Photobioreactor design and illumination systems for H2 production with anoxygenic photosynthetic bacteria: A review

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 9 ( 2 0 1 4 ) 3 1 2 7 e3 1 4 1

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Review

Photobioreactor design and illumination systems for H2 production with anoxygenic photosynthetic bacteria: A review Alessandra Adessi a, Roberto De Philippis a,b,* a

Department of Agrifood Production and Environmental Sciences, University of Florence, Piazzale delle Cascine 24, 50144 Florence, Italy b Institute of Chemistry of Organometallic Compounds (ICCOM), CNR, via Madonna del Piano 10, 50019 Sesto Fiorentino, Florence, Italy

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abstract

Article history:

H2 is a clean, renewable and energy-efficient fuel. However, in order for it to be a fuel

Received 29 July 2013

effectively utilizable at an industrial level, key issues about its economically and envi-

Received in revised form

ronmentally sustainable production have still to be solved. Microbial hydrogen production

11 October 2013

is a process with a low environmental impact and, among microbial processes, photo-

Accepted 10 December 2013

fermentation is considered a promising and sustainable solution. However, the energy

Available online 12 January 2014

input for the biological processes is still higher than the energy output in the form of H2 gas. One possibility for improving this ratio is to increase the efficiency of the process while

Keywords:

at the same time reducing electricity consumption, both of which relate to the issue of an

Biohydrogen

optimal photobioreactor design.

Purple non sulfur bacteria

This review focuses on recent advances made in photobioreactor design towards higher

Photobioreactors

light conversion efficiency, a greater hydrogen production rate and substrate conversion in

Light conversion efficiency

hydrogen production processes carried out with purple non sulfur bacteria, giving partic-

Outdoor photobioreactors

ular attention to the light source and to illumination protocols. Recent achievements in outdoor hydrogen production in large scale photobioreactors are also reviewed. Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

1. Introduction: advantages and hurdles of biohydrogen production Hydrogen has the highest specific energy content per weight of all conventional fuels and is the most abundant element in the universe [1]. It can be produced from a wide variety of primary energy sources and with different production technologies. Approximately 96% of hydrogen

produced comes from fossil fuel-based processes: according to Konieczny and co-workers [2], 48% of hydrogen is currently produced from natural gas, 30% from oil, and 18% from coal. The remaining 4% is produced through water electrolysis [3]. Since the second half of the 1970s, there has been growing interest in biological hydrogen production processes among scientific and industrial researchers, which has led to considerable scientific output on the topic [4].

* Corresponding author. Department of Agrifood Production and Environmental Sciences, University of Florence, Piazzale delle Cascine 24, 50144 Florence, Italy. Tel.: þ39 0553288284; fax: þ39 0553288272. E-mail addresses: [email protected] (A. Adessi), [email protected] (R. De Philippis). 0360-3199/$ e see front matter Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijhydene.2013.12.084

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Four different groups of microorganisms are involved in biological hydrogen production processes: aerobic green microalgae (eukaryotes), cyanobacteria, chemotrophic bacteria and anaerobic photosynthetic bacteria (mostly Gram negative prokaryotes). The last two processes can be combined in a multi-step production process [5,6]. Compared to non-biological hydrogen production processes, biological ones have the advantage of being carried out in ambient conditions and without the use of polluting catalysts; furthermore, they are suitable for decentralized energy production in small-scale installations in locations where biomass or wastes are available, thus avoiding energy expenditure and costs for transport. These processes are considered viable for the future energy market, as they have been evaluated as sustainable, renewable and energy-efficient processes. In particular, Manish and Banerjee [7] performed a Net Energy Analysis of four different biohydrogen production processes: i) dark fermentation, ii) photo-fermentation, iii) combined dark and photofermentation and iv) biocatalyzed electrolysis. They all proved to be renewable, as the hydrogen output was larger than the non-renewable energy input (net energy ratio), and they reduced greenhouse gas emissions by 57e73% as compared to a non biological process (steam methane reforming, taken as a reference as an established hydrogen production technology). Considering the overall balance, the most effective process was the integration of dark and photofermentation, as this had the lowest greenhouse gas emissions, the highest energy efficiency (energy output/energy input) and the highest net energy ratio among the four processes [7]. Having assessed the energetic feasibility and expediency of these kinds of process from a theoretical point of view, it is possible to infer what the benefits might be in a future where biological processes will be used side by side with other production processes. Indeed, in addition to the sustainability mentioned earlier, there are a number of economic benefits, such as the possibility of increasing opportunities for rural employment in this field, the creation of a new investment path for plant and equipment, unprecedented competition in the energy sector and, most interestingly, a noticeable reduction in dependence on fossil fuels. This is in line with the economic focus of many countries and political subjects that are emphasizing the development of the so-called green Economy; for example, the European Community judges the green economy to be a priority in order to combine the need to provide a path to renewed economic growth and job creation, in response to the current severe economic crises, with an environment-friendly outlook [8,9]. Indeed, from the environmental point of view, besides the reduction in CO2 emissions, consideration needs to be given to the biodegradability of microbial biomass, the possibility of coupling the energy production process to waste disposal and the opportunities for improved land and water usage. A no less important point is that biofuels in general and biological hydrogen as well can guarantee greater energy security in terms of supply reliability, capillary and domestic distribution and readiness of availability [10]. Furthermore, localized energy production would have a positive impact on geopolitical stability [11,12].

Among the renewable and sustainable hydrogen production methods, great importance is currently given to so-called solar production, which also includes microbial hydrogen gas production by photosynthetic microorganisms [13]. However, important issues will need to be addressed before it is possible to rely on microbial hydrogen production as an economically viable and efficient H2 production process. As regards the use of phototrophic microorganisms, the main issues for improving the biological hydrogen producing processes are, at the moment, low substrate to H2 yields and low light conversion efficiencies, which respectively entail the need for large volumes of substrates and large surface areas for the photobioreactor [14]. So, besides the importance of dealing with an environmentally sustainable technology, the efficiency and overall cost of this process also have to be considered, as they are crucial for the actual development of a hydrogen-including economy [15]. Recently, a number of reviews have been published about biological and photofermentative hydrogen production, providing a significant general overview of the present state of the art and the future steps to be taken to optimize the process [16e20]. Most of them stress the urgency of developing efficient, cost effective and high performing photobioreactors, but only very few reviews deal with the state of the art regarding reactors for purple bacterial hydrogen production. In fact, when looking more closely at the different types of photobioreactors, they mostly refer to the broad portion of literature devoted to microalgal culturing systems [21e23]. However, in the last few years the number of research papers on various types of bioreactors specifically designed for hydrogen production with purple non sulfur bacteria (PNSB) has blossomed. This review sets out to collect together the recent advances made in photobioreactor design and in the illumination protocols adopted to achieve a higher light conversion efficiency, hydrogen production rate and substrate conversion in hydrogen production processes using PNSB. Thus, reviewing the present state of the art of high efficiency and large scale biohydrogen production processes will help to keep the finger on the pulse of the current and evolving potential of such processes.

2.

Photofermentation

PNSB (see Box 1) are studied intensively as anoxygenic phototrophs that produce H2. This process, called photofermentation, enables the conversion of substrate to hydrogen, carbon dioxide and biomass; near-theoretical maximum H2 yields (mole H2 per mole substrate) are possible where the reaction is carried out by non-growing cultures [17]. Hydrogen evolution is catalyzed by the ATP-requiring nitrogenase enzyme, capable of reducing protons to molecular hydrogen (see Box 2 for details). As nitrogenase is repressed by fixed nitrogen, suitable substrates must have a high C/N ratio [17,39,40]. PNSB mainly acquire electrons from short chain fatty acids (e.g. acetate and lactate), which may also be available in fermentation products found in agricultural and food waste. Some PNSB can also use sugars [41,42]. This process is generally considered very promising, due to the high substrate to H2 conversion yields that can be achieved, the possibility to use a wide spectrum of sunlight, the

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Box 1 Purple non sulfur bacteria. Generalities

Purple non-sulfur bacteria (PNSB) are anoxygenic phototrophic bacteria that contain photosynthetic pigments and are able to perform anoxygenic photosynthesis under anoxic conditions [24]. Indeed, PNSB are a very diverse group as regards morphology, internal membrane structure, carotenoid composition, utilization of carbon sources and electron donors, cytochrome c structures, lipid composition, quinone composition, lipopolysaccharide structure and fatty acid composition. Eutrophic ponds are the most common habitat where they inhabit the anoxic or low-oxygen tension layers. A suitable light irradiation is preferred, even if not strictly necessary [25]. The publication of the first complete genome sequence of a purple non-sulfur bacterium, Rhodopseudomonas palustris [26], pointed out the metabolic versatility of these bacteria; they can grow both aerobically and anaerobically, autotrophically or heterotrophically, chemotrophically or phototrophically. The specificity of purple bacteria is their ability to form their energy carrier (ATP) in the absence of oxygen by using sunlight as a source of energy via cyclic photosynthesis (represented in Fig. B1). H2 is produced only in anaerobic photoheterotrophic conditions, when light is the energy source and the reducing power is derived from the catabolism of carbon compounds through the TCA cycle. PNSB are able to use a wide variety of organic carbon compounds, namely the intermediates of the tricarboxylic acid cycle, but some species can also use one-carbon atom compounds such as methanol and formate, while some other species grow by using aromatic organic compounds such as benzoate, cinnamate, chlorobenzoate, phenylacetate or phenol [27]. The preferred way to assimilate nitrogen is fixation through nitrogenase, which reduces nitrogen to ammonia. A further discussion of this enzyme and its role in the H2 production process can be found in Box 2. In Figure B1 the main metabolic features, linked to hydrogen production, of purple bacteria are schematically represented.

