Bioreactor and Bioprocess Design for Biohydrogen Production

C H A P T E R

13 Bioreactor and Bioprocess Design for Biohydrogen Production Kuan-Yeow Show*, Duu-Jong Lee† *

Department of Environmental Science and Engineering, Fudan University, Shanghai, China † Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan

INTRODUCTION The current global economy and energy supply rely extensively on fossil fuels. This has resulted in an unprecedented increase in the atmospheric carbon dioxide concentration from the hypercombustion of fossil fuels and alarming depletion of nonrenewable fossil fuel resources. The fast escalating greenhouse gas emissions due primarily to carbon dioxide are considered the main culprit of global warming and climate change. To mitigate global warming and other environmental issues, substantial effort is being put in at the global level to explore renewable energy sources that could replace fossil fuels. Hydrogen is a promising alternative biofuel to conventional fossil fuels because it releases explosive energy while producing water as the only by-product. It has been envisaged as the ultimate transport fuel for vehicles because of its nonpolluting nature with the use of fuel cells to convert chemical energy to electricity efficiently and gently (Forsberg, 2007). Most of the current hydrogen is produced from fossil fuels through thermochemical processes (hydrocarbon reforming, coal gasification, and partial oxidation of heavier hydrocarbons) (Das and Veziroglu, 2001; Levin et al., 2004). As the current hydrogen fuel is based on the use of nonrenewable fossil fuel resources with an escalating cost of production, a major concern of conventional hydrogen production is its sustainability. Biohydrogen production is deemed to be a key development to a sustainable global energy supply and a promising alternative to fossil fuels, as it has the potential to overcome most of the problems and challenges that fossil fuels brought about. Using appropriate technologies, biohydrogen can become a desired green energy product of natural microbial conversion.

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Current development of biological hydrogen (biohydrogen) production is focusing on biophotolysis of water, photodecomposition, and dark fermentation of organic compounds using various species of microorganisms. Dark biohydrogen fermentation using anaerobes appears to be the most favorable, as hydrogen could be generated at higher rates. In addition, the anaerobic conversion can be realized with various organic wastes and carbohydrateenriched liquid effluents, thus achieving sustainable low-cost biohydrogen production with concomitant waste purification and renewable clean hydrogen energy. While encouraging technological development of biohydrogen production has been demonstrated in laboratory studies, they are yet confined to laboratory- and pilot-scale studies. The technology is yet to compete with commercial hydrogen production from fossil fuels in terms of production cost, efficiency, and reliability for full-scale applications (Lee et al., 2011; Show et al., 2008, 2010, 2011). Improving the hydrogen yield and production rate are two major challenges for biohydrogen production. This chapter provides an overview of the state-of-the-art and perspectives of biohydrogen production research in the context of pathways, microorganisms, metabolic flux analysis, process design, and reactor system. Challenges and prospects of biohydrogen production are also outlined.

PATHWAYS OF BIOHYDROGEN PRODUCTION There are a variety of biological processes in which hydrogen can be produced via microorganisms. In essence, the metabolic pathways can be differentiated into two distinct categories: light-dependent and light-independent processes. Light-dependent pathways include direct or indirect photolysis and photofermentation, whereas dark fermentation is the major light-independent process.

Direct Photolysis Direct photolysis capitalizes on the photosynthetic capability of algae and cyanobacteria to split water directly into oxygen and hydrogen. Algae have evolved the ability to harness solar energy to extract protons and electrons from water via water-splitting reactions. Biohydrogen production takes place via direct absorption of light and transfer of electrons to two groups of enzymes—hydrogenases and nitrogenases (Manis and Banerjee, 2008). Under anaerobic conditions or when too much energy is captured in the process, some microorganisms vent excess electrons by using a hydrogenase enzyme that converts the hydrogen ions to hydrogen gas (Sorensen, 2005; Turner et al., 2008). It has been reported that the protons and electrons extracted via the water-splitting process are recombined by a chloroplast hydrogenase to form molecular hydrogen gas with a purity of up to 98% (Hankamer et al., 2007). In addition to producing hydrogen, the microorganisms also produce oxygen, which in turn suppresses hydrogen production (Kapdan and Kargi, 2006; Kovacs et al., 2006). Research work has been carried out to engineer algae and bacteria so that the majority of the solar energy is diverted to hydrogen production, with bare energy diverted to carbohydrate production to solely maintain cells. Researchers are attempting to either identify or engineer less oxygen-sensitive microorganisms, isolate the hydrogen and oxygen cycles, or change the ratio

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of photosynthesis to respiration to prevent oxygen buildup (U.S. DOE, 2007). Addition of sulfate has been found to suppress oxygen production. However, the hydrogen production mechanisms are also inhibited (Sorensen, 2005; Turner et al., 2008). The merit of direct photolysis is that the principal feed is water, which is readily available and inexpensive. While this technology has significant promise, it is also seeing tremendous challenges. Currently, this process requires a significant surface area to collect sufficient light. Another challenge is achieving continuous hydrogen production under aerobic conditions (U.S. DOE, 2007).

