Photosynthetic biomass and H 2 production by green algae: from bioengineering to bioreactor scale-up

Physiologia Plantarum 131: 10–21. 2007 Copyright ª Physiologia Plantarum 2007, ISSN 0031-9317

REVIEW

Photosynthetic biomass and H2 production by green algae:from bioengineering to bioreactor scale-upBen Hankamera, Florian Lehrb, Jens Rupprechta, Jan H. Mussgnugc,Clemens Postenb and Olaf Krusec,*

aInstitute for Molecular Bioscience, University of Queensland, St Lucia Campus, Brisbane, Queensland 4072, AustraliabDivision of Bioprocess Engineering, Institute of Engineering in Life Sciences, University Karlsruhe, 76131 Karlsruhe, GermanycDepartment of Biology, Algae Biotech Group, University Bielefeld, 33501 Bielefeld, Germany

Correspondence

*Corresponding author,

e-mail: [email protected]

Received 22 December 2006; revised 12

March 2007

doi: 10.1111/j.1399-3054.2007.00924.x

The development of clean borderless fuels is of vital importance to human and

environmental health and global prosperity. Currently, fuels make up

approximately 67% of the global energy market (total market¼ 15 TW year21)

(Hoffert et al. 1998). In contrast, global electricity demand accounts for only

33% (Hoffert et al. 1998). Yet, despite the importance of fuels, almost all CO2

free energy production systems under development are designed to driveelectricity generation (e.g. clean-coal technology, nuclear, photovoltaic, wind,

geothermal, wave and hydroelectric). In contrast, and indeed almost uniquely,

biofuels also target the much larger fuel market and so in the future will play an

increasingly important role inmaintaining energy security (Lal 2005).Currently,

themain biofuels that are at varying stages of development include bio-ethanol,

liquid carbohydrates [e.g. biodiesel or biomass to liquid (BTL) products],

biomethane and bio-H2. This review is focused on placing bio-H2 production

processes into the context of the current biofuels market and summarizingadvances made both at the level of bioengineering and bioreactor design.

Biofuel options

Photosynthesisplays a central role inall biofuel production.

It drives the first step in the conversion of light to chemical

energy and is therefore ultimately responsible for the

production of the feedstocks required for all biofuel

synthesis (Fig. 1); protons and electrons (for bio-H2), sugarsand starch (for bio-ethanol), oils (for biodiesel) and biomass

(for BTL products and biomethane). Consequently, any

increase inphotosyntheticefficiency (discussedbelow)will

also enhance the competitiveness of biofuel production.

Bio-ethanol

Bio-ethanol is already well established as a fuel mostnotably in Brazil and the US (Goldemberg 2007). It is

mainly produced from starch (derived from corn and

wheat) and sugar (sugar cane) produced during photo-

synthesis. To produce ethanol, starch is usually mixed

with water and heated briefly, before adding enzymes to

release sugars, if required. Yeast is subsequently added to

ferment the sugars to ethanol, and the ethanol is thenpurified from the mixture by distillation and dehydration.

Biodiesel

Biodiesel is typically produced from the oils extracted from

soybeans, rapeseed and oil palm (Jeong et al. 2004,

Sanchez and Vasudevan 2006). However, recently low-

input high-diversitymixtures of native grasslandperennials(Tilman et al. 2006) and micro-algae (Xu et al. 2006) with

Abbreviations – AOX, alternative oxidase; BTL, biomass to liquid; Cyt b6f, cytochrome b6f; H2ase, hydrogenase; LHC, light-

harvesting antenna complex; PQ, plastoquinone; PS, photosystem.

10 Physiol. Plant. 131, 2007

their high lipidand fattyacidcontent are also considered as

a suitable source. These are converted to biodiesel via

a transesterification reaction with methanol or ethanol in

the presence of a catalyst, to yield mono-alkyl esters and

glycerine, which is subsequently removed.

Biomass to liquid

BTL production (see: Kavalov and Peteves 2005) typically

begins with the grinding and drying of solid biomass (e.g.

wood, straw and corn) before forming it into pellets.

Through a low-temperature gasification process, these

biomass pellets are then converted into a gas (smouldering

gas) and solid fraction (charcoal). These are then trans-

formed into a synthetic gas in a second step. This gas is then

purified and liquefied via the ‘Fischer–Tropsch’reaction, inwhich carbonmonoxide (CO) and hydrogen (H) react and

form carbo-hydrogen chains. The product is a paraffin-like

liquid, which is then isomerised to increase its stability

before it is purified via distillation or ‘hydrotreatment’.