absence of O2-evolving reactions (which would inhibit the H2producing enzymes) and the possibility to couple this kind of H2 production process with waste disposal [43]. Most papers on biological hydrogen production focus on the use of different PNSB and different substrates or culture conditions [4]. A very large field of investigation concerns

the kind of substrate to use in the process, mostly studied on a lab scale. Many papers have been published about the use of wastes and of substrates deriving from other fermentation processes, which have been extensively reviewed in the last few years. Most reviews concerning this aspect report the recent advances made when dealing with

Fig. B1 e Main processes related to hydrogen production, under photoheterotrophic growth in non-nitrogen fixing conditions: anoxygenic photosynthesis, ATP synthesis, TCA cycle, hydrogenase and nitrogenase activities. The straight black arrows indicate the electron flow. The dotted grey arrows indicate proton translocation through the membrane. The lightning symbol indicates light excitation. Abbreviations: Cyt bc1 [ cytochrome bc1 complex; Cyt c2 [ cytochrome c2; Fd [ ferredoxin; RC [ Reaction Center; Succinate e DH [ succinate dehydrogenase; NADH-DH [ NADH dehydrogenase TCA [ tricarboxylic acid (Scheme modified from Ref. [80], with kind permission from Springer Science D Business Media BV).

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Box 2 Enzymes involved in hydrogen production

Nitrogenase is the enzyme that, in all the N2-fixing prokaryotes, including PNSB, is responsible for hydrogen production, catalyzing the Reaction (B2.1) that leads to the production of one H2 molecule per molecule of N2 fixed.

N2 þ 8Hþ þ 8e þ 16ATP / 2NH3 þ H2 þ 16ADP

(B2.1)

As mentioned in Box 1, the ATP used in this reaction is mainly produced by anoxygenic photosynthesis, while the reducing power derives from the catabolism of organic acids and is carried to nitrogenase through ferredoxins. Nitrogenase catalyzes a very expensive reaction in terms of energy, thus it is very strictly regulated by the presence of dissolved ammonium ions. The regulation has been modeled as a three-level control mechanism, as described by Masephol et al. [28] for Rhodobacter capsulatus. This regulatory cascade is often used as a general model for the regulation of nitrogenase in PNSB though there are many variations on this regulatory scheme. The general scheme is as follows: at the first level fixed nitrogen signals (e.g., NHþ 4 ) are sensed through the NtrBC two-component system to prevent the transcription of nifA, a gene encoding for an RNA polymerase sigma 54-dependent transcriptional activator. At the second level, the presence of NHþ 4 affects NifA, inducing structural changes that prevent the transcriptional activator from binding to its binding site and activating nitrogenase (nif) gene transcription. At the third level, the presence of NHþ 4 affects nitrogenase itself, causing a “switch-off” of the enzyme through ADP-ribosylation mediated by DraT [29e32]. In the absence of molecular nitrogen, the enzyme catalyzing the Reaction (B2.2) dissipates the excess of reducing equivalents deriving from other metabolic processes. This is the reaction used for hydrogen production processes.

8Hþ þ 8e þ 16ATP / 4H2 þ 16ADP

(B2.2)

Hydrogen can also be an electron donor for purple bacteria, oxidized by a membrane bound hydrogenase which functions as a ubiquinone oxydoreductase. The overall reaction (B2.3) can take place in both directions, depending on the presence or absence of the substrates.

H2 4 2Hþ þ 2e

(B2.3)

High hydrogenase activities have been observed in cells possessing an active nitrogenase; the hydrogen produced by nitrogenase stimulates the activity of hydrogenase in growing cells even though the synthesis of hydrogenase is not closely linked to the synthesis of nitrogenase [33]. In H2 production processes, active uptake-hydrogenases are undesirable, as they affect gas production: in particular an inactivation of such enzymes (hup- strains) usually leads to enhanced hydrogen production [34e38].

the complexity of using wastes as starting materials for hydrogen production; the necessity for multiple-organism systems to obtain higher H2 yields is often pointed out [19,40,44e49]. A great deal of research work has been carried out in the past few years on reactor design, scale-up and optimization of light distribution for purple bacterial cultivating systems; in the past, most of the studies on large scale photobioreactors concerned the mass cultivation of microalgae, mainly for biomass production [21,50]. In 2001, Tsygankov [51], reviewing lab scale photobioreactors specifically used for H2 production with PNSB, pointed out the difficulty of scaling up the reactors tested until then, but during the last ten years the number of papers dealing with photobioreactors newly designed for hydrogen production with PNSB has increased noticeably. Of these, a significant number of studies are based on large scale reactors. This indicates that the field of photofermentative hydrogen production is becoming more open to scaled-up

applicable processes, which have significantly different requirements compared to the cultivation of microalgae and are one of the major issues to be addressed in order to make a real industrial development of this process possible.

3.

Photobioreactors

Photobioreactors for biohydrogen production with PNSB must be closed systems, owing to the need to maintain anaerobic conditions and prevent H2 gas dispersion. They must have a high illuminated surface to volume ratio and an efficient mixing system to allow the cells to be illuminated as uniformly as possible; they also need a satisfactory gas exchange system for removing the H2 produced, and, possibly, a temperature control. The most important issues to be considered in designing a photobioreactor for the production of

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Table 1 e Types of photobioreactors (PBR) and artificial light distribution systems with the corresponding hydrogen production rates (HPR), light conversion efficiencies (LCE) and substrate conversion (SC). LCE and SC were calculated as indicated in Box 3, with a few exceptions indicated in the footnotes. n.a. [ not available. Type of PBR and light distribution system

Organism

Light source and intensity

Working volume (l)

Mixing

HPR (ml l1 h1)

LCE (%)

SC (%)

Ref.

Tubular Vertical

Rb. sphaeroides

Tungsten lamp 200 W m2

0.4

n.a.

20.0

1.1a

n.a.

[56]

Flat panel Rocking

Rb. sphaeroides

1.0

Oscillation

11.0

3.31

45

[88]

Floating type Induced diffuse light

Rp. palustris Rb. sphaeroides

Tungsten lamp 10.25 W m2 Halogen lamp Halogen lamp

6.0 0.3

Waves None

10.0e12.0 7577c

n.a. 61

[89] [87]

Double-layer

Rb. Sphaeroides (wild type þ reduced pigment mutant) Rb. Sphaeroides (reduced pigment mutant)

Tungsten lamp 500 W m2

0.2 þ 0.2

None

3640c

0.31b 6.12 Peak value: 9.23d 2.18e

n.a.

[90]

Tungsten lamp 300 W m2

0.8

None

2000c

n.a.

n.a.

[91]

Rs. rubrum

Tungsten lamps 500 lux

2.0

107.5f

8.67

n.a.

[58]

With optical fibers

Rp. palustris

Sunlight þ tungsten lamp

1.8

Dual impeller Rushton turbine (500 rpm) Impeller

22.7

n.a.

62.3

[84]

Other geometries Annular triple jacketed

Rb. sphaeroides

Luminine tubular light 15 W m2

1.0

Magnetic stirring

6.5

3.7

75

[59]

Biofilm Cover glass slides

Rp. palustris

LED (590 nm) 5000 lux LED (590 nm) 6.75 W m2 metalehalide lamp 12 W m2 LED (590 nm) 6.75 W m2

0.072

Medium flow

26.9

8.9

2.1

[86]

0.1

Medium flow

86.46c,f

3.8b

6.25

[71]

0.125

Medium flow

39.2f

9.3

75

[72]

1.2

Medium flow

38.9

56

1.7

[70]

Tungsten lamp 34 mEg Halogen þ Tungsten lamp 95 W m2 LED (590 nm) 6000 lux Incandescent lamp 150 W m2

0.01

None

201.6c,f

n.a.

n.a.