Indirect Photolysis Alternatively, biohydrogen can be prepared through the use of some microorganisms (algae) that can directly produce hydrogen under certain conditions (Manis and Banerjee, 2008). Anaerobic cultures of green algae with energy derived from light under deprivation of sulfur would induce the “hydrogenase pathway” to produce hydrogen photosynthetically (Melis, 2002). In the course of such a hydrogen production condition (sulfur deprivation), cells consumed significant amounts of internal starch and protein (Zhang et al., 2002). Such catabolic reactions apparently sustain the hydrogen production process indirectly. Indirect photolysis biohydrogen production via algae is deemed feasible if photon conversion efficiency can be improved via large-scale algal bioreactors. Algal bioreactors can provide an engineering approach to regulate light inputs to the culture to improve the photon conversion efficiency of algal cells. The improvement of photosynthesis efficiency is too difficult to achieve for conventional crop plants (Hankamer et al., 2007). Research has reported a substantial increase in light utilization efficiency of up to 15% compared with the previous utilization of around 5% (Laurinavichene et al., 2008; Tetali et al., 2007). Some researchers claim that efficiency between 10 and 13% is attainable by engineering the microorganisms to better utilize the solar energy (Turner et al. 2008). Biohydrogen production can be improved via bioengineering (Hankamer et al., 2007). Several algae strains have been isolated and manipulated. Mutant algae with less chlorophyll can allow more light penetrating into deeper algae layers in the bioreactor. Hence sunlight is made available for more algal cells to generate hydrogen, thus improving the production rate. It was estimated that if the entire capacity of the photosynthesis of algae could be diverted toward hydrogen production, 80 kg of hydrogen could be produced commercially per acre per day. With a realistic 50% capacity, the cost of producing hydrogen comes close to $2.80 a kilogram (Melis and Happe, 2001). Biohydrogen could compete with gasoline at this price, assuming 1 kg of hydrogen is equivalent to a gallon of gasoline. Currently, less than 10% of the algae photosynthetic capacity was utilized for biohydrogen production. Research is underway in improving further algae photosynthetic capacity using a molecular engineering approach. With technology advancement, biohydrogen production via algae bioreactors will offer a sustainable alternative energy resource in the future.

Photofermentation In the photofermentation approach, photosynthetic microorganisms are able to convert solar energy to hydrogen directly from organic substrates. Despite the relatively lower yields

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of hydrogen photodecomposition of organic compounds by photosynthetic bacteria, the fermentative route is a promising method of biohydrogen production due to its higher rate of hydrogen evolution in the absence of any light source, as well as the versatility of the substrates used. The photosynthetic system of purple bacteria is relatively simpler compared with green algae. It consists of only one photosystem, which is fixed in the intracellular membrane. The photosystem itself is not powerful enough to split water. Under anaerobic circumstances, however, these bacteria are able to use simple organic acids, such as acetic acid, or even dihydrogen sulfide as an electron donor (Akkerman et al., 2003). Electrons released from the organic carbon are driven through substantial electron carriers, during which protons are pumped through the membrane. A proton gradient is developed and the ATP synthase enzyme generates ATP through the gradient. Extra energy in the form of ATP can be used to transport the electrons further to the electron acceptor ferredoxin. Under nitrogen-deficient circumstances, these electrons can be used by the nitrogenase enzyme with the extra energy from ATP to reduce molecular nitrogen into ammonium. In the absence of molecular nitrogen, this enzyme can, again with the extra ATP energy, reduce protons into hydrogen gas with the electrons derived from the ferredoxin (Akkerman et al., 2003). In this manner, various organic acids can be transformed into hydrogen and carbon dioxide. Photofermentation by purple nonsulfur (PNS) bacteria is a major field of research through which the overall yield for biological hydrogen production can be improved significantly by the optimization of growth conditions and immobilization of active cells. Various processes of biohydrogen production using PNS bacteria along with several current developments had been examined. However, suitable process parameters, such as carbon and nitrogen ratio, illumination intensity, bioreactor configuration, and inoculum age, may lead to higher yields of hydrogen generation using PNS bacteria (Basak and Das, 2007).

