Meng Ni, Dennis Y.C. Leung *, Michael K.H. Leung, K. Sumathy, “An overview of hydrogen production from biomass”, Fuel Processing Technology 87 (2006) 461 – 472

The phenomenon of biological hydrogen production was observed one century ago. When the

oil crisis broke out in 1970s, the technology started receiving attention, especially in

hydrogen production by photosynthetic process. However, these works are in laboratory scale

and the practical applications still need to be demonstrated. Biological hydrogen

production can be classified into five different groups: (i) direct biophotolysis, (ii) indirect

biophotolysis, (iii) biological water–gas shift reaction, (iv) photo-fermentation and (v)

darkfermentation [66]. All processes are controlled by the hydrogen-producing enzymes, such

as hydrogenase and nitrogenase. The major components of nitrogenase are MoFe protein and

Fe protein. Nitrogenase has the ability to use magnesium adenosine triphosphate (MgATP)

and electrons to reduce a variety of substrates (including protons). This chemical reaction

yields hydrogen production by a nitrogenase-based system [67]:

where ADP and Pi refer to adenosine diphosphate and inorganic phosphate, respectively.

Hydrogenases exist in most of the photosynthetic microorganisms and they can be classified

into two categories: (i) uptake hydrogenases and (ii) reversible hydrogenases. Uptake

hydrogenases, such as NiFe hydrogenases and NiFeSe hydro-genases, act as important

catalysts for hydrogen consumption as follows:

Reversible hydrogenases, as indicated by its name, have the ability to produce H2 as well as

consume H2 depending on the reaction condition,

Koku et al. [68] have reported that though a variety of substrates can be used for Rhodobacter

sphaeroides growth, only some of them are suitable for hydrogen production. The properties

of nitrogenase and hydrogenase are summarized in Table 4.

4.2. Direct biophotolysis

Direct biophotolysis of hydrogen production is a biological process using microalgae

photosynthetic systems to convert solar energy into chemical energy in the form of hydrogen:

Two photosynthetic systems are responsible for photosynthesis process: (i) photosystem I

(PSI) producing reductant for CO2 reduction and (ii) photosystem II (PSII) splitting water and

evolving oxygen. In the biophotolysis process, two photons from water can yield either CO2

reduction by PSI or hydrogen formation with the presence of hydrogenase. In green plants,

due to the lack of hydrogenase, only CO2 reduction takes place.

On the contrary, microalgaes, such as green algae and Cyanobacteria (blue-green algae),

contain hydrogenase and, thus, have the ability to produce hydrogen. In this process, electrons

are generated when PSII absorbs light energy. The electrons are then transferred to the

ferredoxin (Fd) using the solar energy absorbed by PSI. The hydrogenase accepts the

electrons from Fd to produce hydrogen as shown in Fig. 4.

Since hydrogenase is sensitive to oxygen, it is necessary to maintain the oxygen content at a

low level under 0.1% so that hydrogen production can be sustained [67]. This condition can

be obtained by the use of green algae Chlamydomonas reinhardtii that can deplete oxygen

during oxidative respiration [69]. However, due to the significant amount of substrate being

respired and consumed during this process, the efficiency is low. Recently, mutants derived

from microalgae were reported to have good O2 tolerance and thus higher hydrogen

production. The works reported on mutants for hydrogen production are listed in Table 5 [70–

74]. It can be seen that, using mutants for hydrogen production, the efficiency can be

increased significantly. Benemann [75] estimated the cost of direct biophotolysis for

hydrogen production to be $20/GJ assuming that the capital cost is about US$60/m2 with an

overall solar conversion efficiency of 10%. Hallenbeck and Benemann [67] performed

similar cost estimation and reported that the capital cost of US$100/m2. In their estimation,

some practical factors were neglected, such as gas separation and handling.