[66]

0.8

Magnetic stirring

43.8

2.34

90.8

[92]

0.8

Medium flow

58.4f

82.3

5.17

[69]

0.08

Stirring

32.9

0.96

77.0

[67]

Multi-layer

Fermentor type

Groove-type (small incisions) Rough surface optical fibers Glass beads

Rp. palustris Rp. palustris Rp. palustris

Innovative immobilization Latex Rp. palustris Clay þ optical fibers

Rp. palustris

PVA þ carrageenan þ alginate Activated Carbon Fibers

Rp. palustris

a b c d e f g

Rp. faecalis

calculated by Ref. [57]. LCE ¼ combustion energy of H2/light energy, no further details given by the Authors. Volume expressed as ml m2 h1. LCE ¼ gross combustion heat of H2 produced/input light energy  100. LCE ¼ combustion enthalpy/net light energy absorbed (Delta of light). Value in mmoles of H2 converted to ml of H2 by 22.4 multiplying factor. mE ¼ mmol photons m2 s1.

hydrogen with PNSB are related to light. Indeed, light intensity, light quality and sources, light distribution into the photobioreactors are key issues for the optimization of H2 production.

3.1.

Geometries of photobioreactors

Many different geometries have been tested for hydrogen production with PNSB (Tables 1 and 2). The two main types of

[84] 62.3 n.a.

[53] [93] [96] n.a. 76.7 n.a. n.a. n.a. 1.4

[52] 10.3

c

Value in mmoles of H2 converted to ml of H2 by 22.4 multiplying factor. Volume expressed as ml m2 h1. The absorbed energy is calculated as Global solar radiation (meteorological data)  surface area  0.64 (ratio of PAR for R.palustris). b

22.7 Rp. palustris

Impeller 1.8 Sunlight þ tungsten lamp

Rb. capsulatus Rb. sphaeroides Rb. sphaeroides

Horizontal Flat panel Vertical 30 inclined With light shading bands Fermentor type With optical fibers

a

8.0 10.0 0.87b By the gas produced Ar sparging n.a. 25.0  4 6.5 1.0 Sunlight Sunlight Sunlight

10.7 50.0

8.9a 4.5a

Rb. Capsulatus hup- mutant Rp. palustris

90.0

Rotary pump (5 min per hour) Centrifugal PVC pump

1.0 16.6a 6.9a

Sunlight (þinitial artificial light) Sunlight (þinitial artificial light) Sunlight (shielded) Rb. capsulatus

80.0

27.2

Avg 0.63 Max 0.92c

[55] 12 0.2

[54] 16

n.a. 3.3

Rotary pump (5 min per hour) Rotary pump 65.0 Sunlight Rb. capsulatus

Avg

Max Tubular Near horizontal

Organism

Light source

Working volume (l)

Mixing

HPR (ml l1 h1)

LCE (%)

n.a.

SC (%)

[53]

Ref.

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Type of PBR and light distribution system

Table 2 e Types of photobioreactors (PBR) sunlight irradiated with the corresponding hydrogen production rates (HPR), light conversion efficiencies (LCE) and substrate conversion (SC). LCE and SC were calculated as indicated in Box 3, with a few exceptions indicated in the footnotes. n.a. [ not available; Avg [ average; Max [ maximum.

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photobioreactors studied are tubular reactors and flat panel bioreactors. Tubular photobioreactors consist of transparent tubes that are either horizontal [52] or with an inclination of 10 e30 , south oriented [53e55]; vertical tubular reactors consist of a single column [56]. The mixing is mechanical and the gas is collected at one side (the top, if inclined) of the reactor. Collecting the gas on one side of the tube imposes a limit in length; indeed, the longer the tube, the longer the time the gas bubble stays inside the reactor where H2 could be taken up by cells (see Box 2), thus theoretically decreasing the hydrogen productivity. The case described by Boran et al. [54], where a lower productivity was obtained with a hup- strain (mutant with inactivated uptake hydrogenase) compared to the wild type strain, can be ascribed to a non optimal mixing of the culture rather than to a low efficiency of the strain. Most experiments carried out with serpentine-tubular reactors were conducted in scaled-up systems, using natural sunlight as the main or the sole light source. Recent experiments with tubular reactors have showed interesting hydrogen production rates per illuminated surface (maximum of 5.89 l m2 d1 in Ref. [52]); however, a disadvantage of this kind of reactors might be the ground area occupancy, which also affects the cost of the process. Flat panel reactors are rectangular transparent boxes that can be either vertically placed or inclined in the direction of the sun. They are only a few centimeters thick (1e5 cm, according to [57]), and this exposes cells to very short mixing-induced light/dark cycles, which can decrease hydrogen productivity. Flat panel photobioreactors are difficult to scale up over a few liters of volume, but they offer the possibility to arrange a set of reactors one behind the other, at an appropriate distance, to increase the ground area productivity [53]. Furthermore, flat panels are easy to construct and modify, and are therefore suitable for base-studies on light distribution and mixing; a large proportion of the literature about the use of this kind of reactor refers to small scale reactors, with innovative systems to get higher yields. Fermentor type photobioreactors are glass cylinders mixed with impellers, which can vary the type of geometry. They are rarely used due to the long light path through the reactor, whose diameter is bigger than in the tubular reactors. Interesting solutions, though, have been presented regarding the type of mixing [58] and the possibility of illuminating the reactor from its inside with optical fibers in order to optimize light distribution and overcome the lightpath length hurdle. Various other types of geometries, such as, alveolar panels or helical, alpha-shaped and torus shaped photobioreactors, have been investigated, mainly with microalgal cultures [22], but an interesting example of alternative geometry used with PNSB has been reported by Ref. [59]. An annular PBR consisting of three concentric chambers was used and, due to the high surface-to-volume ratio, a quite interesting light conversion efficiency (LCE) (3.7%) was obtained.

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3.2.

Immobilized photobioreactors

Research on immobilized cells reactors started at the beginning of the 1980s [60e62] and blossomed during the 1980s and the 1990s [63e65]; it was then put aside for almost a decade. Here we are only reviewing publications from the last few years that have reported innovations to the previously tested immobilized systems. At the present time, alternative materials for immobilization are under investigation, such as latex [66], carbon fibers [67] or a mixture of different immobilizing materials [68,69]. Biofilm reactors are starting to be studied intensively as well [70e72], mainly because cells in biofilms are usually characterized by enhanced resistance to the presence of toxic components or other extreme culture conditions as compared to cells in suspension [73,74]. However, at present all the immobilized systems have been studied under lab conditions, with relatively small-volume photobioreactors. The main advantages in using immobilized cells are the stability of the process and the possibility of carrying out continuous feeding, which would be the best solution in particular when working with wastes, whose organic matter content needs to be reduced. Moreover, in every immobilized system there is a natural separation of solid, liquid and gaseous phases; this not only facilitates gas recovery, but also repeated use of biomass. However, the promising features of the immobilized systems need to be tested and validated on a larger scale, under outdoor light conditions.

3.3.

Light sources and distribution systems

Culture irradiation is a major concern in hydrogen producing systems [75], and thus many different light distribution solutions and different light sources have been under intensive study. The absorption spectrum of purple bacteria (Fig. 1) covers the two ends of the visible spectrum. Carotenoids absorb the radiation between 450 and 550 nm, giving the typical “three fingered” shape to the spectrum. Bacteriochlorophylls (BChls) absorb at 590 and at 800, 850 and 880 nm in the near infrared region; the peaks at 850 and 880 often merge in one larger peak,

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having the maximum at an intermediate wavelength in between, but shoulders can be observed occasionally [76e79]. There is also the absorption peak of the Soret band in the near UV (around 390 nm), not shown in Fig 1. When using artificial light, the most frequently employed light sources are tungsten lamps, as their emission spectrum covers the whole absorption spectrum of PNSB [80]. Particularly important is the high near infrared emission, where the absorption maximum of bacteriochlorophylls is located. Halogen and incandescent lamps are quite frequently used as well. As tungsten-filament lamps are energy-expensive light sources, an interesting alternative is offered by Light Emitting Diodes (LEDs). Kawagoshi et al. [81] state that LEDs have a life time ranging between 20,000 and 30,000 h, while a tungsten lamp lasts for 1000e2000 h, and they predict a 98% reduction in energy costs by using LEDs instead of tungsten lamps. In a cost effective scaled-up system, though, the best solution would appear to be the use of natural solar light. However, even though PNSB are able to use a wide range of the solar light spectrum, in fact only 65.8% is actually part of the PAR (Photosynthetic Active Radiation) for purple bacteria [57]. Miyake et al. [82] have pointed out how the intrinsic variability of solar light results in the varying of the rates along with light intensity during the day, making it impossible to attain constant production rates. Furthermore, they observed a probable photoinhibition at noon, under the highest irradiation of the day (0.9 kW m2). Therefore, integrated artificial and solar light systems need to be taken into consideration; Ogbonna et al. [83] proposed a system, for microalgal cultures, to overcome the solar light variations during the day, the bad weather periods and the night periods: solar light was collected by Fresnel lenses equipped with a light-tracking sensor; the solar light collection device was connected to optical fibers that brought light into light radiators which homogeneously diffused light into the photobioreactor. This system was equipped with a light intensity sensor that in the event of insufficient solar light intensity switched on an artificial light, to meet the culture’s light needs. This idea has been developed by Chen and collaborators [84] and applied to a small volume (1.8 l) flat panel photobioreactor. Hydrogen yields comparable to solely artificial light illuminated systems were obtained.