Dark Fermentation Among the biological biohydrogen production processes, dark fermentation or heterotrophic fermentation under anaerobic conditions seems to be more favorable. Hydrogen is yielded at a high rate, and various organic compounds and wastewaters are enriched with carbohydrates as the substrate results in low-cost hydrogen production (Hallenbeck and Ghosh, 2009). The conventional anaerobic fermentation process has been well known since the production of methane, a useful energy carrier, and is associated with the degradation of organic pollutants in two distinct stages: acidification and methane production. Each stage is carried out by specific microorganisms through syntrophic interactions. Hydrogen is produced in the first stage as an intermediate metabolite, which in turn is used as an electron donor by many methanogens at the second stage of the process. It might be feasible to harvest hydrogen at the acidification stage of anaerobic fermentation, leaving the remaining acidification products for further methanogenic treatment. Therefore, it is possible that only acidogens are left to produce hydrogen gas, CO2, and volatile fat acids (VFAs) if the final stage or methanogenesis and other hydrogen-consuming biochemical reactions are inhibited during the dark fermentation. Such a purpose is able to achieve through regulating biohydrogen cultures at a low pH

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and/or short hydraulic retention time (HRT) conditions (Kim et al., 2005; Mizuno et al., 2000) or through inactivating hydrogen consumers by heat treatment (Lay, 2001; Logan et al., 2002) and chemical inhibitors (Sparling et al., 1997; Wang et al., 2003b). Currently, dark fermentation technologies are under the development at a laboratory scale to produce biohydrogen from wet biomass (e.g., molasses, organic wastes, sewage sludge) using anaerobic hydrogen-fermenting bacteria. Wastes and biomass rich in sugars and/or complex carbohydrates turn out to be the most suitable feedstocks for biohydrogen generation (Ntaikou et al., 2010). During dark fermentation, various organic acids are also produced. These compounds can subsequently be converted to hydrogen by a process denoted as photofermentation. During dark fermentation, carbohydrates are converted into hydrogen gas and VFAs and alcohols, which are organic pollutants and energy carriers. For the purpose of energy production and protection of the water bodies, a second-stage process is necessary to recover the energy residues remaining in the effluent in the form of VFAs and alcohols. Thus the fermentative reactor becomes part of a process wherein the effluent post-treatment process and hydrogen utilization should also be included. A possible second-stage process is photofermentation, anaerobic digestion, or microbial fuel cells, which has been assessed elsewhere (Hawkes et al., 2007). For hydrogen produced from dark fermentation to be used alone in an internal combustion engine or a fuel cell, some issues, such as biohydrogen purification, storage, and transport, need to be addressed. The gas produced is a mixture of primary hydrogen (generally less than 70%) and CO2, but may also contain other gases such as CH4, H2S, ammonia (NH4), and/or moisture. Purification of the hydrogen is essential before the hydrogen utilization can be practical. Relevant suggestions on hydrogen separation and purification can be found in the review literature (Das and Veziroglu, 2001; Hallenbeck and Benemann, 2002). Although positive features of dark fermentation technology, such as high production rate, low energy demand, easy operation, and sustainability, have been demonstrated in laboratory studies (Claassen et al., 1999; Hallenbeck and Benemann, 2002; Hawkes et al., 2002; Kapdan and Kargi, 2006; Nandi and Sengupta, 1998; Nath and Das, 2004,), this technology is yet to compete with commercial hydrogen production processes from fossil fuels in terms of cost, efficiency, and reliability. Enhancing the hydrogen production efficiency is thus a major challenge to dark hydrogen fermentation. To achieve such a purpose, numerous studies have been conducted, and several hundreds of public reports were published during the past decade.

HYDROGEN-PRODUCING MICROORGANISMS Biological processes are carried out largely at ambient temperatures and pressures, and hence are less energy-intensive than chemical or electrochemical ones. A large number of microbial species, including significantly different taxonomic and physiological types, can produce hydrogen. Biological processes use the enzyme hydrogenase or nitrogenase as hydrogen-producing protein. This enzyme regulates the hydrogen metabolism of uncountable prokaryotes and some eukaryotic organisms, including green algae. The function of nitrogenase, as well as hydrogenase, is linked with utilization of the products of