Biomethane (biogas)

Organic material like crop biomass or liquid manure canbe used to produce biogas via anaerobic digestion and

fermentation (Farquhar and Rovers 1973). Mixtures of

bacteria are used to hydrolyse and break down the

organic biopolymers (i.e. carbohydrates, lipids and

proteins) into monomers, which are then converted into

a methane-rich gas via fermentation (typically 50–75%

CH4). Carbon dioxide is the second main component

found in biogas (approximately 25–50%) and, like otherinterfering impurities, has to be removed before the

methane is used for electricity generation.

Bio-H2

Certain green algae and cyanobacteria (beyond the

scope of this review) have evolved the ability to harness

solar energy to extract protons and electrons from water

via the water-splitting reaction of PSII. In green algae,

these protons and electrons are then subsequently

recombined by a chloroplast hydrogenase (H2ase;

Happe and Naber 1993, Melis et al. 2000, Zhang et al.

2002) to form molecular H2, which is released from the

algal culture as a gas with a purity of up to 98% (Kruse

et al. 2005b).

Advantages of bio-H2 production

Given the choice of biofuels, it is worth briefly

summarizing the theoretical advantages of future bio-H2

fuel production systems, before describing recent devel-

opments of this biotechnology and the improvements

required to realize economic viability.

H2 – a promising future fuel

H2 has been identified as one of the most promising fuels

for the future (Abraham 2002, E.U. Commission 2002,

Koizumi 2002). The US, European Union and Japan have

already embarked on establishing H2 fuel stations, and in

parallel, car manufacturers have invested extensively in

the development of H2 fuel cell-powered cars.

Bio-H2: a sustainable alternative toconventional H2 production

Currently, the developing H2 economy is almost entirely

dependant upon the use of carbon-based non-renewable

resources [steam reformation of natural gas (approxi-mately 48%), petroleum refining (approximately 30%),

coal gasification (18%) and nuclear powered water

electrolysis (4%); Gregoire-Padro 2005]. Although these

resources are likely to drive the initial transition towards

a H2 economy, new, clean, sustainable and economic

ways of generating H2, such as solar powered bio-H2

production, will be required.

Fig. 1. The role of photosynthesis in biofuel production. Photosynthesis drives the first step in the conversion of light to chemical energy and is therefore

ultimately responsible for the production of the feedstocks required for all biofuel synthesis protons and electrons (for bio-H2), sugars and starch (for

bio-ethanol), oils (for biodiesel) and biomass (for BTL products and biomethane).

Physiol. Plant. 131, 2007 11

High-photon conversion efficiencies

The photon conversion efficiency (light to H2) cantheoretically be as high as approximately 10–16% (Kruse

et al. 2005a, Prince and Kheshgi 2005). Currently, how-

ever, such a high efficiency can only be approached for

transient periods, and this must be improved upon, for

economic viability to be achieved.

Carbon sequestration

Theoretically, solar powered bio-H2 production provides

a unique approach to CO2 sequester. During the aerobic

growth phase of the fuel production cycle, substantial

amounts of CO2 are absorbed and converted to biomass.

H2 is subsequently produced during the anaerobic phase,

with a large proportion of the sequestered carbon re-

maining in the form of biomass. In contrast, the com-

bustion of bio-ethanol, biodiesel, biomethane and BTLcarbohydrates release CO2, making these fuels at best

CO2 neutral, although the importance of this should not

be underestimated.

Fuel cells

Advances in fuel cell technology and the fact that the

combustion of H2 produces only H2O further increasesthe attractiveness of bio-H2.

Significance and innovation ofalgal biofuel systems

Another important factor to consider in the developing

biofuel area is whether to use conventional crops or algal

bioreactor systems. The acceptance of one approach over

the other will likely ultimately come down to a matter of

economics; however, algal bioreactors do offer a number

of important advantages.

Increased photon conversion efficiencies

Most plants have evolved genetic strategies to assemble

large light-harvesting antenna complexes (LHCI and II)

that give them a competitive advantage in terms of light

capture under low-light conditions. The downside of this

‘large antenna’ strategy is that at high light levels (e.g. up

to approximately 500 W m22), 80–95% of the absorbedphotons can be dissipated by these LHC proteins as

fluorescence and heat, via photoprotective mechanisms

(Polle et al. 2002). This occurs despite the fact that the

antenna is naturally, but only partially, downregulated in

size under these conditions (Escoubas et al. 1995, Melis

et al. 1999). Consequently, higher plants and wild-type

algae are not ideally suited for biofuel production.