4. Maximization of hydrogen production rate, light conversion efficiency and substrate conversion

Fig. 1 e Absorption spectrum typical for PNSB. Absorption maxima indicated with * are due to carotenoids, while ** indicate the absorption maxima due to bacteriochlorophylls.

Three parameters for the evaluation of hydrogen production performances were taken into account in this review: Hydrogen Production Rate (HPR), Light Conversion Efficiency (LCE) and Substrate Conversion (SC) (see Box 3 for details about the calculation of the last two parameters). The results for artificial light illuminated photobioreactors are reported in Table 1, and those for sunlight illuminated photobioreactors in Table 2. Only experiments using synthetic media were taken into account in this review, in order to better evaluate LCE optimizations, as non synthetic media are often not

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Box 3 Light conversion efficiency and substrate conversion

Two of the most relevant parameters to be considered for the optimization of the production of H2 via photofermentation are the photochemical efficiency, or light conversion efficiency (LCE) of the system, and the substrate-to-hydrogen conversion (SC). The light conversion efficiency (LCE) for hydrogen production processes is defined as the energy stored as hydrogen produced per unit of light energy absorbed (B3.1).

LCEð%Þ ¼

Free energy of the total amount of H2 produced  100 Total energy of the light incident on the culture

(B3.1)

Most of the Authors of the works reviewed here and reported in Tables 1 and 2 use the Formula (B3.2) reported by Miyake and Kawamura [85]. The free energy is calculated as the amount of hydrogen produced by the standard enthalpy of combustion of H2: hð%Þ ¼

33:61  rH2  VH2  100 IAt

(B3.2)

where h is the LCE, 33.61 is the energy density of hydrogen (W h g1), rH2 is hydrogen density (g l1), VH2 is the volume of hydrogen produced (l), I is light intensity (W m2), A is the irradiated area (m2) and t is the duration of the process (h). Different multiplying factors can be found in some papers owing to different measure units chosen by the Authors. For example, Ref. [58], calculated the enthalpy as 0.0672 (W h mol1), while Ref. [69] used the value of 286.0 J mmol1; those choices entail slight differences in the results obtained but the approach in the calculation of LCE of these Authors can be considered substantially the same. This calculation provides all the information needed to evaluate the energy output as hydrogen divided by the amount of light energy input. Therefore, it should be stressed that this calculation only takes into account the amount of energy derived from light and ignores the amount of energy deriving from the substrate used for hydrogen production. In actual fact, a total energy balance of the metabolic process should also include in the denominator the energy associated with the organic substrate consumed during the hydrogen production process. When calculating LCE, differences might appear in the final results depending on the way the light energy is measured. Indeed, some Authors indicate the amount of light energy impinging on the reactor either emitted by the lamps or sunlight, measured with luxmeters (which usually measure the intensity only of PAR radiation); other Authors [86] indicate the amount of net light energy absorbed by measuring the delta between light in the front and the light on the back of the reactor; a few Authors [52,87], working with sunlight illuminated photobioreactors, calculate the light absorbed as the sunlight radiation corrected by the PAR portion of the organism; some other Authors do not indicate the details of their calculations. Indeed, the way light intensity is measured is not always clearly indicated in the papers reported in Tables 1 and 2, even if such an important parameter in a process that involves photosynthesis should be clearly and thoroughly defined to allow a fair comparison between data. PAR corrected light intensity should always be used (whether measured by PAR measuring luxmeters or manually corrected). Calculating the amount of energy effectively absorbed by a photobioreactor might not be a simple task, depending on the technical characteristics and geometry of the reactors. Consequently, for the sake of clarity, we suggest using the term LCEa when this parameter is calculated from the amount of light energy absorbed and LCE when it is calculated on the amount of light impinging only on the surface of the reactor. As a more general observation, we believe that LCE should always be reported in studies that are meant to maximize light absorption and the efficiency of hydrogen production, but unfortunately this is not always the case. It is worth mentioning that the complexity in the evaluation of this parameter in the case of H2 production by using PNSB is due to the fact that the route leading to H2 from light energy is not direct, but goes through ATP generation and nitrogenase activity (see Box 2). For instance, Go¨bel [88] determined that at a wavelength of 860 nm 1.5 photons are required to generate one ATP. Since at least 4 ATP molecules are needed to generate one molecule of H2 (B2.2), this means that at least 6 moles of photons are required to generate 1 mole of hydrogen. Moreover, in PNSB the ferredoxin mediated electron transfer to nitrogenase is an energy consuming process [89]. Indeed, Miyake [90] calculated that for the production of a single molecule of H2, 11 photons at 860 nm are required (Energy of 1 mole of photons at 860 nm ¼ 0.139 MJ). Akkerman et al. [57] calculated that 14e15.8 photons at 522 nm are required for producing one molecule of H2 (Energy of one mole of photons at 522 nm ¼ 0.229 MJ). From these data, it has been estimated that the maximum conversion efficiencies are 19% [0.290 MJ/ (11  0.139 MJ)] and 8.4% [0.290 MJ/(15  0.229 MJ)] at wavelengths of 860 and 522 nm, respectively. The same Authors [57] stated that the overall theoretical light conversion efficiency (LCE) (B3.1) under solar irradiation should be at least 10%, considering all the utilizable wavelengths of the solar spectrum (Fig. 1) and their respective energies. On the other hand, Ghirardi et al. [91] estimated the maximum light conversion efficiency for different photosynthetic organisms, stating that the maximum LCE achievable with non-oxygenic photosynthetic microorganisms would be 6% under sunlight.

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Recently, Sakurai et al. [89] calculated in a very scrupulous way the quantum requirements for PNSB. They defined that the quanta required to generate 4 ATP molecules and 2 reduced Ferredoxins (all needed to generate one molecule of H2) range from 3.7 to 8.7. From these data, they estimated that the maximum LCE reachable with PNSB ranges from 10.9% to 25.6%, using as a reference the energy of one mole of photons at the mean wavelength of 650 nm (0.184 MJ). As a matter of fact, all these energy conversion efficiency calculations are based to some extent on assumptions and approximations. In our opinion, estimates based on deduced or presumed data, although all correct, cannot set a benchmark but can be still used to define the range within which the LCE can stand. However, unless an experimental data set of all photon requirements for each wavelength is available, it is not possible to exactly define the maximum energy conversion efficiency obtainable with PNSB. However, it can be taken for granted that it is not possible to achieve a complete conversion of light energy to hydrogen energy, due to the energy losses occurring in the large number of energy transfer processes leading to hydrogen production from the light absorption by the photosystem of PNSB. Moreover it is a matter of fact that, to the best of our knowledge, reports in literature only rarely indicate an LCE in excess of 10%. As a conclusion, to date the only effectively interesting evaluation of the efficiency of such processes is to measure the total light energy facing the reactor (regardless of the energy of single photons) and to use it to obtain the hydrogen energy yield. The conversion yield of the carbon substrate to H2 (SC) is represented in (B3.3). SCð%Þ ¼

mol H2 obtained  100 mol H2 theoretical

(B3.3)

SC is the ratio (%) between the amount of hydrogen really obtained and the amount of H2 theoretically obtainable from the amount of substrate consumed. As an example, for lactate, acetate, malate and butyrate the theoretical reactions used are respectively (B3.4eB3.7):

C3H6O3 þ 3H2O / 6H2 þ 3CO2

(B3.4)

C2H4O2 þ 2H2O / 4H2 þ 2CO2

(B3.5)

C4H6O5 þ 3H2O / 6H2 þ 4CO2

(B3.6)

C4H8O2 þ 6H2O / 10H2 þ 4CO2

(B3.7)

perfectly bright and thus affect light penetration inside the reactor. Data available in literature that permitted the calculation of LCE or that provide significant innovations to the hydrogen producing process are reported in Tables 1 and 2. In the following sections the discussion about the results obtained by illuminating photobioreactors with artificial light or with sunlight is carried out separately, as we consider sunlight illuminated systems to be a wholly different matter to controlled light illumination systems.

4.1.