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photosynthetic reactions that generate reductants from water. In fact, hydrogen is utilized by the consortia as it is produced, mainly by methanogenic archaea, acetogenic bacteria, and sulfate-reducing bacteria (Adams et al., 1980). Several algae mutant strains related to indirect photolysis biohydrogen production have been isolated, for example, the Chlamydomonas reinhardtii strain can increase biohydrogen production under high starch conditions (Hankamer et al., 2007). It is also reported that an algae mutant with less chlorophyll was manipulated for a large-scale commercial bioreactor. The algae mutant will absorb less light to allow more light penetrating into deeper algae layers. Hence sunlight is made available for more algal cells to generate hydrogen, thus improving the overall hydrogen production rate. Research studies on dark fermentative organisms have been developed intensively in recent years, and some new or efficient bacterial species and strains for dark hydrogen fermentation have been isolated and recognized. A wide variety of dark fermentative bacteria are capable of hydrogen production, which are distributed across 10 of the 35 bacterial groups (Fang et al., 2002b). In a review on hydrogen-producing microorganisms (Nandi and Sengupta, 1998), such bacterial groups include strict anaerobes (Clostridia, Methylotrophs, methanogenic bacteria, Rumen bacteria, archaea), facultative anaerobes (Escherichia coli and Enterobacter), aerobes (Alcaligenes and Bacillus), photosysnthetic bacteria, and Cyanobacteria. In general, the isolated and identified mesophiles are mainly affiliated with two genera: facultative Enterobactericeae (Kumar and Das, 2000; Penfold et al., 2003; Tanisho and Ishiwata, 1995; Yokoi et al., 1995) and strictly anaerobic Clostridiaceae (Collet et al., 2004; Evvyernie et al., 2001; Wang et al., 2003a), whereas most thermophiles belong to genus Thermoanaerobacterium (Ahn et al., 2005; Ueno et al., 2001; Zhang et al., 2003). In addition to the use of anaerobes in dark fermentation, aerobes such as Bacillus (Kalia et al., 1994; Kumar et al., 1995; Shin et al., 2004), Aeromonons spp., Pseudomonos spp., and Vibrio spp. (Oh et al., 2003) have been cultivated or isolated, but their hydrogen yields are generally less than 1.2 mol-H2/mol-glucose under anaerobic conditions. Pure cultures of seed have been used to produce hydrogen from various substrates. Clostridium and Enterobacter were used most widely as inoculum for fermentative hydrogen production (Wang and Wan, 2009; Zhang et al., 2006b, 2008c). Most of the studies using pure cultures of bacteria for fermentative hydrogen production were conducted in batch mode and used glucose as the substrate (Wang and Wan, 2009). Because facultative anaerobes can be grown more easily than obligate ones, attempts have been made to work with pure cultures of facultative anaerobes of genus Enterobactericeae (Kumar and Das, 2001; Kumar et al., 2000; Rachman et al., 1997; Tanisho and Ishiwata, 1995). Among the fermentative anaerobes, Clostridia have been well known and studied extensively, not for their hydrogen production capability, but for their role in the industrial solvent production from various carbohydrates. Some species such as C. acetobutylicum (Andersch et al., 1983; Holt et al., 1984; Kim et al., 1984) and C. beijerinckii (Chen and Blaschek, 1999) were used previously as industrial solvent producers. Use of Clostridia for hydrogen production attracts much attention. Hydrogen yields of pure cultures belonging to the genus Clostridiaceae have been examined, including C. paraputrificum (Evvyernie et al., 2001), C. lentocellum (Ravinder et al., 2000), C. thermosuccinogenes (Sridhar and Eiteman, 2001), C. bifermentans