Algal bioreactors can provide an engineering means to

regulate light levels to the culture not available to

conventional crop plants. The use of bioreactors therefore

opens up the possibility of using algal cell lineswith small

LHC antenna systems in which the amount of captured

light more closely matches the requirement of the cells athigh light levels. This in turn minimizes energy losses, as

fluorescence and heat. It also simultaneously increases

the photosynthetic efficiency of the cells at the illumi-

nated surface of the bioreactor and facilitates improved

light penetration into the algal culture. The latter allows

higher cell densities to be used to increase overall process

efficiency towards its theoretical maximum.

Reducing arable land use for biofuel production

A common concern related to biofuels is that as their

production capacities increase, so will the competition

with agriculture for arable land. In contrast, algal

bioreactors can be sited on non-arable land, eliminating

this competition and opening up new economic oppor-

tunities for arid regions.

Reducing H2O use in agriculture

Conventional crops used for biofuel production require

substantial amounts of fresh water. Because of the use of

closed bioreactors, considerable savings in the net water

use can be achieved.

Coupling clean fuel production to desalination

The use of marine algae has the theoretical potential to

couple bio-H2 production to desalination as the com-

bustion of hydrogen and oxygen extracted from seawater

yields fresh water. This approach requires stationary fuel

cells that use hydrogen and oxygen to feed electricity into

the national grid.

Advances in bioengineering of the solar bio-H2

process

As early as the 1930s, Gaffron and coworkers discovered

that under certain conditions unicellular green algae are

able to produce hydrogen during illumination (Gaffron

1939, Gaffron and Rubin 1942). Subsequently, the H2aseenzymes,which catalyse the recombination ofH1 and e2

to formmolecular H2, were shown to have a high specific

activity (approximately 1000 units mg protein21) (Florin

et al. 2001, Happe andNaber 1993). This level of specific

activity is approximately 100-fold higher than many of

otherH2ases (Adams 1990) and highlights the potential of

algal systems such as Chlamydomonas reinhardtii for


future solar poweredH2 production.However, in contrast

to cyanobacterial NiFe H2ases (Schutz et al. 2004,

Tamagnini et al. 2002), the algal H2ase was found to be

highly sensitive to O2 inhibition. In fact, it was not until

the groundbreaking work of Melis et al. (2000) that this

challenge was overcome through the cyclical depletionand repletion of liquidC. reinhardtii cultureswith sulphur

(Melis et al. 2000). The principle underlying this process

is that solar powered H2 production from water can be

divided into an aerobic and an anaerobic stage (1S stage:

PSII actively producing H1, e2 and O2, H2ases inactive;

2S stage: PSII inhibited, H2ases actively recombine H1

and e2 to produceH2). This discovery formed the basis for

an expanding area of research focused on the engineeringof a more efficient bio-H2 production process. Melis and

coworkers, e.g. developed a sulphate transporter mutant

to restrict S supply for de novo synthesis of the D1 protein

of PSII (Melis andChen 2005). The rationale for thiswas to

engineer a mutant that could support low-level PSII

turnover during a continuous micro-oxic H2 production

process.

Recently, several micro-algae mutant strains related tobio-H2 production have been isolated in different

laboratories. These include strains with smaller LHC

antenna systems (Polle et al. 2003) leading to more

efficient light energy conversion, strains with low starch

content showing the direct correlation between H2

production and starch supply (Posewitz et al. 2004),

and a C. reinhardtii strain with high starch content and

a block in LHC state transitions resulting in a substantialincrease in H2 production capacity (Kruse et al. 2005b).

The latter strain was calculated to have a photon to H2

conversion efficiency of approximately 2% (from light to

H2) at 20 Wm22 in the presence of acetate, with a purity

of more than approximately 98%. The purity of the gas

was sufficient to power a fuel cell without further

purification (data not shown). Modified production

systems have since resulted in H2 production levels of850 ml H2 l

21.

The regulation and expression of the chloroplast H2ase

hydA isoforms are also an area of intense research, as is

their engineering to induce overexpression and a reduc-

tion of O2 sensitivity (M.C. Posewitz, A. Dubini, J.E.

Meuser, M.L. Ghirardi, in preparation).

To further improve the production efficiency, new

targets for bioengineering of the biophotolysis process inH2-producing micro-organisms need to be identified.