Artificial light illuminated photobioreactors

Light conversion efficiency in artificial light illuminated photobioreactors can vary considerably even among photobioreactors designed with similar geometries. The geometry of the bioreactor has to be considered together with the light distribution system and the light source, because these parameters all together can play a major role in enhancing LCE. A vertical tubular photobioreactor illuminated by a tungsten lamp [56] was used with Rhodobacter sphaeroides: a high

HPR was obtained, but with a low LCE. Furthermore, this geometry of photobioreactor is difficult to scale up: a single column has a limitation in height, due to the increase of the liquid pressure and to a non optimal mixing. Flat type reactors showed interesting LCEs when working with sophisticated light distribution systems, like specific wavelength LEDs [92] or with light diffusion systems [93], along with noticeable HPRs. The rocking [94] and floating [95] type photobioreactors yielded interesting HPRs (10e12 ml l1 h1), but in the latter the LCE was almost ten times lower than with other reactors of the same geometry, meaning that light distribution in that kind of reactor was not optimal. A double-layer photobioreactor [86] was designed in order to capitalize the performances of strains with different pigmentation (i.e. a wild type and a reduced-pigment mutant of Rb. sphaeroides) overlapping the two separate cultures. This system led to a high HPR per surface area, but to a not very significant LCE. Multi-layer reactors were also investigated [96] with a single strain of Rb. sphaeroides, either wild type or a reducedpigment mutant. The novelty of this kind of photobioreactor is the presence of permeable membranes dividing the layer containing cells and the layer containing the medium; the clear

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medium layers thus serve as a light diffusion system, improving light distribution inside the reactor. However, the data reported in the paper were insufficient to calculate HPR and LCE. Fermentor type photobioreactors are not frequently used, unless for light distribution experiments as in Chen et al. [84], or for stirring studies as in Ref. [58]. Chen et al. designed a light distribution system that was capable of integrating artificial and solar light, the latter distributed through optical fibers; this led to a significant HPR (22.7 ml l1 h1) and good substrate conversion (SC). Ismail et al. [58] tested different mixing speeds with Rhodospirillum rubrum, getting the best results at a speed mixing of 500 rpm with a dual rushton turbine impeller; the HPR and the LCE proved to be unexpectedly high (107.5 ml l1 h1 and 8.67%, respectively) for this kind of system, indicating that fermentor type photobioreactors can be taken into further account if provided with efficient mixing systems. Generally speaking, hydrogen production experiments with photobioreactors containing immobilized cells result in very high yields and HPRs, in particular when working with LEDs as illuminating systems, but to date these systems have all been tested only in small volume photobioreactors. Considering the immobilized systems, the highest HPR (201.6 ml l1 h1) was obtained with latex immobilized cells [66], but also a groove type immobilized photobioreactor [71] yielded a very high HPR (86.46 ml l1 h1) by increasing the cell-attaching surface without enlarging the overall surface of the reactor. Zhang et al. [71] tested different LEDs emitting at specific wavelengths (namely, 470, 520, 590, 620 nm), and the best was the one emitting at 590 nm, a wavelength very close to one of the absorption peaks of bacteriochlorophylls in Rp. palustris (see Fig. 1). Kawagoshi et al. [81] used long-wavelength emitting LEDs with an emission spectrum having a maximum at 850 nm for illuminating a halotolerant Rhodobacter sp. but they obtained poor hydrogen production. Indeed, they obtained about 70 mmol H2 l1 in 300 h, which roughly corresponds to 5.2 ml l1 h1. However, in this case the low hydrogen production in comparison with the other results reported in Table 1 was most probably due not to the light source but to the suboptimal culture conditions. Specific LED illumination permits the attainment of high LCEs, when observing the yields reported in Table 1. However, a number of observations should be made. The use of specific 590 nm LEDs with a biofilm reactor attached on cover glass slides [92] resulted in an LCE of 8.9%, despite the use of very high light intensities, which is a very interesting result as up until now high LCEs have only been obtained with low light intensities using wavelength-aspecific lamps [57,97]. This result might have been obtained because of the optimization of the light used by the cells, namely that only the energy cells are capable of utilizing was used to illuminate the culture. When using the same specific LED illumination in PBRs operating with cells immobilized on glass beads or gel granules, outstanding LCEs were reported, as high as 56 and 82% ([70] and [69], respectively). These values stand really far from the typical range for LCE (see Box 3). They were calculated with the same Equation (B3.2) used for most of the other processes reported in this review. However, it should be stressed that Equation (B3.2) calculates LCE by dividing the energy of the amount of hydrogen produced by the energy input from the light, which is the product of light intensity to

irradiated area. In the above-cited papers [70] and [69], it is not clear whether the irradiated area was considered just as the reactor surface. If that was the case, the surface was underestimated, because the effective irradiated surface is actually the sum of the illuminated parts of the surface of each glass bead [70] or gel granule [69] contained in the PBR. The use of the reactor surface in the calculation of LCE would lead to a certain overestimation of this parameter. As far as substrate conversion (SC) is concerned, the results are quite variable and seem to be influenced by the mixing system chosen for the experiment. From the results reported in Table 1, it emerges that the lowest SCs were obtained when not very efficient mixing systems were used, as in Refs. [71,72,92], even if the hydrogen production was significant. Where efficient mixing systems are applied, SC ranges between 62.3 and 75% for suspension cultures. El-Shishtawy et al. [93] reported significant substrate conversion (61%) even if no stirring was applied, but in this case it can be attributed to the efficiency of the light distribution system in the culture. The highest result so far obtained (90.8%) was achieved with clay immobilized cells, magnetically stirred and illuminated by artificial lights distributed through optical fibers [98].

4.2.

Sunlight illuminated PBRs

As stressed above, light irradiance is very important when using photosynthetic bacteria, and in a cost effective system the use of natural solar light becomes mandatory. However, there are a number of known problems deriving from the use of natural irradiation that have recently been tackled by several Authors (Table 2). The use of sunlight is often an obligatory choice when working with large-volume systems, because artificial irradiation would represent a cost in economic and energy terms. Therefore, all the semi-pilot scale systems for hydrogen production with PNSB investigated up to now were mainly irradiated by natural sunlight, and most of them were tubular reactors. Large scale tubular reactors have been used both horizontally or inclined, but the results (Table 2) seem to demonstrate that inclining the reactor is not crucial for obtaining high hydrogen yields [52e55]. Flat panel reactors produced HPRs comparable to those attained with tubular reactors, but the highest SC was obtained by Eroglu et al. [99] with an inclined panel mixed by argon gas sparging. Though the substrate conversion seems very promising, the choice of Ar gas dramatically increases the cost of the process. Alternative gases are not very easy to find, as the most common and cheaper gases have other drawbacks. Indeed, compressed air cannot be used owing to the need to maintain anaerobic conditions inside the reactor; CO2 can be fixed by purple bacteria [31,100], deviating the electrons from the production of H2; N2 is not appropriate as nitrogenase fixes it by using the electrons otherwise needed for hydrogen production (see Box 2). Recirculating the gas produced by the cells themselves has been evaluated as a possible solution, in artificial lab scale conditions by Ref. [101]; however, it should be taken into account that H2 can be taken up by cells, thus diminishing the total production. Moreover, in a process carried out by sparging a gas different from H2 it is necessary to have a gas purification

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step. On the contrary, the gas produced naturally by purple bacteria consists predominantly of H2 (80e95% v/v). The remaining part of the gas produced is mostly CO2, which can be easily removed by bubbling the gas in an alkaline solution. For these reasons, gas sparging systems seem not to be a viable solution for mixing the cultures. Generally speaking, when H2 production is carried out outdoors using solar irradiation, LCEs are around 1%. The highest LCE reported with a solar photobioreactor containing purple bacteria (1.4%) was obtained with a flat plate reactor equipped with light shading bands [102]. The efficiency is considerably low if compared to optimized light irradiation systems like the ones reported in Table 1. However, the complete absence of energy and economic costs as a result of using sunlight has to be considered. The special feature of the system used by Wakayama and Miyake [102] was the reduction of the total amount of light irradiation reaching the cells through the use of shading bands. Miyake et al. [82] hypothesized the presence of photoinhibition in outdoors purple bacteria culturing systems, having observed a delay of 2e4 h in the maximum H2 production rate after the maximum irradiation at noon. In the same regard, Adessi et al. [52] tried to avoid photoinhibition by cutting sunlight intensity by 50% using a lightshield. HPRs were not negatively affected by this light intensity decrease; on the contrary, the maximum HPR (27.2 ml l1 h1), reached 2 h after noon, was comparable to artificial lab condition results. However, this delay in achieving the maximum production rate compared to the peak of irradiation might still indicate photoinhibition at noon. Nonetheless, this study also showed that the bacterial photosystem was able to maintain a constant photosynthetic efficiency throughout the experiment (21 days), indicating that long term exposure to solar irradiation was not affecting the functionality of the photosystem, and it was not degraded by the excess of solar radiation. This shows that purple bacteria have a very high capability to acclimate to conditions that could potentially be a source of stress, such as excessive light irradiance. Chen et al. [23] attributed to the solar photobioreactors a scarce operating stability, due to inconstant light availability; however, they are cost effective in terms of electricity consumption. A possible way to overcome the instability of light irradiation is to combine it with an artificial light supply, activated only in case of need as at night time or if scarce light irradiance occurs [83,84]; a further initial capital investment that might result in higher yields is to distribute the collected sunlight inside the reactor with optical fibers [84].

5.