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(Wang et al., 2003a), C. thermolacticum (Collet et al., 2004), C. butyricum (Chen et al., 2005), C. saccharoperbutylacetonicum (Ferchichi et al., 2005), C. acetobutylicum (Zhang et al., 2006a), and C. pasteurianum (Dabrock et al., 1992). The optimum hydrogen yields observed of each species vary between 1.1 and 2.6 mol-H2/mol-hexose, dependent on the organism per se as well as environmental conditions. Use of undefined mixed cultures in nonsterile conditions would be more attractive for anaerobic hydrogen production, as the pure culture is contaminated easily by other bacteria, including hydrogen consumers such as methane-producing bacteria and sulfate-reducing bacteria (Adams et al., 1980; Ueno et al., 1995). There is a risk of hydrogen consumers coexisting in the mixed hydrogen-producing cultures. Several types of anaerobic bacteria are able to use hydrogen as a source of energy by coupling its oxidation to the reduction of a variety of electron acceptors. This group also includes facultative anaerobes such as E. coli, which is capable of hydrogen-dependent fumarate reduction, and Paracoccus denitrificans, which, although classified as an aerobic hydrogen bacterium, uses either O2 or nitrate as a terminal electron acceptor during respiratory hydrogen oxidation. All these reactions are characterized by the uptake of hydrogen via a hydrogenase (Adams et al., 1980). Mixed culture derived from anaerobic reactor treating molasses-based wastewater subjected to multiple pretreatment processes showed better hydrogen production than the inoculum collected from a laboratory-scale anaerobic biofilm reactor (Venkata Mohan et al., 2009; Zhang et al., 2007a,c, 2008b). Hydrogen yields by mixed thermophilic cultures are reported in a range of 0.7–3.2 molH2/mol-hexose (Liu et al., 2003; Monmoto et al., 2004; Shin et al., 2004; Ueno et al., 1995, 1996; Valdez-Vazquez et al., 2005). The highest yield was obtained from organic solid wastes (60% fruit/vegetable waste and 40% office paper waste, based on dried weight) by a mixed culture at 55
C (Valdez-Vazquez et al., 2005), whereas the lowest one was achieved on fermenting starch-containing wastewater at 55
C (Zhang et al., 2003). Several reports on microbial hydrogen production using hyperthemophiles have been reported. For example, van Niel et al. (2002) obtained a hydrogen yield of 3.33 mol-H2/mol-hexose using a pure culture of extremophiles—either Caldicellulosiruptor saccharolyticus on sucrose (70
C) or Thermotoga elfi on glucose (65
C)—and the same yield was reported by Kanai et al. (2005) from starch fermentation with the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 at 85
C. Moreover, it is stressed that a yield of 4 mol-H2/mol-hexose, the maximal theoretical value, was reported by Schro¨der et al. (1994) with a batch eubacterial culture of Thermotoga maritima on glucose at 80
C. However, the authors pointed out that both microbial growth of the culture and glucose utilization are low. In addition, compared with mesophilic cultures, hyperthermophiles exhibit much lower production rates of hydrogen—generally ranging from 0.01 to 0.2 liter/liter  h—which is largely attributed to the slow-growing characteristics of hyperthermophiles. Aerobic Alcaligenes eutrophus has been shown to grow heterotrophically on gluconate and fructose and produced hydrogen when exposed to anaerobic conditions (Kuhn et al., 1984). It contains a soluble NAD-reducing hydrogenase (Pinchukova et al., 1979). Aerobic hydrogenproducing Bacillus licheniformis was isolated from cattle dung (Kalia et al., 1994). It produced 0.5 mol H2/mol glucose (Kumar et al., 1995). Immobilized cells had an average hydrogen yield of 1.5 mol per mole glucose and cells were stable for 60 days.

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PROCESS DESIGN OF BIOHYDROGEN PRODUCTION Batch Processes In general, hydrogen-producing cultures were found to perform inefficiently when conducted under batch mode laboratory conditions (Li and Fang, 2007; Zhang et al., 2005) with relatively inferior hydrogen production rates (0.06–0.66 liter/literh). Enhancing hydrogen production efficiency is thus a major challenge to batch biohydrogen production systems. A semicontinuous mode for anaerobic hydrogen production from food waste was developed (Han and Shin, 2004). Pretreated seed sludge and food waste were fed into an anaerobic-leaching bed reactor, and dilution water was pumped continuously into the reactor at different dilution rates. Microbial conversion was deemed accomplished when biogas production ceased in about a week. It was found that appropriate control of the dilution rate could enhance fermentation efficiency by improving the degradation of nondegradable fractions of the food waste. The dilution rate may also delay the shift of predominant metabolic flow from a hydrogen- and acid-forming pathway to a solvent-forming pathway. In another study involving municipal solid wastes, it was reported that hydrogen can be produced steadily in reactors fed with substrate twice a week in a draw-and-fill mode in an anaerobic chamber and operated continuously at 35 and 55
C for 40 days (Valdez-Vazquez et al., 2005). A bioreactor operated under sequencing batch mode was used to evaluate the hydrogen productivity of an acid-enriched sewage sludge microflora at 35
C (Lin and Chou, 2004). A 4-h cycle, including feed, reaction, settle, and decant stages, was employed to operate the 5-liter anaerobic sequencing batch reactor. The sucrose substrate concentration was kept at 20 g chemical oxygen demand (COD)/liter, and HRT was maintained initially at 12–120 h and thereafter at 4–12 h. The reaction to settling time ratio was maintained at 1.7. The hydrogenic activity of biomass microflora was HRT dependent, with peak activity recorded at an HRT of 8 h and an organic loading rate (OLR) of 80 kg COD/m3 reactor liquid per day. Each mole of sucrose converted produced 2.8 mol of hydrogen, whereas each gram of biomass produced 39 mmol of hydrogen per day. An excessively low HRT might deteriorate hydrogen productivity. The concentration ratios of butyric acid to acetic acid, as well as volatile fatty acids and soluble microbial products to alkalinity, can be used as monitoring indicators for hydrogenic activity. Proper pH control was necessary for stable operation of the sequencing batch reactor.