4.3. Indirect biophotolysis

According to Gaudernack [76], the concept of indirect biophotolysis involves the following

four steps as illustrated in Fig. 5: (i) biomass production by photosynthesis, (ii) biomass

concentration, (iii) aerobic dark fermentation yielding 4 mol hydrogen/mol glucose in the

algae cell, along with 2 mol of acetates, and (iv) conversion of 2 mol of acetates into

hydrogen. In a typical indirect biophotolysis, Cyanobacteria are used to produce hydrogen via

the following reactions [66]:

Markov et al. [77] investigated the indirect biophotolysis with Cyanobacterium anabaena

variabilis exposed to light intensities of 45–55 Amol_1 m_2 and 170–180 Amol_1 m_2

in the first stage and second stage, respectively. Photoproduction of hydrogen at a rate of

about 12.5 ml H2/gcdw h (cdw: cell dry weight) was found. In the study on indirect

biophotolysis with Cyanobacterium gloeocapsa alpicola by Troshina et al. [78], it was found

that maintaining the medium at pH value between 6.8 and 8.3 yielded optimal hydrogen

production. Increasing the temperature from 30 -C to 40 -C can increase the hydrogen

production twice as much. The hydrogen production rate through indirect biophotolysis is

comparable to hydrogenase-based hydrogen production by green algae. The estimated overall

cost is US$10/GJ of hydrogen [67]. However, it should be pointed out that indirect

biophotolysis technology is still under active research and development. The estimated

cost is subject to a significant change depending on the technological advancement.

4.4. Biological water–gas shift reaction

Some photoheterotrophic bacteria, such as Rhodospirillum rubrum can survive in the dark by

using CO as the sole carbon source to generate ATP by coupling the oxidation of CO to the

reduction of H+ to H2 [79]:

In equilibrium, the dominating products are CO2 and H2. Therefore, this process is favorable

for hydrogen production. Organisms growing at the expense of this process are the gram-

negative bacteria, such as R. rubrum and Rubrivax gelatinosus, and the gram-positive

bacteria, such as Carboxydothermus hydrogenoformans [80]. Under anaerobic conditions, CO

induces the synthesis of several proteins, including CO dehydrogenase, Fe–S protein and CO-

tolerant hydrogenase. Electrons produced from CO oxidation are conveyed via the Fe–S

protein to the hydrogenase for hydrogen production [81].

Biological water–gas shift reaction for hydrogen production is still under laboratory scale and

only few works have been reported. The common objectives of these works were to identify

suitable microorganisms that had high CO uptake and to estimate the hydrogen production

rate. Kerby et al. [79] observed that under dark, anaerobic conditions in the presence of

sufficient nickel, the doubling time of R. rubrum was less than 5 h by the oxidation of CO to

CO2 coupled with the reduction of protons to hydrogen. However, R. rubrum requires light to

grow and hydrogen production is inhibited by medium CO partial pressure above 0.2 atm. An

alternative new chemoheterotrophic bacterium Citrobacter sp. Y19 was tested by Jung et al.

[82] for hydrogen production using water–gas shift reaction. The maximum hydrogen

production activity was found to be 27 mmol/g cell h, which is about three times higher

than R. rubrum. Recently, Wolfrum et al. [83] have conducted a detailed study to compare the

biological water–gas shift reaction with conventional water–gas shift processes. Their analysis

showed that biological water–gas shift process was economically competitive when the

methane concentration was under 3%. The hydrogen production cost from biological water–

gas shift reaction ranged from US$1.75/kg (US$14.6/GJ) to around US$2.25/kg

(US$18.8/GJ) for a methane concentration between 1% and 10%. Compared with

thermochemical water– gas shift processes, the cost of biological water–gas shift processes

are lower due to the elimination of reformer and associated equipment.

4.5. Photo-fermentation

Photosynthetic bacteria have the capacity to produce hydrogen through the action of their

nitrogenase using solar energy and organic acids or biomass. This process is known as photo-

fermentation, as shown in Fig. 6. In recent years, some attempts have been made for hydrogen

production from industrial and agricultural wastes to effect waste management.

As summarized in Table 6 [84–87], hydrogen can be produced by photo-fermentation of

various types of biomass wastes. However, these processes have three main drawbacks: (i)

use of nitrogenase enzyme with high-energy demand, (ii) low solar energy conversion

efficiency and (iii) demand for elaborate anaerobic photobioreactors covering large areas [84].

Hence, at the present time, photo-fermentation process is not a competitive method for

hydrogen production.