Consequently, many laboratories in the field of

microbial hydrogen production target this important

research area using systems biology approaches includ-

ing transcriptomics, proteomic andmetabolomic studies.

However, so far no data are available on analyses, which

directly refer to effects on H2 production pathways.

Nevertheless, in the near future, combined transcrip-

tomics, proteomics and metabolomic data will assist in

the in silico modelling of the complete H2 production

process and in targeting physiological bottlenecks for

bioengineering. These improvements will ultimately

interlink with parallel bioreactor development streams.

Efficiency considerations for scale-up

Before embarking upon scale-up, it is prudent to evaluatewhether bioengineering and bioreactor design streams

can at least in theory yield an economically viable solar

bio-H2 production process.

Current photon conversion efficiencies at20 W m22 illuminations

The culture flasks used in our laboratory have a surfacearea of 0.0142 m2 and are illuminated with 20 W m22

white light, resulting in an illumination level of 0.285 J

s21. The regular overall culturing time is 9 days (2 days

aerobic growth phase1 7 days anaerobic H2 production

phase) (Kruse et al. 2005b). During this time (777 600 s),

221 kJ radiation energy was transferred to the surface of

the culture, and in this experiment, 350 ml of pure

hydrogen gaswas produced on average (0.0156mol). Thecombustion reaction of hydrogen and oxygen has

a reaction enthalpy of 286 kJ mol21. The produced

hydrogen can therefore yield a maximum of 4.46 kJ of

energy, which corresponds to 2.0% of the transferred

irradiation energy.

Current photon conversion efficiencies atnatural light levels

In Europe, the average daytime irradiation levels are

approximately 230Wm22, which is more than a 10-fold

increase in illumination level over of that typically usedunder laboratory conditions (approximately 20 W m22)

(Kruse et al. 2005b). In addition, day and night cycles

reduce the illumination time to approximately 12 h

day21. Thus, a maximum of 9.94 � 103 kJ solar energy

can be absorbed per square metre per day at this

illumination level. If we assume that the photosynthetic

rate (and therefore the H2 production rate) correlates

linearly with the irradiation intensity up to natural lightlevels, this solar energy input could be converted into

0.695 mol H2 by an algal Stm6 culture with a photon

conversion efficiency of 2%. This corresponds to a H2

production rate of 1.39 g m22 day21. Melis et al. (1999)

showed a near linear increase in O2 production (a

measure of photosynthetic efficiency) of up to 1000mmol

m22 s21 illumination in high-light adapted Dunaliella


salina cells, which had a reduced antenna size. However,

it should be noted that a linear efficiency increase to

higher light levels (e.g., 2700mmolm22 s21) is only likely

to be approached using LHCII-deficient mutants, which

have already been constructed (Polle et al. 2003) but have

not yet been introduced into high-H2-producing strains.

Plant size at current efficiency levels

Based on the considerations above, a production plant

with a culture surface area of 1000 m2 (array of plate

reactors) could, e.g., yield 1.39 kg H2 day21, which is

equivalent to 15 568 l at standard conditions. If it is

possible to enhance the photon conversion efficiency to5% by means of genetic engineering and process

optimization, this would result in a hydrogen production

rate of 3.48 g m22 day21. Thus, the plant would produce

38 976 l H2 day21. If the upper photon conversion

efficiency limit of 10.6% could be reached, the pro-

duction rate could be as high as 7.37 g m22 day21,

equivalent to 82 523 l H2 day21.

Further production improvements

It should be taken into account that these calculations are

based on an irradiation intensity of 230Wm22. For other

geographical areas with higher irradiation levels (e.g.

Australia with approximately 545 W m22), the pro-

duction rates could potentially be higher if the light

saturation point is not reached and if no other negativereactions as result of high light levels occur (Barber and

Andersson 1992). In addition, the use of small antenna

mutants, which eliminate much of the 80–95% of energy

lost through fluorescence in the normal system, could

result in major yield improvements. This is because of

both improved photocapture efficiencies as well as light

penetration properties. These in turn will also allow

higher cell densities to be used in the bioreactor,potentially facilitating further improvements.

Bioreactor design

The two-phase process

Currently, bio-H2 frommicro-algae is produced in a two-

phase process, in which photosynthetic growth and H2

production are physically separated (Fig. 2). The advan-

tage of this approach is that it separates O2 (Fig. 2A) and

H2 production (Fig. 2B), preventing O2 inhibition of the

H2ase during the H2 production phase (Happe et al.