Continuous Processes Continuous stirred tank reactors (CSTRs) are used frequently for continuous hydrogen production (Chen and Lin, 2003; Majizat et al., 1997; Yu et al., 2002). Compared with batch reactor systems, hydrogen-producing bacteria in a CSTR are well suspended in mixed liquor and suffer less from mass transfer resistance. Because of its intrinsic structure and operating pattern, a CSTR is unable to maintain high levels of biomass inventory. Depending on the operating HRT, biomass inventory in a range between 1 and 4 g-VSS/liter is commonly reported (Chen and Lin, 2003; Horiuchi et al., 2002; Lin and Chang, 1999; Zhang et al., 2006b). Biomass washout is likely at short HRTs, resulting in deterioration in hydrogen

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production rates. Hydrogen production rates are thus restricted considerably by a low CSTR biomass retention and low hydraulic loading (Lay et al., 1999; Yu et al., 2002). The highest hydrogen production rate of CSTR culture fermenting sucrose with a mixed hydrogenproducing microflora was reported as 1.12 liter/liter  h (Chen and Lin, 2003). Spontaneous granulation of hydrogen-producing bacteria can occur with shortened HRT in CSTR (Fang et al., 2002a; Oh et al., 2004b; Yu and Mu, 2006). Show et al. (2007) and Zhang et al. (2007c) found that the formation of granular sludge significantly increased overall biomass inventory to as much as 16.0 g-VSS per liter of reactor volume, enabling the CSTR to be operated at higher OLRs of up to 20 g-glucose/liter  h, which hence enhanced hydrogen production. In another study, it was reported that granular sludge disappeared within 3 weeks when CSTRs were incubated statically instead of being shaken (Vanderhaegen et al., 1992). A reactor coupled with a membrane has been used to increase biomass concentration in the system. At a HRT of 3.3 h, Oh et al. (2004a) demonstrated that biomass concentration increased from 2.2 g/liter in a control reactor (without membrane fitted) to 5.8 g/liter in an anaerobic membrane bioreactor (MBR). This was achieved by controlling sludge retention time (SRT) at 12 h, corresponding to a marginal increase in the hydrogen production rate from 0.50 to 0.64 liter/literh. Increasing SRT can enhance biomass retention, which favors substrate utilization, but may result in a decrease in the hydrogen production rate. Li and Fang (2007) summarized that hydrogen production rates ranged between 0.25 and 0.69 liter/literh in the MBR systems reviewed. While the biomass concentration can be enhanced in a MBR, there is no clear advantage of a MBR over other types of efficient hydrogen production systems. In addition, membrane fouling and a high operating cost may limit the use of the MBR process in hydrogen fermentation.

Immobilized Cell Processes In order to achieve higher biomass concentration and dilution rates without biomass washout, immobilized cell systems in the form of granules, biofilm, or gel-entrapped biomass particles have been developed (Show et al., 2007; Zhang et al., 2008c). Immobilized pure or mixed cultures of hydrogen-producing bacteria can be achieved by entrapment as biogels such as C. butyricum strain IFO13949 in agar gel (Yokoi et al., 1997a), E. aerogenes strain HO-39 in k-carrageenan, calcium alginate or agar gel, sewage sludge in calcium alginate beads, or alginate beads with added activated carbon powder, polyurethane, and acrylic latex/silicone (Wu et al., 2002), and sewage sludge and activated carbon powder fixed by ethylene vinyl acetate copolymer (Wu et al., 2003). Peak hydrogen production rates of continuous gel-immobilized sludge ranged from 0.090 to 0.93 liter/liter  h in a stirred chemostat and a fluidized bed reactor, respectively (Wu et al., 2005). In comparison with gel-entrapped bioparticles, biofilm attached on solid and porous support carriers seems to be superior for continuous hydrogen production. Hydrogen production and glucose consumption rates (Yokoi et al., 1997a) with C. butyricum immobilized on porous glass beads were higher than corresponding values with cells immobilized in agar gel at HRTs of 3 and 5 h in continuous cultures without pH control. In another study (Yokoi et al., 1997b), the hydrogen production rate of agar gel and porous glass beads developed with E. aerogenes strain HO-39 culture increased with decreasing HRT, which was not