4.6. Dark fermentation

Fermentation by anaerobic bacteria as well as some microalgaes, such as green algae on

carbohydrate-rich substrates, can produce hydrogen at 30 -C to 80 -C especially in a dark

condition [88]. This process using dark fermentation for hydrogen production is illustrated in

Fig. 7. Unlike a biophotolysis process that produces only H2, the products of dark

fermentation are mostly H2 and CO2 combined with other gases, such as CH4 or H2S,

depending on the reaction process and the substrate used. With glucose as the model

substrate, maximum 4 mol H2 is produced per mole glucose when the end product is acetic

acid:

However, in practice, the 4 mol H2 production/mol glucose cannot be achieved because the

end products normally contain both acetate and butyrate [89].

The amount of hydrogen production by dark fermentation highly depends on the pH value,

hydraulic retention time (HRT) and gas partial pressure. For the optimal hydrogen production,

pH should be maintained between 5 and 6 [90– 92]. Partial pressure of H2 is yet another

important parameter affecting the hydrogen production. When hydrogen concentration

increases, the metabolic pathways shift to produce more reduced substrates, such as lactate,

ethanol, acetone, butanol or alanine, which in turn decrease the hydrogen production [93].

Besides the pH value and partial pressure, HRT also plays an important role in hydrogen

production. Ueno et al. [94] have reported that an optimal HRT of 0.5 day could effect

maximum hydrogen production (14 mmol/g carbohydrate) from wastewater by anaerobic

microflora in the presence of chemostat culture. When HRT was increased from 0.5 day to 3

days, hydrogen production rate was reduced from 198 to 34 mmol l_1 day_1, while the

carbohydrates in the wastewater were decomposed at an increasing efficiency from 70% to

97%. Due to the fact that solar radiation is not a requirement, hydrogen production by dark

fermentation does not demand much land and is not affected by the weather condition. Hence,

the feasibility of the technology yields a growing commercial value.

C.C. Wang a, C.W Chang a, C.P. Chu a, D.J. Lee a,*, B.-V. Chang b, C.S. Liao b „ Producing hydrogen from wastewater sludge by Clostridium Bifermentans „ , Journal of Biotechnology 102 (2003) 83_/92

Excess wastewater sludge collected from the recycling stream of an activated sludge process

is biomass that contains large quantities of polysaccharides and proteins. However, relevant

literature indicates that the bio-conversion of wastewater sludge to hydrogen is limited and

therefore not economically feasible. This work examined the anaerobic digestion of

wastewater sludge using a clostridium strain isolated from the sludge as inoculum. A much

higher hydrogen yield than presented in the literature was obtained. Also, the effects of five

pre-treatments*/ultrasonication, acidification, sterilization, freezing/thawing and adding

methanogenic inhibitor*/on the production of hydrogen were examined. Freezing and thawing

and sterilization increased the specific hydrogen yield by 1.5_/2.5 times to that of untreated

sludge, while adding an inhibitor and ultrasonication reduced the hydrogen yield.

G. ANTONOPOULOU,I. NTAIKOU, H.N. GAVALA,, I.V. SKIADAS,K. ANGELOPOULOS, G. LYBERATOS, „BIOHYDROGEN PRODUCTION FROM SWEET SORGHUM BIOMASS USING MIXED ACIDOGENIC CULTURES AND PURE CULTURES OF RUMINOCOCCUS ALBUS” , Global NEST Journal, Vol 9, No 2, pp 144-151, 2007

The present study focuses on the exploitation of sweet sorghum biomass as a source for

hydrogen in continuous and batch systems. Sweet sorghum is an annual C4 plant of tropical

origin, well-adapted to sub-tropical and temperate regions and highly productive in biomass.

Sweet sorghum biomass is rich in readily fermentable sugars and thus it can be considered

as an excellent raw material for fermentative hydrogen production. Extraction of free sugars

from the sorghum stalks was achieved using water at 30°C. After the extraction process, a

liquid fraction (sorghum extract), rich in sucrose, and a solid fraction (sorghum

cellulosichemicellulosic residues), containing the cellulose and hemicelluloses, were obtained.