2002, Melis et al. 2000). An additional advantage is that

temporal separation is a useful way of improving H2

purity.

Light energy is essential for the process because it

drives photosynthetic cell growth as well as the hydrogen

production by photolytic water splitting. Compared with

fermentative hydrogen production by non-photosyn-

thetic bacteria, the biophotolytic process can in theory

be far more efficient (Rupprecht et al. 2006). This isbecause here light energy is directly coupled to hydrogen

production (via H2ase enzymes), whereas fermentative

bacteria have to metabolize fixed carbon sources.

In the first phase of this two-phase bio-H2 process

(Fig. 2A), the cells are grown aerobically to accumulate

biomass. To induce H2 production in the second phase,

the cells are transferred into a sulphur-depleted medium.

Alternatively, the sulphur concentration of the initialgrowthmedium can be adjusted appropriately for a given

cell density (Kosourov et al. 2002). Sulphur is needed for

amino acid synthesis, and so in sulphur-depleted

medium, protein synthesis cannot function properly and

degraded proteins cannot be replaced. This particularly

affects proteins with high turnover rates, such as the

D1 protein, a key component of PSII (Ohad et al. 1984).

Thus, in the absence of sulphur, the water-splitting reac-tion of PSII (2 H2O / 2 H2 1 O2) can therefore not be

maintained as the damaged D1 proteins cannot be

replaced. Consequently, O2 production decreases over

time. Simultaneously, O2 consumption via oxidative

respiration, alternative oxidase (AOX) activity in the

mitochondria as well as chlororespiration result in an

overall reduction in cellular O2 concentration in the

illuminated culture, and this ultimately leads to anaero-biosis and the onset (Fig. 2B) of H2 production after

approximately 24 h (Melis et al. 2000).

The efficiency of the two-phase process is dependant

on factors such as light level and distribution, pH,

temperature, media composition and substrate feeding

(Fedorov et al. 2005, Kosourov et al. 2002), all of which

are directly affected by the design of the bioreactor.

As an advancement on this theme, a continuoushydrogen photoproduction process was reported by

Fedorov et al. (2005). In this system, two stirred photo-

bioreactorswerebuilt to operate in parallel in a chemostat

mode, thereby separating the aerobic biomass produc-

tion from anaerobic H2 production phases.

The problem with using stirred tank reactors for

establishing such a continuous bioprocess is that they

are characterized by a high degree of back-mixing, re-sulting in a residence-time distribution of the cells in the

reactor. This in turn leads to a statistical age distribution

and thus a lowering of productivity. For biomass pro-

duction, back-mixing effects are favourable, but for H2

production, plug flow reactors are likely more suitable. In

such systems, the residence time can be adjusted more

precisely bymeans of reactor lengths andvolumetric flow


rate, and this in turn allows the cultures to bemaintained in

the optimal log-phase range for longer. Also, othernegative

aspects of stirred tank reactors related to photobiotechnol-

ogy, like high-light gradients, can be minimized usingtubular or plate systems (Janssen et al. 2003).

Further advantages of the two-stage system are that

semisterile photobioreactors can be used for the H2

production phase, thereby lowering the overall pro-

duction costs, particularly as the purity of the produced

H2 gas is already high enough for direct electricity pro-

duction in fuel cells (Kruse et al. 2005b). By eliminating

the need for sterilization, the material requirements are

much lower (e.g. reduced temperature and pressure

stability). This in turn extends the range of materials that

can be used into a lower cost bracket, and the reactor

design can simultaneously be simplified.

One-phase versus two-phase systems

The two-phase system (i.e. aerobic plus anaerobic stage)

does, however, carry a penalty in terms of the maximum

attainable photon conversion efficiency. This is because

three photons are required to transfer one e2 from H2O

to H2ase (photon 1: extracts e2 from H2O; photon

Fig. 2. Photosynthetic H1/e2 flow in C. reinhardtii under aerobic and anaerobic conditions in WT and H2 high-production mutant Stm6. (A) Under

aerobic conditions, e2 derived from the water-splitting reaction of PSII are passed along the photosynthetic e2 transport chain (solid black arrows) via

plastoquinone (PQ), cytochrome b6f (Cyt b6f), PSI and ferrodoxin (Fd). H1 released into the thylakoid lumen by PSII and the PQ/PQH2 cycle (H1 flow

indicated by solid grey arrows), generate a H1 gradient, which drives ATP production via ATP synthase. Dashed lines indicate H1 and e2 transfer pathways

inhibited in Stm6. (B) Under anaerobic conditions, H1/e2 stored in starch and NADPH are fed to H2ase for H2 production. In Stm6, cyclic e2 transport

(dashed line connecting Fd and Cyt b6f) is inhibited. The addition of an uncoupler (i.e. carbonylcyanide-m-chlorophenylhydrazone) results in an increased

H1 supply to the H2ase (HydA), resulting in an 9� increased rate of H2 production (Kruse et al. 2005b). This suggests that with cyclic electron transport

inhibited in Stm6, e2 supply has been increased to such an extent that H1 supply has become limiting.