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observed with free cells culture. In the same study, the hydrogen production rate of glass beads was superior to that of agar gels at HRTs below 3 h, reaching the maximum rate at 0.85 liter/liter  h with a HRT of 1 h. Biofilm culture had an advantage in hydrogen production rate compared to that of gel-entrapped bioparticles with lower mass transfer efficiency and cell stability. It can be deduced that gel-entrapped biomass particles may not be the technology of choice for fermentative hydrogen production. However, there are also several operational and economic drawbacks of biofilm systems. Washout of support carriers might be an intrinsic problem of biofilm processes, especially when operating under low HRTs. Moreover, a large amount of support carriers is normally required to support microbial growth in biofilm processes. This leads to a considerable reduction in reactor effective volume for efficient biomass–substrate interactions, thereby resulting in lower reactor performance. A system upset might occur once interstitial void spaces in a pack bed reactor are clogged with biomass. The process stability of such a biofilm-based system may be challenged by long-term operation. As support carriers need to be replaced periodically due to wear and tear, the cost of carrier replacement can be a major economic consideration in maintenance. Microbial self-aggregation, in which microbial cells are organized into dense and fastsettling granules, is studied extensively due to its practical importance in biohydrogen production. Granular sludge has some advantages over biofilm sludge in continuous dark hydrogen fermentation. Possible advantages of microorganisms in anaerobic granule in comparison with flocculated or suspended counterparts are a higher retention of granulated biomass in a reactor, diversity of physiological functions of microorganisms in the granule, and resistance of the microorganisms within the granule to toxic substances. It is favorable for the microorganisms to be very close to each other in the granule in order to achieve a high substrate conversion to hydrogen. Aggregation leads to a heterogeneous community and facilitate syntrophic relationships, especially interspecies metabolic transfer, and under unfavorable conditions for growth (e.g., toxicity), a more favorable microenvironment can be maintained within the aggregates so that metabolism can be sustained. Biogranulation is a gradual process from seed sludge to compact aggregates, then to granular sludge, and finally to mature granules. A major drawback of granulation is its long startup period and development of active biogranules for efficient functioning of the process. A complete development of hydrogen-producing granules may take several months (Fang et al., 2002a; Yu and Mu, 2006). During startup of an UASB hydrogen-producing reactor, Mu and Yu (2006) found that small granules (diameter 400–500 mm) were formed at the reactor bottom after 140 days of operation. Granules developed further to sizes larger than 2.0 mm upon 200 days. Although the reactor reached steady-state hydrogen production and substrate degradation after 5 months of startup operation, development and accumulation of mature and stable granular sludge were only completed beyond 8 months of operation. Chang and Lin (2004) noted that a UASB reactor took 39 days to achieve constant gas production at a HRT of 24 h and granules became visible after 120 days of operation. A longer period (180 days), however, was required for further development of granules. Granulation of hydrogen-producing cultures can be accelerated markedly. Packing of a small quantity of carrier matrices significantly accomplished sludge granulation within 80–290 h in a novel carrier-induced granular sludge bed bioreactor (Lee et al., 2006). Granulation of seed sludge could take place in all carrier-packed reactors as HRTs were shortened to 4–8 h, dependent on carrier type. By adding cationic polymer (cationic polyacrylamide) and anionic polymer (silica sol), rapid granulation of hydrogen-producing culture could

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be accomplished within 5 min (Kim et al., 2005). Zhang et al. (2008a) developed an approach of acid incubation to initiating formation of hydrogen-producing granules rapidly in a CSTR. Hydrogen-producing granules were formed rapidly within 114 h as the seed microbial culture was subjected to a 24-h period of acid incubation at a pH of 2.0. Changing culture pH would result in an improvement in surface physicochemical properties of culture favoring microbial granulation.

BIOHYDROGEN REACTOR SYSTEMS Photobioreactors Different types of photobioreactors have been reviewed by Janssen (2002). Small-scale flat panel reactors consisting of a rectangular transparent box were mixed with gas introduced via a perforated tube at the bottom of the reactor. In order to create a high degree of turbulence, 3 to 4 liter of air per liter of reactor volume per minute has to be provided. The panels were illuminated from one side by direct sunlight and the panels were placed vertically, or inclined toward the sun. Light/dark cycles were short in flat panel reactors, which is probably the key factor leading to the high photosynthetic efficiency. A disadvantage of flat panel reactors systems is that the power consumption of aeration (or mixing with another gas) is high, although mixing is always necessary in any reactor. The large-scale flat plate reactor consisted of a rectangular air-lift photo bioreactor with a large number of light redistributing plates fixed a few centimeters from each other. Mixing was provided by air injected between adjacent plates and the culture liquid rises in between. Tubular photo bioreactors consist of long transparent tubes with diameters ranging from 3 to 6 cm and lengths ranging from 10 to 100 m (Janssen 2002). The culture liquid is pumped through these tubes by means of mechanical or air-lift pumps. The tubes can be positioned in many different ways: in a horizontal plane as straight tubes with a small or large number of U bends; vertical, coiled as a cylinder or a cone; in a vertical plane, positioned in a fence-like structure using U bends or connected by manifolds; or horizontal or inclined parallel tubes connected by manifolds; in addition, horizontal tubes can be placed on different reflective surfaces with a certain distance between the tubes. Although the tubular reactor design is rather diverse, the predominant effect of the specific designs on the light regime is a difference in the photon flux density incident on the reactor surface. The shape of the light gradient in the tubes is similar in most designs. Also with respect to liquid mixing, the circumstances in most designs are similar. The length of the tubes is limited because of accumulation of gas, although this might not be so important for nitrogenase-based processes, as they may be less inhibited by hydrogen. The way to scale up is to connect a number of tubes via manifolds. Flat panel reactors normally show a high photochemical efficiency or biomass yield on light energy, while biomass density is also high. Tubular reactors in theory should show better efficiencies because of the shorter average light/dark cycles.