Hydrogen production from sorghum extract was investigated using mixed acidogenic

microbial cultures, coming from the indigenous sorghum microflora and Ruminococcus albus,

an important, fibrolytic bacterium of the rumen. Hydrogen productivity of sorghum residues

was assessed as well, using R. albus.

The highest hydrogen yield obtained from sorghum extract fermented with mixed microbial

cultures in continuous system was 0.86 mol hydrogen per mol of glucose consumed, at a

hydraulic retention time of 12 hours. This corresponded to a hydrogen productivity of 10.4 l

hydrogen per kg of sorghum biomass and was comparable with those obtained from batch

experiments. On the other hand, the hydrogen yield obtained from sorghum extract treated

with R. albus was as high as 2.1-2.6 mol hydrogen per mol of glucose consumed. Hydrogen

productivity of sorghum residues fermented with R. albus reached 2.6 mol hydrogen per mol

of glucose consumed. In total, the productivity of sorghum biomass (that of sorghum extract

plus that of sorghum residues) could be 60 l hydrogen per kg of sorghum biomass if R. albus

is used.

Laurent Beckers, Serge Hiligsmann, Christopher Hamilton, Julien Masset, Philippe Thonart, „Fermentative hydrogen production by Clostridiumbutyricum CWBI1009 and Citrobacter freundii CWBI952in pure and mixed cultures”, Biotechnol. Agron. Soc. Environ. 2010 14(S2), 541-548

Hydrogen (H2), whether burned or used directly in a fuel cell, is a very promising clean

energy vector for the decrease of our environmental impact since its utilization generates only

water vapor. Nevertheless, H2 is still mainly produced by steam reforming of methane, a

process releasing large amount of fossil CO2 in the atmosphere. In the last few years, there

has been an increasing interest to find new H2 production processes with almost no carbon

emission (Balat, 2009; Holladay et al., 2009; Moriarty et al., 2009). One of the most

promising and investigated prospects is the biological production of hydrogen through the

degradation of a large spectrum of carbon sources by anaerobic bacteria in a process called

“dark fermentation” (Das et al., 2001; Levin et al., 2004; Nath et al., 2004a; Das, 2009;

Hallenbeck, 2009).The best described mesophilic strains are, on the one hand, strict anaerobic

bacteria from the genus Clostridium that have the potential to reach high experimental

hydrogen yields (about two moles of hydrogen per mole of hexose consumed). And on the

other hand, facultative anaerobes such as Enterobacteriaceae that present lower experimental

yields (~ 1 molH2.molhexose) but can achieve higher production rates (Hallenbeck et al.,

2002; Hawkes et al., 2002; Kotay et al., 2008). The main purpose to enhance fermentative

hydrogen production is to improve hydrogen yields for an efficient energy recovery from the

substrate. The two species investigated in this work, Clostridium butyricum CWBI1009

(Masset et al., 2010) and Citrobacter freundii CWBI952 (Hamilton et al., 2010), have a

maximum theoretical hydrogen yield of 4 and 2 molH2.molhexose -1 respectively depending

on the metabolic pathway followed for the fermentation of the carbon source (Nandi et al.,

1998; Nath et al., 2004b; Kraemer et al., 2007; Oh et al., 2008a). Clostridia are however

extremely sensitive to the presence of oxygen which strongly inhibits H2 evolving enzymes

(Heinekey, 2009). This can be avoided with the addition of an expensive reducing agent such

as L-cysteine. However, the use of such an agent is not suitable for a large-scale cost effective

biohydrogen production process (Das et al., 2008; Yuan et al., 2008).

Hydrogen may evolve through the fermentation processes of simple carbohydrates such as

glucose, sucrose, lactose and maltose or more complex ones such as starch or even cellulose

(Ueno et al., 1995; Davila- Vazquez et al., 2008; Magnusson et al., 2008). Only a few studies

have investigated the hydrogen production with these different substrates on pure cultures in

comparison with co-cultures (Yokoi et al., 1995; Nath et al., 2006; Chen et al., 2008; Pan et

al., 2008). This is why this work compares the hydrogen and major metabolites production

(i.e. acetate, butyrate, formate, lactate, ethanol and succinate) in pure C. freundii and Cl.