2: transports the e2 through PSI, allowing it to form

NADPH and starch; photon 3: is required to transfer the

e2 from starch through PSI to H2ase).

In contrast, a one-step direct H2 production process (in

which PSII turnover and HydA-mediated H2 production

occur simultaneously under micro-oxic conditions) onlyrequires two photons (photon 1: extracts e2 from H2O;

photon 2: transports the e2 through PSI to the H2ase).

Consequently, this system could in theory yield a 33%

increase in efficiency. To achieve this, however, either

O2 insensitivity HydA mutants will be needed to be

developed (Ghirardi et al. 2006) or the rate of O2 con-

sumption must be higher or equal to the rate of O2

production, so that micro-oxic conditions that do notinhibit H2ase are maintained. Stm6, with its high O2

consumption capacity, is well suited for this purpose

(Kruse et al. 2005b) as it has an increased AOX activity,

which assists in the rapid activation of the H2ase.

Furthermore, it induced H2 production approximately

4 h earlier than in the wild-type (WT) with higher

subsequent production rates.

In a parallel approach, it has been shown that PSIIactivity can be maintained at low levels when very low

amounts of sulphur are added to the culture medium

(Kosourov et al. 2002). Using mutants with a high O2

consumption capacity under such conditionsmay indeed

offer further improvements as the rate of PSII-drivenwater

splitting could continue at higher levels and so support

a faster rate of substrate supply without inducing O2

inhibition. Such an approach will likely be necessary as

part of a strategy to achieve photon conversion efficiency

from light to H2 of 10%.

Light levels and distribution

Perhaps one of themost important issues to investigate on

the path towards commercial H2 production is the

linearity of the relationship between light intensity and

the hydrogen production rate. Such measurements

require specially designed photobioreactors in which

light gradients are minimized and light intensity canbe controlled over a wide range. A photobioreactor has

been developed (personal communication) especially for

kinetic studies under highly controlled light levels

(Csogor et al. 2001). The reactor is a 3-l stainless steel

stirred draft tube reactor (Fig. 3), which combines the

advantages of a stirred reactor (homogeneity) and a plate

reactor (short-light path length). Currently, this reactor

model is being modified to attain light intensities up toan approximately 1300 mmol m22 s21. A new internal

light system with high-performance light-emitting diodes

arranged on a cylindrical double-sided printed circuit

board has been installed inside the draft tube (Fig. 3B).

The electrical circuits of both sides can be controlled

Fig. 3. (A) Scheme of the 3-l stirred draft tube reactor system, developed for kinetic studies under highly controlled light levels. A propeller stirrer is used

to homogenize the biosuspension, which results in an axial liquid flow, streaming downwards inside the draft tube and upwards in the gap between the

vessel wall and the outside of the tube. The reactor can be operated in turbidostat mode. (B) Design drawing of the draft tube with internal light-emitting

diode system, which is used in the photobioreactor, to generate an almost homogenous light distribution.


separately, so that a homogenous light distribution inside

the reactor can be realized. Such systems are required to

measure the general kinetics of hydrogen production at

natural light levels and under conditions of light fluctua-

tions (e.g. light–dark cycles).

Light quality and distribution within the bioreactoritself is also of fundamental importance, as all photo-

trophic micro-organisms need light as an energy source.

Because of the absorption and shading effects, the light

intensity drops exponentially from the surface to the

centre of the reactor. This results in an uneven light

distribution inside the reactor. Amajor design criterion for

photobioreactors is therefore to achieve a high surface to

volume ratio, which minimizes light gradients. For thisreason, tank reactors have generally been considered less

suitable for photobiotechnological applications, as mix-

ing results in individual cells being exposed to constantly

changing light regimes, which are likely to reduce

production efficiency.