Conclusions

An efficient utilization of light in photoheterotrophic hydrogen production with PNSB is still the most critical problem that needs to be solved to turn this promising biological process into a real industrial technology. This review shows that in the last years there has been an increase in the number of studies aimed at improving the design of photobioreactors for optimizing hydrogen production, especially towards large scale, cost effective production systems. However, at the same time there emerged a number of constraints that still have to be overcome to industrially exploit PNSB for photoproduction of hydrogen.

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Most of the studies published so far describe experiments carried out by using small volume, indoor photobioreactors, in which the researchers tested many ingenious solutions for increasing LCE. Unfortunately, most of these systems can only be considered as good tools for enhancing knowledge about the relationships between the processes of light absorption and hydrogen production in PNSB. Such solutions are hardly reproducible in large volume outdoor PBRs. At the same time, the reduction in light intensity aimed at increasing LCE tested by a number of Authors looks more like an ingenious trick than a real solution. Indeed, the decrease in the intensity of the light impinging on the PBR, even if it actually increases the LCE, is always coupled with a reduction in HPR and consequently in H2 productivity. In the last few years, high LCE and HPRs have been obtained by using illumination systems equipped with LEDs emitting at wavelengths corresponding to the absorption maxima of the pigments of PNSB. Even if the LCE and the HPRs reported for these illumination systems are high, it has to be stressed that these systems again can only be considered as useful tools for studying the interactions between light and hydrogen production in PNSB. Indeed, it is thermodynamic nonsense to spend energy in the form of electricity to obtain energy in the form of H2, owing to the low conversion efficiency of the process relating to the increase of the entropy of the system. On the other hand, the few papers reporting outdoor H2 production on large scale PBRs clearly show the feasibility of processes that exploit sunlight for hydrogen production with PNSB. However, considering the results reviewed in this paper and the extensive literature available on outdoor mass cultivation of microalgae and cyanobacteria (see the recent review by Molina Grima et al. [103]), in our opinion two are the main problems that still have to be addressed in order to increase LCE in the large scale outdoor processes for H2 production with PNSB. The first problem is the way to obtain a good light distribution in the PBR (defined as light dilution by Giannelli and Torzillo [104]) and the second one is the definition of efficient light dark cycles for the single cells in PBR. The first issue is related to the observation that, under natural light conditions, some layers of cells, generally those facing the light source, are usually subjected to an excess of light, while most of the other cells receive low or null illumination. Thus, working towards the so called dilution of light [104] could represent a significant contribution to increasing LCE by providing all the cells with a suitable amount of light. The second issue relates to the fact that bacterial cells, flowing in PBR, are usually subjected to a turbulent flow, which means that for part of the time they remain close to the surface of the PBR, under light conditions, and part of the time far from the surface, under dark conditions. The optimization of the light distribution in the PBR and of the light dark cycles of the single cells are, in our view, two key issues to be addressed in the near future if the production of hydrogen through photofermentation with PNSB in outdoor, large scale PBRs is to become competitive. Finally, it needs to be stressed that all the achievements in optimizing photofermentation have to be considered in a wider picture that comprises other upstream H2 production processes to be coupled with photofermentation, which cannot be regarded as a stand-alone process [105]. Moreover,

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it should also be emphasized that the choice and the costs of the materials for building the photobioreactors, in particular for large scale systems, and the costs for the management of the plants, need to be thoughtfully considered in order to make an appropriate cost benefit analysis of this process at an industrial level [106].

Acknowledgments The Authors gratefully acknowledge, for the studies coming from their lab and reported in this review, the Italian Ministry of Agricultural, Food and Forest Politics (MIPAAF; project IMERA), the Italian Ministry of the Environment (MATTM; project PIRODE), MIUR and CNR (Italian National Research Centre) (EFOR project), ECRF (Project HYDROLAB2). The Authors would also like to mention the contribution to the development of their researches on biological hydrogen given by the activities carried out by RDP in the frame of the IEA-HIA (International Energy Agency eHydrogen Implementation Agreement), Annex 21 “Bioinspired and biological hydrogen”. The Authors wish also to thank the anonymous Reviewers who contributed, with their constructive comments, to improve the text of the manuscript.

references

[1] Campen A, Mondal K, Wiltowski T. Separation of hydrogen from syngas using a regenerative system. Int J Hydrogen Energy 2008;33:332e9. [2] Konieczny A, Mondal K, Wiltowski T, Dydo P. Catalyst development for thermocatalytic decomposition of methane to hydrogen. Int J Hydrogen Energy 2008;33:264e72. [3] Balat M, Balat M. Political economic and environmental impacts of biomass-based hydrogen. Int J Hydrogen Energy 2009;34:3589e603. [4] Sen U, Shakdwipee M, Banerjee R. Status of biological hydrogen production. J Sci Ind Res India 2008;67:980e93. [5] Das D, Veziroglu TN. Advances in biological hydrogen production processes. Int J Hydrogen Energy 2008;33:6046e57. [6] Kotay SM, Das D. Biohydrogen as a renewable energy resource - prospects and potentials. Int J Hydrogen Energy 2008;33:258e63. [7] Manish S, Banerjee R. Comparison of biohydrogen production processes. Int J Hydrogen Energy 2008;33:279e86. [8] Balat M. An overview of biofuels and policies in the European Union. Energy Sources B 2007;2:167e81. [9] European Environment Agency. Environmental indicator report 2012; ecosystem resilience and resource efficiency in a green economy in Europe. Luxembourg: Publications Office of the European Union; 2012. [10] Demirbas A. Politics, economic and environmental impacts of biofuels: a review. Appl Energy 2009:S108e17. [11] Dunn S. Hydrogen futures: toward a sustainable energy system. Int J Hydrogen Energy 2002;27:235e64. [12] Yergin D. The prize: the epic quest for oil, money, and power. New York: Simon and Schuster; 1991. [13] Ngoh SK, Njomo D. An overview of hydrogen gas production from solar energy. Renew Sust Energ Rev 2012;16:6782e92.

[14] Westermann P, Jorgensen B, Lange L, Ahring BK, Christensen CH. Maximizing renewable hydrogen production from biomass in a bio/catalytic refinery. Int J Hydrogen Energy 2007;32:4135e41. [15] European hydrogen and fuel cell technology platformStrategic research agenda; 2005. [16] Lee H-S, Vermaas WFJ, Rittmann BE. Biological hydrogen production: prospects and challenges. Trends Biotechnol 2010;28:262e71. [17] McKinlay JB, Harwood CS. Photobiological production of hydrogen gas as a biofuel. Curr Opin Biotechnol 2010;21:244e51. [18] Eroglu E, Melis A. Photobiological hydrogen production: recent advances and state of the art. Bioresour Technol 2011;102:8403e13. [19] Hallenbeck PC, Abo-Hashesh M, Ghosh D. Strategies for improving biological hydrogen production. Bioresour Technol 2012;110:1e9. [20] Show KY, Lee DJ, Tay JH, Lin CY, Chang JS. Biohydrogen production: current perspectives and the way forward. Int J Hydrogen Energy 2012;37:15616e31. [21] Ugwu CU, Aoyagi H, Uchiyama H. Photobioreactors for mass cultivation of algae. Bioresour Technol 2008;99:4021e8. [22] Dasgupta CN, Gilbert JJ, Lindblad P, Heidorn T, Borgvang SA, Skjanes K, et al. Recent trends on the development of photobiological processes and photobioreactors for the improvement of hydrogen production. Int J Hydrogen Energy 2010;35:10218e38. [23] Chen C-Y, Liu C-H, Lo Y-C, Chang J-S. Perspectives on cultivation strategies and photobioreactor designs for photo-fermentative hydrogen production. Bioresour Technol 2011;102:8484e92. [24] Imhoff JF. The phototrophic alpha-proteobacteria. In: Dworkin M, Falkow S, Rosenberg E, Schleifer KH, Stackebrandt E, editors. The prokaryotes. New York: Springer; 2006. pp. 41e64. [25] Imhoff JF. The anoxygenic phototrophic purple bacteria. In: Boone DR, Castenholz RW, Garrity GM, editors. Bergey’s manual of systematic bacteriology. II ed. New York: Springer; 1995. pp. 631e7. [26] Larimer FW, Chain P, Hauser L, Lamerdin J, Malfatti S, Do L, et al. Complete genome sequence of the metabolic versatile photosynthetic bacterium Rhodopseudomonas palustris. Nat Biotechnol 2004;22:55e61. [27] Harwood CS. Degradation of aromatic compounds by purple nonsulfur bacteria. In: Hunter CD, Daldal F, Thurnauer MC, Beatty JT, editors. Advances in photosynthesis and respiration. The purple phototrophic bacteria, vol. 28. The Nederlands: Springer; 2008. pp. 577e94. [28] Masephol B, Drepper T, Paschen A, Groß S, Pawlowski A, Raabe K, et al. Regulation of nitrogen fixation in the photoautotrophic purple bacterium Rhodobacter capsulatus. J Mol Microbiol Biotechnol 2002;4:243e8. [29] Rey FE, Heiniger EK, Harwood CS. Redirection of metabolism for biological hydrogen production. Appl Environ Microbiol 2007;73:1665e71. [30] Kars G, Gunduz U. Towards a super H2 producer: Improvements in photofermentative biohydrogen production by genetic manipulations. Int J Hydrogen Energy 2010;35:6646e56. [31] McKinlay JB, Harwood CS. Carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria. PNAS 2010;107:11669e75. [32] Heiniger EK, Oda Y, Samanta SK, Harwood CS. How posttranslational modification of nitrogenase is circumvented in Rhodopseudomonas palustris strains that produce hydrogen gas constitutively. Appl Environ Microbiol 2012;78:1023e32.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 9 ( 2 0 1 4 ) 3 1 2 7 e3 1 4 1