butyricum cultures with five different substrates. These experiments were carried out in serum

bottles batch cultures based on the biochemical hydrogen potential (BHP) test procedure

described by Lin (Lin et al., 2007). Furthermore, a co-culture of C. freundii and Cl. butyricum

in the same BHP culture may enhance hydrogen production. Such a culture would not require

the addition of any reducing agents since C. Freundii consumes oxygen and provides the

anaerobic conditions required for Cl. butyricum growth. This has already been shown in a

mixed culture of Enterobacter sp. and Cl. butyricum growing on starch (Yokoi et al., 1998;

2002). However, little is known about this consortium on other substrates. In this work, co-

cultures were monitored on glucose and also on starch for comparison. They are discussed in

comparison with the results found in the literature.

X. Gómez !, C. Fernández, J. Fierro, M.E. Sánchez, A. Escapa, A. Morán, „Hydrogen production: Two stage processes for waste degradation”, Bioresource Technology 102 (2011) 8621–8627

Conventional methods for producing H2 gas include: steam reforming of methane and hydrocarbons, non-catalytic partial oxidation of fossil fuels and auto-thermal reforming. These methods are all energy intensive processes requiring high temperatures (>850 "C) (Kapdan and Kargi, 2006). In this way, when it is taken into account that a continuous increase is expected in the demand for the use of H2, the main goal in the near future should be to attain cost-effective production processes from renewable sources. Among the processes currently available to fulfil this objective, it is worthwhile mentioning the production of H2 by thermal processing of biomass, as also biomass pyrolysis and gasification through the steam reforming of gases by water–gas shift reactions. These methods present the main advantage of being capable of treating biomass with a high lignocellulosic content, thus avoiding market distortions, which were observed in the past because of an expectation of increasing demand for raw materials. Biological methods of hydrogen production are preferable over chemical methods because they offer the possibility of using sunlight, CO2 and organic wastes as substrates. These methods are considered environmentally benign conversions, which take place under moderate conditions (Redwood et al., 2009). The biological methods for generating H2 include light-dependent methods, such as direct and indirect biophotolysis and photo-fermentation. The other routes for biological production are not light-dependentmethods. In this category are included the dark fermentation process, bioelectrochemical systems (BES) and water–gas shift reaction mediated by photoheterotrophic bacteria. However, the need of light of this latter process for microbial growth and the use of CO as a carbon source may seem to be technical and economic barriers with regard to the reactor design and the thermal pre-treatment step necessary for the generation of CO. Ustak et al. (2007) compared two different biological methods for hydrogen production: fermentative and photosynthetic based upon the modality of batch cultures. For testing fermentative bio-hydrogen production, four mixed cultures representing anaerobic microorganisms (dominant

strain Clostridium) were selected. The testing of green algae proved that the most effective was the algae species Scenedesmus. High bio-hydrogen purity was analytically verified. Thefermentative method of H2 production was more efficient; it does not need light, has a longer efficiency of a single charge and enables effective use of different biological wastes.However, production of hydrogen from either of the methods previously commented upon is characterised by low efficiency, this acting as a barrier for commercial application. Several disadvantages of these methods have been stated by Das and Vezirog˘lub (2001). A fact of great relevance is the small scale at which most of these technologies have been tested, with the dark fermentation process being the only one with a prospect of being rapidly scaled upowing to its similarity to the well-known anaerobic digestion process. In addition, this process can use residual carbohydraterich biomass as substrate, which makes the process an alternative for the conventional biological methods for treating and taking full advantage of this biomass. The process itself may be seen as a pretreatment step in a complete stabilisation chain. In the present review, a description of successful experiences for the production of hydrogen through dark fermentation from residual biomass was undertaken. As dark fermentation has a maximum yield of 33% (on sugars), a description is also presented of possible second stage processes for further degradation of dark fermentation effluents (mainly containing fatty acids). Alternatives for the second stage considered were photofermentation and BES as processes capable of converting fermentation sub-products into H2. Anaerobic digestion as a final stabilisation stage was also considered, owing to the wide application of this technology in the treatment of bio-wastes.The process is then characterised by low efficiency and this also becomes the main limiting factor for commercial application. Only 33% of the chemical oxygen demand (COD) contained in the wastecan be transformed into hydrogen (considering glucose). The remainder is mainly composed of volatile fatty acids (VFAs – acetate, butyrate) (Bartacek et al., 2007). Nonetheless, the formation of caproate has also been reported in dark fermentation systems,with the presence of this acid being coupled to the production of H2. The generation of caproate does not translates into a higher H2 yield since its formation has been explained by the secondary fermentation of two substrates, either ethanol and acetate or ethanol and butyrate. Thus, the appearance of caproate in the final products is an indication that significant solventogenesis has occurred and thus the yield of the fermentative H2 production is poor (Ding et al., 2010). Thus, considering the composition of the effluent stream of the dark fermentation process, direct biological production of H2 appears to be restricted to a pre-treatment step in a larger bio-energy or biochemical production concept (Angenentet al., 2004).