Reactor costs also significantly affect the price of the

produced hydrogen. It is therefore important to simplify

the design and minimize the cost towards <V10 m22

(Amos 2004). Newly developed plastic compounds, e.g.,

offer a great variety of alternative construction materials

for photobioreactors and are able to replace costly glass

and steel. Special properties that must be taken into

account include strength, durability, spectral properties

and its diffusion coefficient for hydrogen.

Systems currently under consideration include thin-

layer bioreactor systems (Pulz 2001), plate reactors,biocoil technology (Borowitzka 1999) and a range of

related immobilized systems (Laurinavichene et al. 2006,

Rupprecht et al. 2006).

Of the above, plate reactors offer themost homogenous

light distribution as a result of their high surface to volume

ratio. Indeed, an almost homogenous light distribution

can be realized byminimizing the plate depth. This has to

be accompanied by an increase of the cell density tominimize photon loss. Thin-layer bioreactor systems take

both of these considerations into account by using the

natural adhesion power between two hydrophilic materi-

als (i.e. foils, plastics and glass) to produce a thin liquid

film between them. Culture growth occurs to high density

within this film or on its surface (immobilized micro-

algae). Suspension and immobilization processes using

biocompatible nano-adhesive surfaces are anotherrelated alternative.

Minimizing energy use

To increase the energy efficiency of the bioreactor, the

energy required to pump or stir the culture must be

minimized. Energy efficiency can be increased by using

the H2 produced as a pneumatic energy source to mix the

culture. If this energy input is not sufficient to reach the

necessary mixing quality, a split stream of the produced

gas can be recycled to improve the mixing properties.

Using the H2-rich process, gas may in fact have the

additional benefit of maintaining anaerobic conditions.One such quasicontinuous hydrogen production system

with minimized energy input can be realized by establish-

ing an array of plate reactors. Each single plate reactor runs

through a two-stage process (aerobic growth/recovery

phase and anaerobic H2 production phase). The quasicon-

tinuous hydrogen production is achieved by a controlled

reversal between the single plates, similar to a quasicontin-

uous chromatography system consisting of several col-umns. By using immobilized cells, the system’s quality can

be further enhanced. Compared with the continuous

process described by Fedorov et al. (2005), such a set-up

potentially has three three theoretical advantages:

(1) optimal reactor geometries for light capture,

(2) enhanced production efficiencies based on uni-

form cell ageing profiles and

(3) modular scale-up.

Converting biomass waste stream to fuels

Biomass is one of the main byproducts of the solar bio-H2

process. There are several ways of producing biogases

from biomass. These include anaerobic fermentation,

gasification or pyrolysis (Demirbas and Balat 2006, Li

et al. 2004, Matsumura et al. 2005, Shizas and Bagley2005). The produced biogas can itself be used for H2

production through steam reforming. There are also

approaches to convert high-hydrous biomass directly

into hydrogen through hydrothermal gasification by using

supercritical water (Matsumura et al. 2005). Based on the

DWof the residual algal biomass after a laboratory-based

H2 production run and reported H2 production yields

frombiomass gasification,we estimate that gasification ofthe biomass waste stream could yield a further 5%

increase in total H2 production (unpublished results).

However, given the advance of BTL technology, new

avenues of waste to fuel conversion can be explored.

Nutrient optimization

Nutrient optimization has also proven to be a criticalcomponent for efficient biomass and bio-H2 production.

For example, within the first day of S-deprivation (and

nutrient deprivation in general) C. reinhardtii reacts by

accumulating large amounts of starch in the chloroplast,

while photosynthesis and subsequent Calvin cycle

reactions appear to remain active (Grossman 2000). The

accumulation of starch is an important precondition for


indirect H2 (i.e. the three-photon process described

above). This correlation was demonstrated by the low

H2 production phenotype of an isoamylase knockout

mutant (Posewitz et al. 2004), by the high H2 production

phenotype of Stm6, which is enriched in starch (Kruse et

al. 2005b) and through PSII inhibition experiments duringthe S-deprivation process (Fouchard et al. 2005).

Acetate is another critical component of the mixotro-

phic Tris acetate phosphate (TAP)medium as it is reported

to promote starch synthesis, thereby drastically extending

the period of starch availability for H2 production

(Fouchard et al. 2005). Consequently, acetate promotes

the cellular H2 production capacity.

The TAP medium typically used for H2 productionexperiments with C. reinhardtii was developed in 1965

(Gorman and Levine 1965). Early attempts of improve-

ment concentrated on the effect of the nitrogen sources on

H2 production. For example, it was demonstrated that

NH41 as the sole nitrogen source had an enhancing effect

onH2production (Aparicio et al. 1985). Recently, a 150%

increase in the hydrogen production ratewas observed by

modifying the standard TAP medium to NH41, 9.20 mM;

PO432, 2.09 mM and pH 7.00.