[33] Colbeau A, Kelley BC, Vignais PM. Hydrogenase activity in Rhodopseudomonas capsulata: relationship with nitrogenase activity. J Bacteriol 1980;144:141e8. [34] Ooshima H, Takakuwa S, Katsuda T, Okuda M, Shirasawa T, Azuma M, et al. Production of hydrogen by a hydrogenase deficient mutant of Rhodobacter capsulatus. J Ferment Bioeng 1998;85:470e5. [35] Franchi E, Tosi C, Scolla G, Penna DG, Rodriguez F, Pedroni MP. Metabolically engineered Rhodobacter sphaeroides RV strains for improved biohydrogen photoproduction combined with disposal of food wastes. Marine Biotechnol 2004;6:552e65. [36] Kim MS, Baek JS, Lee JK. Comparison of H2 accumulation by Rhodobacter sphaeroides KD131 and its uptake hydrogenase and PHB synthase deficient mutant. Int J Hydrogen Energy 2006;31:121e7. ¨ ztu¨rk Y, Yu¨cel M, Daldal F, Mandacı S, Gu¨ndu¨z U, Tu¨rker L, [37] O et al. Hydrogen production by using Rhodobacter capsulatus mutants with genetically modified electron transfer chains. Int J Hydrogen Energy 2006;31:1545e52.  lu I, Kovacs LK. [38] Kars G, Gu¨ndu¨z U, Rakhely G, Yu¨cel M, Erog Improved hydrogen production by hydrogenase deficient mutant strain of Rhodobacter sphaeroides O.U.001. Int J Hydrogen Energy 2008;33:3056e60. [39] Androga DD, Ozgur E, Eroglu I, Gunduz U, Yucel M. Significance of carbon to nitrogen ratio on the long-term stability of continuous photofermentative hydrogen production. Int J Hydrogen Energy 2011;36:15583e94. [40] Keskin T, Abo-Hashesh M, Hallenbeck PC. Photofermentative hydrogen production from wastes. Bioresour Technol 2011;102:8557e68. [41] Abo-Hashesh M, Ghosh D, Tourigny A, Taous A, Hallenbeck PC. Single stage photofermentative hydrogen production from glucose: an attractive alternative to two stage photofermentation or co-culture approaches. Int J Hydrogen Energy 2011;36:13889e95. [42] Keskin T, Hallenbeck PC. Hydrogen production from sugar industry waste using single-stage photofermentation. Bioresour Technol 2012;112:131e6. [43] Basak N, Das D. The prospect of purple nonesulfur (PNS) photosynthetic bacteria for hydrogen production: the present state of the art. World J Microb Biot 2007;23:31e42. [44] Kapdan IK, Kargi F. Bio-hydrogen production from waste material. Enzyme Microb Technol 2006;38:569e82. [45] Redwood MD, Paterson-Beedle M, Macaskie LE. Integrating dark and light bio-hydrogen production strategies: towards the hydrogen economy. Rev Environ Sci Biotechnol 2009;8:149e85. [46] Gomez X, Fernandez C, Sanchez ME, Escapa A, Moran A. Hydrogen production: two stage process for waste degradation. Bioresour Technol 2011;102:8621e7. [47] Argun H, Kargi F. Bio-hydrogen production by different operational modes of dark and photo-fermentation: an overview. Int J Hydrogen Energy 2011;36:7443e59. [48] Adessi A, De Philippis R, Hallenbeck PC. Combined systems for maximum substrate conversion. In: Hallenbeck PC, editor. Microbial technologies in advanced biofuels production. New York USA: Springer ScienceþBusiness Media; 2012. pp. 107e26. [49] Wu TY, Hay JXW, Kong LB, Juan JC. Recent advances in reuse of waste material as substrate to produce biohydrogen by purple non-sulfur (PNS) bacteria. Renew Sust Energ Rev 2012;16:3117e22. [50] Janssen M, de Bresser L, Baijens T, Tramper J, Mur LR, Snel JFH, et al. Scale-up aspects of photobioreactors: effects of mixing-induced light/dark cycles. J Appl Phycol 2000;12:225e37.

3139

[51] Tsygankov AA. Laboratory scale photobioreactors. Appl Biochem Microbiol 2001;37:333e41. [52] Adessi A, Torzillo G, Baccetti E, De Philippis R. Sustained outdoor H2 production with Rhodopseudomonas palustris cultures in a 50 L tubular photobioreactor. Int J Hydrogen Energy 2012;37:8840e9. [53] Gebicki J, Modigell M, Schumacher M, Van Der Burg J, Roebroeck E. Development of photobioreactors for anoxygenic production of hydrogen by purple bacteria. Chem Eng Transactions 2009;18:363e6. ¨ zgu¨r E, Van der Burg J, Yu¨cel M, Gu¨ndu¨z U, Eroglu I. [54] Boran E, O Biological hydrogen production by Rhodobacter capsulatus in solar tubular photobioreactor. J Clean Prod 2010;18:S29e35. ¨ zgu¨r E, Yu¨cel M, Gu¨ndu¨z U, Eroglu I. Biohydrogen [55] Boran E, O production by Rhodobacter capsulatus Hup- mutant in pilot solar tubular photobioreactor. Int J Hydrogen Energy 2012;37:16437e45. [56] Eroglu I, Aslan K, Gunduz U, Yucel M, Turker L. Continuous hydrogen production by Rhodobacter sphaeroides O.U.001. In: Zaborsky OR, editor. Biohydrogen. London: Plenum Press; 1998. pp. 143e51. [57] Akkerman I, Janssen M, Rocha J, Wijffels RH. Photobiological hydrogen production: photochemical efficiency and bioreactor design. Int J Hydrogen Energy 2002;27:1195e208. [58] Ismail KSK, Najafpour G, Younesi H, Mohamed AR, Kamaruddin AH. Biological hydrogen production from CO: bioreactor performance. Biochem Eng J 2008;39:468e77. [59] Basak N, Das D. Photofermentative hydrogen production using purple non-sulfur bacteria Rhodobacter sphaeroides O.U.001 in an annular photobioreactor: a case study. Biomass Bioenergy 2009;33:911e9. [60] Vincenzini M, Balloni W, Mannelli D, Florenzano G. A bioreactor for continuous treatment of waste waters with immobilized cells of photosynthetic bacteria. Experientia 1981;37:710e1. [61] Hallenbeck PC. Immobilized microorganisms for hydrogen and ammonia production. Enzyme Microb Technol 1983;5:171e80. [62] Francou N, Vignais PM. Hydrogen production by Rhodopseudomonas capsulata cells entrapped in carrageenan beads. Biotechnol Lett 1984;6:639e44. [63] Tsygankov AA, Hirata Y, Miyake M, Asada Y, Miyake J. Photobioreactor with photosynthetic bacteria immobilized on porous glass for hydrogen photoproduction. J Ferment Bioeng 1994;77:575e8. [64] Fißler J, Kohring GW, Giffhorn F. Enhanced hydrogen production from aromatic acids by immobilized cells of Rhodopseudomonas palustris. Appl Biochem Biotechnol 1995;44:43e6. [65] Nakada E, Nishikata S, Asada Y, Miyake J. Hydrogen production by gel-immobilized cells of Rhodobacter sphaeroidesddistribution of cells, pigments, and hydrogen evolution. J Mar Biotechnol 1996;4:38e42. [66] Gosse JL, Engel BJ, Rey FE, Harwood CS, Scriven LE, Flickinger MC. Hydrogen production by photoreactive nanoporous latex coatings of nongrowing Rhodopseudomonas palustris CGA009. Biotechnol Prog 2007;23:124e30. [67] Xie G-J, Liu B-F, Ding J, Xing D-F, Ren H-Y, Guo W-G, et al. Enhanced photo-H2 production by Rhodopseudomonas faecalis RLD-53 immobilization on activated carbon fibers. Biomass Bioenerg 2012;44:122e9. [68] Wang Y-Z, Liao Q, Zhu X, Tian X, Zhang C. Characteristics of hydrogen production and substrate consumption of Rhodopseudomonas palustris CQK 01 in an immobilized-cell photobioreactor. Bioresour Technol 2010;101:4034e41. [69] Wang Y-Z, Liao Q, Zhu X, Chen R, Guo C-L, Zhou J. Bioconversion characteristics of R.palustris CQK 01