Improving H2 yieldsStrategies for improving H2 yields have been reviewed by Kraemer and Bagley (2007) analysing diverse alternatives, such as inoculum pre-treatment, gas sparging, reduction of H2 and CO2 levels in the liquid phase, and varying organic loading rates. Several options successfully tested for obtaining active hydrogen producing microflora are listed in Table 2. These pre-treatments may regularly be applied to reactors in order to maintain the activity of H2 producing microorganisms. However, when considering large scale implementations, some of these pre-treatment options may increase operating costs, as it is the case of heat shock on startingup the reactor or from ongoing heat treatment as proposed by Khanal et al. (2006) for maintaining an active H2 producing population during extended operation. In addition, differences in hydrogen yields have proved to disappear in continuous experimentations which indicate that pre-treatment methods have only short-term effects on hydrogen production (Luo et al., 2010). Another relevant aspect that should be considered is the inhibition caused by the accumulation of fermentation products, with this being

detrimental to H2 yield. Operating alternatives associated with the removal of such products will favour process efficiency. In this sense, either gas sparging or vigorous mixing will aid in reducing H2 and CO2 concentrations inside the reactor (Kraemer and Bagley, 2006) and thus favour stability for extended operating periods. Gómez et al. (2009) reported stable operationduring continuous fermentation of household waste when mixing was provided to the reactor in contraposition to what occurs in static reactors. This same effect may be attained by gas sparging inside the reactor. Nguyen and co-workers (Nguyen et al., 2010) demonstrated that the removal of the H2 produced from the gas headspace during batch fermentation improved H2 yields. However, accumulation of VFAs still poses a problem. An increase in the organic loading rate, in an attempt to increase the treatment capacity of reactors may lead to inhibition of the fermentation system, owing to VFA accumulation. Wang and Zhao (2009) demonstrated that a significant reduction in H2 yield was caused by an increase of the OLR to 30.2 kg VS m!3 d!1, when food wastes were treated as the substrate. In this way, adaptation of the inoculum may play a crucial role in attaining stable operation at high concentrationsof VFAs, as demonstrated by Valdez-Vazquez et al. (2005). These authors reported successful performance from a reactor treating organic wastes during solid state fermentation.The commercialization of industrial hydrogen fermentation makes imperative to achieve steady operation and also to carry out the process under non-sterile conditions using readily available complex feed stocks with only minimal pre-treatment. Microbial consortia may address these issues if they are selected for growth and dominance under non-sterile conditions (Hallenbeck and Ghosh, 2009). Steady operation over long periods of reactorsproducing H2 has been reported by several authors (Valdez-Vazquez et al., 2005; Chu et al., 2008). However, fluctuating H2 production has also been reported. This is the case of results obtained by Zhu et al. (2008) when fermenting potato waste in a two-phase configuration. Similar results were reported by Gómez et al. (2009) when fermenting food waste under static conditions. These divergences in results obtained may indicate that the selection of optimum parameters is a key factor for attaining stable performance. Laboratory and pilot scale fermentation experiences Centralised collection of urban solid wastes and chemical characteristics of the organic fraction contained in these wastes are two factors that make them a suitable substrate for the dark fermentation process. In addition, the fermentation of wastes for H2 production may be considered as a pre-treatment option which may be integrated with minimum modifications into centralised solid waste treatment plants where an anaerobic digester is already operating. On these lines, fermentation of household wastes hasbeen studied by several authors (Gómez et al., 2009; Liu et al., 2008; Kim et al., 2009; Lee et al., 2010) under different temperature conditions, increases in H2 yields being reported when there were increases in temperature to thermophilic regimes. Substrates such as wastewaters with high carbohydrate content have also been demonstrated to be suitable for the dark fermentationfermentation process. This is the case for molasses (Li et al., 2007) and cheese whey (Ferchichi et al., 2005), which have been studied using completely stirred tank reactors (CSTRs) and reactors with immobilised biomass. Another feedstock of interest is the use of marine algae. Jung et al. (2011) tested various algae for fermentative hydrogen production, with Laminaria japonica presenting the highest H2 yields. The presence of particulate material set limits to the application of high-rate systems. For this reason CSTR systems are often applied for the treatment of household wastes, while in general, attached growth systems are used for the fermentation of high strength wastewater. Immobilized systems are an effective and stable approach for continuous hydrogen production allowing efficient utilization of carbon substrates (Jo et al., 2008).Experiments with dark fermentation processes are rapidly increasing on a laboratory scale. Several reports in litter-scale can be found in literature. Table 3 shows a list of published results for different fermentation systems working under continuous operation in reactors of