The influence of the extracellular pH onH2 production

has also been investigated at different stages of the

production cycle (Kosourov et al. 2003). Overall, the op-

timal pH for WT cultures has been reported to be pH 7.7.

Raising the pH to 8.2 delayed the onset of H2 production

by almost 100 h compared with the onset at pH 6.5.

Furthermore, towards the later part of the H2 productionphase, the rate dropped more markedly at pH values

below 6.5 and above 8.2 (Kosourov et al. 2003).

Another point of optimization is the reduction ofmedia

costs. In the case of TAP medium, the Tris buffer

component accounts for approximately 70% of the total

cost. We have already shown that Tris concentrations can

be reduced by 50%, with clear implications for reduced

production costs (unpublished results).As discussed earlier, acetate is important for the

establishment of anaerobiosis and is reported to be

relatively expensive, based on current market prices

(Fouchard et al. 2005). However, its concentration can be

lowered. Furthermore, acetate is a general waste product

from many biotechnological bacterial processes and

indeedwaste water treatment. This opens up the potential

for the linkage to pretreated waste streams.

Medium exchange

Medium exchange (e.g. S1/S2) is also a costly process,

and centrifugation of cells for this purpose is impractical

for scale-up. In this context, it is of note that Laurina-

vichene et al. (2002) successfully demonstrated a dilution

process that takes into consideration the consumption of

sulphur through cell amplification. This procedure also

reduces the risk of contamination.

Water recycling

An additional advantage of photobiotechnological

hydrogen production, compared with bio-ethanol and

biodiesel production, is that the subsequent combustion

of H2 andO2, e.g., by an on-site fuel-cell system linked to

the electricity grid, would not only recycle water but also

purify it as the combustion of H2 and O2 is pure water.

This is also an important advantage compared with the

cultivation of crops as energy feedstocks, where up to1000 l of water are reported to be required per kg

biodiesel produced.

Down stream gas purification

Another important issue for optimization is the gas purity

because most fuel-cell types need highly pure hydrogen.

In contrast to other hydrogen production processes likesteam gas reforming of fossil fuels, there are no critical

sulphur or carbon monoxide impurities (which act as

catalyst poison in fuel cells) in the gas produced by algae,

which consists mainly of hydrogen (approximately 98%)

and smaller amounts of N2, O2 and CO2 during the

hydrogen production process. Such high purities are

necessary even in continuous large-scale processes to

avoid expensive gas purification steps like pressure swingadsorption (Iyuke et al. 2000, Sircar and Golden 2000) or

membrane-basedmethods (Adhikari and Fernando 2006,

Hagg and Quinn 2006, Nenoff et al. 2006).

Conclusion

This review has outlined the importance of H2 and in

particular sustainably produced bio-H2 as part a future

clean energy mix and highlighted a selection of many

developments at the level of bioengineering a bioreactordesign on theway to developing economically viable bio-

H2 production systems. Substantial progress has been

made in facilitating continuous bio-H2 production and

enhancing production efficiencies through the engineer-

ing of light-harvesting antenna systems, optimizing

photon conversion efficiencies (direct vs. indirect bio-

H2 production), substrate supply to the H2ase, reducing

oxygen sensitivity of the system and many adaptations tobioreactor design. With H2-powered cars now being

developed bymost of the large car manufacturers and the

number of H2 fuel stations increasing, there is a clear

demand to improve clean and cost-effective bio-H2

production systems. This will likely require that H2


production levels will have to be increased from their

current level of approximately 2% at 20 W m22 illu-

mination (e.g. Kruse et al. 2005b) towards approximately

7–10% at normal daylight levels. The fact that reduced

antenna mutants are already reported to have photosyn-

thetic efficiencies (to O2) of approximately 10% (Melis etal. 1999) supports the view that economically viable H2

production is indeed a realizable goal. The value of

reaching this goal was clearly stated by Sir Nicholas Stern

in his recent report on the Economics of Climate Change

(Stern 2007): ‘markets for low-carbon energy products are

likely to be worth at least $500 billion per year by 2050,

and perhaps much more. Individual companies and

countries should position themselves to take advantage ofthese opportunities’.

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Edited by E.-M. Aro


Photosynthetic biomass and H 2 production by green algae: from bioengineering to bioreactor scale-up

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