3140

[70]

[71]

[72]

[73]

[74]

[75]

[76]

[77]

[78]

[79]

[80]

[81]

[82]

[83]

[84]

[85]

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 9 ( 2 0 1 4 ) 3 1 2 7 e3 1 4 1

entrapped in a photobioreactor for hydrogen production. Bioresour Technol 2012;135:331e8. Tian X, Liao Q, Zhu X, Wang Y, Zhang P, Li H, et al. Characteristics of a biofilm photobioreactor as applied to photo-hydrogen production. Bioresour Technol 2010;101:977e83. Zhang C, Zhu X, Liao Q, Wang Y, Li J, Ding Y, et al. Performance of a groove-type photobioreactor for hydrogen production by immobilized photosynthetic bacteria. Int J Hydrogen Energy 2010;35:5284e92. Guo C-L, Zhu X, Liao Q, Wang Y-Z, Chen R, Lee D-J. Enhancement of photo-hydrogen production in a biofilm photobioreactor using optical fiber with additional rough surface. Bioresour Technol 2011;102:8507e13. Dagher SF, Ragout AL, Sineriz F, Bruno-Barcena JM. Cell immobilization for production of lactic acid: biofilms do it naturally. In: Laskin AI, Sariaslani S, Gadd GM, editors. Advances in applied microbiologyVol 71. San Diego, USA: Elsevier Academic Press Inc.; 2010. pp. 113e48. Smirnova TA, Didenko LV, Azizbekyan RR, Romanova YM. Structural and functional characteristics of bacterial biofilms. Microbiology 2010;79:413e23. Gadhamshetty V, Sukumaran A, Nirmalakhandan N. Photoparameters in photofermentative biohydrogen production. Environ Sci Technol 2011;41:1e51. Hoff AJ, Deisenhofer J. Photophysics of photosynthesis. Structure and spectroscopy of reaction centers of purple bacteria. Phys Rep 1997;287:1e247. Frank HA, Polı´vka T. Energy transfer from carotenoids to bacteriochlorophylls. In: Hunter CD, Daldal F, Thurnauer MC, Beatty JT, editors. Advances in photosynthesis and respiration. The purple phototrophic bacteria, vol. 28. The Nederlands: Springer; 2008. pp. 213e30. Loach PA, Parker-Loach PS. Structure-function relationships in bacterial light-harvesting complexes investigated by reconstitution techniques. In: Hunter CD, Daldal F, Thurnauer MC, Beatty JT, editors. Advances in photosynthesis and respiration. The purple phototrophic bacteria, vol. 28. The Nederlands: Springer; 2008. pp. 181e98. Robert B. Spectroscopic properties of antenna complexes from purple bacteria. In: Hunter CD, Daldal F, Thurnauer MC, Beatty JT, editors. Advances in photosynthesis and respiration. The purple phototrophic bacteria, vol. 28. The Nederlands: Springer; 2008. pp. 199e212. Adessi A, De Philippis R. Hydrogen production: photofermentation. In: Hallenbeck PC, editor. Microbial technologies in advanced biofuels production. New York USA: Springer ScienceþBusiness Media; 2012. pp. 53e75. Kawagoshi Y, Oki Y, Nakano I, Fujimoto A, Takahashi H. Biohydrogen production by isolated halotolerant photosynthetic bacteria using long-wavelength light-emitting diode (LW-LED). Int J Hydrogen Energy 2010;35:13365e9. Miyake J, Wakayama T, Schnackenberg J, Arai T, Asada Y. Simulation of the daily sunlight illumination pattern for bacterial photo-hydrogen production. J Biosci Bioeng 1999;88:659e63. Ogbonna JC, Soejima T, Tanaka H. An integrated solar and artificial light system for internal illumination of photobioreactors. J Biotechnol 1999;70:289e97. Chen C-Y, Saratale GD, Lee C-M, Chen P-C, Chang J-S. Phototrophic hydrogen production in photobioreactors coupled with solar energy excited optical fibers. Int J Hydrogen Energy 2008;33:6686e95. Miyake J, Kawamura S. Efficiency of light energy conversion to hydrogen by the photosynthetic bacterium Rhodobacter sphaeroides. Int J Hydrogen Energy 1987;3:147e9.

[86] Kondo T, Arakawa M, Wakayama T, Miyake J. Hydrogen production by combining two types of photosynthetic bacteria with different characteristics. Int J Hydrogen Energy 2002;27:1303e8. [87] Carlozzi P, Sacchi A. Biomass production and studies on Rhodopseudomonas palustris grown in an outdoor, temperature controlled, underwater tubular photobioreactor. J Biotechnol 2001;88:239e49. [88] Go¨bel F. Quantum efficiencies of growth. In: Clayton RK, Sistrom WR, editors. Photosynthetic bacteria. New York: Plenum Press; 1978. pp. 907e25. [89] Sakurai H, Masukawa H, Kitashima M, Inoue K. Photobiological hydrogen production: bioenergetics and challenges for its practical application. J Photochem Photobiol C 2013;17:1e25. [90] Miyake J. The science of biohydrogen. In: Zaborsky OR, editor. Biohydrogen. London: Plenum Press; 1998. pp. 7e18. [91] Ghirardi ML, Dubini A, Yu J, Maness P-C. Photobiological hydrogen-producing systems. Chem Soc Rev 2009;38:52e61. [92] Liao Q, Wamg Y-J, Wang Y-Z, Zhu X, Tian X, Li J. Formation and hydrogen production of photosynthetic bacterial biofilm under various illumination conditions. Bioresour Technol 2010;101:5315e24. [93] El-Shishtawy RMA, Kawasaki S, Morimoto M. Biological H2 production using a novel light-induced and diffused photoreactor. Biotechnol Techniq 1997;11:403e7. [94] Gilbert JJ, Ray S, Das D. Hydrogen production using Rhodobacter sphaeroides (O.U.001) in a flat panel rocking photobioreactor. Int J Hydrogen Energy 2011;36:3434e41. [95] Otzuki T, Uchiyama S, Fujiki K, Fukunaga S. Hydrogen production by a floating-type photobioreactor. In: Zaborsky OR, editor. Biohydrogen. London: Plenum Press; 1998. pp. 369e74. [96] Kondo T, Wakayama T, Miyake J. Efficient hydrogen production using a multi-layered photobioreactor and a photosynthetic bacterium mutant with reduced pigment. Int J Hydrogen Energy 2006;31:1522e6. [97] Yamada A, Hatano T, Matsunaga T. Conversion efficiencies of light energy to hydrogen by a novel Rhodovulum sp. And its uptake-hydrogenase mutant. In: Zaborsky OR, editor. Biohydrogen. London: Plenum press; 1998. pp. 167e237. [98] Chen C-Y, Chang J-S. Enhancing phototrophic hydrogen production by solid-carrier arrested fermentation and internal optical-fiber illumination. Process Biochem 2006;41:2041e9. [99] Eroglu I, Tabanoglu A, Gunduz U, Eroglu E, Yucel M. Hydrogen production by Rhodobacter sphaeroides O.U.001 in a flat plate solar bioreactor. Int J Hydrogen Energy 2008;33:531e41. [100] Tabita FR. The biochemistry and metabolic regulation of carbon metabolism and CO2 fixation in purple bacteria. In: Blankenship RE, Madigan MT, Bauer CE, editors. Anoxygenic photosynthetic bacteria. The Nederlands: Kluwer Academic Publishers; 1995. pp. 885e914. [101] Hoekema S, Bijmans M, Janssen M, Tramper J, Wijffels RH. A pneumatically acitated flat-panel photobioreactor with gas re-circultaion: anaerobic photoheterotrophic cultivation of a purple non-sulfur bacterium. Int J Hydrogen Energy 2002;27:1331e8. [102] Wakayama T, Miyake J. Light shade bands for the improvement of solar hydrogen production efficiency by Rhodobacter sphaeroides RV. Int J Hydrogen Energy 2002;27:1495e500. [103] Molina Grima E, Ferna´ndez Sevilla JM, Acien Fernandez FG. Microalgae, mass culture methods. In: Flickinger MC, editor. Encyclopedia of industrial biotechnology: bioprocess, bioseparation, and cell technology. USA: Wiley and Sons Inc; 2010. pp. 1e24.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 9 ( 2 0 1 4 ) 3 1 2 7 e3 1 4 1

[104] Giannelli L, Torzillo G. Hydrogen production with the microalga Chlamydomonas reinhardtii grown in a compact tubular photobioreactor immersed in a scattering light nanoparticle suspension. Int J Hydrogen Energy 2012;37:16951e61. [105] Patel SKS, Kumar P, Kali VC. Enhancing biological hydrogen production through complementary microbial

3141

metabolisms. Int J Hydrogen Energy 2012;37:10590e 603. [106] Lijunggren M, Zacchi G. Techno-economic evaluation of a two-step biological process for hydrogen production. Biotechnol Prog 2010;26:496e504.