. CHO refers to organic material witha hexose-type elemental ratio, and a calorific value of 469 kJ mol1 C (15.6 kJ g1). CHON refers to the calculated carbon-normalised major elementcomposition (C1H1.64O0.44N0.13) for a cell containing 42% protein, 28% carbohydrate, 25% lipid and 5% nucleic acid, with a calorific value of 541 kJmol1 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 Units CHO CHON (NH3) CHON (NO3)

Bioenergetic yield (J%) % (kJ kJ1) 26.7 [12] 17.7 [8] 14.3 [6.4]

Energetic yield (JkJ) g kJ1 17 103 [7.7 103] 7.2 103 [3.2 103] 5.8 103 [2.6 103]

Quantum yield (JE) g einstein1 3.8 2.1 1.7

Photons/mol C fixed einstein mol C1 8 10.2 12.6



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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

andthe more cautious quantum requirement of 10, then, allowing

for respiration, the maximum theoretical yields of a carbohy-

drate 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.

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 efficiency of energy

transfer from photons to biomass. We discuss two types of algal

biomass production systems, closed bioreactors and open

ponds.

4.1. Algal growth and production

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 efficient system. Bioreactors (see

Fig. 17) are enclosed systems of various geometries, character-

istically, 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 flat

plate structures. Many of the financial 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 flow in long lengths

of narrow tubing and the possibility of inhibition of

Table 6 Observed and projected yields for crop plants and microalgae.Figures for annual insolation are calculated as for Fig. 18A; theoreticalvalues are given for three latitude zones, the same as in Fig. 24. * allowingfor respiration, ** assuming a calorific value of 20 kJ g1 dry weight forthe crop as a whole, *** assuming a biomass calorific value of 24.7 kJ g1

dry weight (see Table 5) and a respiratory loss of 20%

Crop

Maximumbiomassyield/tonnes dry

weight ha1

a1

Source

Higher plantsTheoretical* (C3 plants)c% 4.6%

Low 210, mid 170,high 140**

54

Theoretical* (C4 plants)c% 6.0%

Low 270, mid 220,high 190**

54

Sugar cane 7495 6768Switchgrass 820 19,67Corn (grain) 834 66Poplar wood chips 11 69Soya 4.65.5 66Rape seed 4.56 66Oil palm 8.7 66MicroalgaeTheoretical c% 12% (Table 5) Low 410, mid 330,

high 280***Present

accountProjected raceway, algae

unspecified110220 16

Bioreactor raceway, algaeunspecified

175 4

Projected raceway, algaeunspecified

127 4

Best case raceway, algaeunspecified

120153 9

Achieved bioreactor(Phaeodactylum)

182 70

Achieved raceway (Pleurochrysis)over 10 months

60 71

Fig. 17 Photobioreactors (Greenfuels image courtesey of http://

www.flickr.com/photos/jurvetson/58591531/).

Fig. 16 Open ponds (image courtesy of Nature Beta Technologies Ltd,

Eilat, Israel, subsidiary of Nikken Sohonsha Co. Gifu, Japan).



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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 flooded intolarge area raceways, which serve as the grow-on phase. Such

systems have been successfully used for growth of the astax-

anthin-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 ofmodifying 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.

4.2. Controls on algal growth

Growth of microorganisms is usually modelled as a species-

specific 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 first 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

activation energy of ca. 50 000 kJ mol1. Biologists customarily

use a simple logarithmic alternative to the Arrhenius equation

the Q10 defined as the change in rate over 10C 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 responsesto temperature and light, as the photochemical system is essen-

tially 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

(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 partic-

ular 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 irra-

diancemay be calculated foreach hour angle of the dayand forthe

cycle of the solar declination angle through the year. Finally

a correction is made for reflective 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, field data of total

incoming radiation, derived from satellite observations, are given

in Fig. 19. The point made by this latter figure is that local atmo-

spheric conditions notably cloudcover give riseto 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 estab-

lished 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 moleculeto dinitrogen, whereas the starting and end nitrogen compound inmicroalgal growth is commonly ammonia. Strictly a correction shouldbe made to the reported bomb calorimitry values for the heat offormation of ammonia from dinitrogen (46 kJ mol1) otherwise the DGof 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 iscustomarily ignored.



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mass of hexose required to produce a given mass of biochemical

and the calorific value of hexose itself (taken as 15.7 kJ g1, ref.

63, 64). The former can be e

micro algae biofuel energetic

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