size bigger than 20 L. Although results are promising, on feature that needs to be addressed is the requirement for alkalis. Controlling the pH of the process requires large amounts of alkaline solutions, which not only increases operating costs, but also leads to an effluent with high conductivity. When concentrated solutions of NaOH are used, the effects on a potentialsecond treatment process should be considered. Kraemer and Bagley (2005) proposed the recycling of a fraction of the methanogenic effluent in order to take advantage of the alkalinity generated in the digestion phase which is needed in the dark fermentation phase for pH control. Although results reported were not satisfactory, since lower H2 productivity was attained during recycling, this strategy has been tested with satisfactory results by other authors. This was the case for the two-phase process (thermophilic-mesophilic) with sludge recirculation tested by Lee et al. (2010) in a 10 L H2 reactor and a 40 L CH4 reactor. Addition of precipitated sludge at the bottom of a storage tank was used for pH control. These authors obtained stable operation over a period of 150 days with a H2 yield of 205 mL g!1 VS added. This configuration presented two main advantages. The first was that the hydrogen-producing bacteria which exist in digested sludge could replenish the hydrogen production reactor by recirculation. The second advantage was that acidity in the hydrogen production stage was neutralized by recirculation of the digested sludge, which presented high alkalinity, so that the reagent for pH adjustment could be saved. Another option for reducing the amount of alkaline solutions needed would be the use of co-substrates. The co-fermentation of manures and carbohydrate-rich wastes would avoid acidification of the reactor, although high VFA concentrations might occur. However, single fermentation of swine manure resulted in low tonegative H2 yields. There are several factors that may contribute to low hydrogen production from swine manure, such as the lack of suitable sugars in the wastewater, since most of the hydrogen evolved in high-rate hydrogen fermentation tests is a result of sugar in the sample. It appears that hydrogen recovery from swine wastewater will not be feasible by fermentation processes unless some breakthrough is made in changing the nature of the wastewater or the conditions for microbial growth that inhibit the utilization of H2 by microorganisms in the wastewater (Wagner et al., 2009). On these same lines, Perera and Nirmalakhandan (2010) obtained successful results under batch conditions when co-fermenting sucrose with heat-treated cattle manure. These authors reported a 10% improvement in the H2 yield and also demonstrated the capacity of these wastes to reduce buffering needs. Zhu et al. (2009) studied the fermentation of swine manure supplemented with glucose in a 4 L working volume reactor under mesophilic conditions at varying pH and HRT. These authors reported that to increase hydrogen content in the offgas, methane production had to be limited below 2%. On the basis of the results recorded above, it seems reasonable to assume that different strategies intended to increase fermentation yields and lower operating costs should also consider the use of acid-rich effluents, either to enhance treatment efficiency or to make the overall process economically viable by the generation of a high-value product in a second fermentation stage (Mohan et al., 2010).