Dark Fermentation Bioreactors The influence of reactor configuration on hydrogen production was evaluated by examining CSTR and anaerobic fluidized-bed reactor (AFBR) systems with granular sludge for hydrogen production (Show et al., 2007, 2010; Zhang et al., 2007b, 2008d). Maximum

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hydrogen production rates of the suspended sludge, CSTR granular sludge, AFBR granular sludge, and biofilm sludge were essentially correlated to the biomass concentration, indicating that the system performance is largely influenced by reactor biomass retention. Compared to the CSTR, much more biomass retention was retained in the AFBR reactor (35 g-VSS/liter vs 16 g-VSS/liter), which might be attributed to the reactor intrinsic structure. A larger height/section diameter ratio of the column reactor, such as AFBR, may be an advantage favoring solid/liquid separation and biomass retention, and hence hydrogen production. It should be stressed that Wu et al. (2006) reported the highest hydrogen production rate of up to 15 liter/literh, which was achieved by a granule-based CSTR system at a hydraulic retention time of 0.5 h. The biomass concentration of the CSTR culture maintained at a level of 32.5 g-VSS/liter, which was comparable to that of the present AFBR system, but was two times higher than that of the present CSTR. Additionally, because the recirculation flow rate was kept consistently at 200 ml/min, its effect on mass transfer and reactor performance was not examined in the present study. Nevertheless, it is possible that an increasing liquid recirculation ratio might improve mass transfer of the immobilized culture and substrate in a column reactor. A fixed- or packed-bed reactor is operated under conditions with a lesser extent of hydraulic turbulence, thus its immobilized cultures usually encounter mass transfer resistance, which would result in inferior rates of substrate conversion and hydrogen production. Rachman et al. (1998) found that a high hydrogen molar yield could not be maintained consistently in a packed-bed reactor, although the pH in the effluent was controlled at more than 6.0. This was due to pH gradient distribution along the reactor column, resulting in a heterogeneous distribution of microbial activity. In order to overcome the mass transfer resistance and pH heterogeneous distribution, they recommended that a fluidized-bed or an expanded bed reactor system with recirculation flow is more appropriate for further enhancing the hydrogen production rate and yield. In a later study, Kumar and Das (2001) observed that hydrogen production and substrate conversion rates of a packed-bed reactor both increased with the recycling ratio and obtained the maximum hydrogen production rate of 1.69 l/liter  h at a recirculation ratio of 6.4. This means that the mass transfer resistance in a packed-bed reactor can be reduced by increasing the slurry recycle ratio. An appropriate reactor configuration would help increase hydraulic turbulence of the column reactors and result in an increase in hydrogen production. Kumar and Das (2001) investigated hydrogen production by attaching E. cloacae on coir in packed bed reactors with different configurations, that is, tubular, tapered, or rhomboid shape at a HRT of 1.08 h. The comparative study indicated that the rhomboid bioreactor with a convergent–divergent configuration has a maximum hydrogen production rate of 1.60 liter/liter  h as compared with 1.46 liter/liter  h in a tapered reactor and 1.40 liter/liter  h in a tubular reactor. The enhancement in the hydrogen production rate could be attributed to higher turbulence caused by reactor geometry favoring mass transfer and reduced gas hold-up.

Microbial Electrolysis Cells Microbial-aided electrolysis cells (MEC), also called a bioelectrochemically assisted microbial reactor, use electrohydrogenesis to directly convert biodegradable material into

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hydrogen (Call and Logan, 2008; Cheng and Logan, 2007; Ditzig et al., 2007). The MEC is a modified microbial fuel cell. In a microbial fuel cell, special microbial exoelectrogens decompose organic material and transfer electrons to the anode. The electrons combine at the cathode, after traveling through an external load, with protons and oxygen forming water (Holladay et al., 2009). An MEC operates in anaerobic state, and an external voltage is applied to the cell rather than generated by it. The added energy is required, as acetate substrate decomposition is not spontaneous under standard conditions (Call and Logan, 2008; Cheng and Logan, 2007; Ditzig et al., 2007). Hydrogen gas production occurs at the cathode via reaction of hydrogen ion with electrons. Operation of MECs is, in fact, versatile in the sense that suitable microbial cultures can be selected during operation, and a variety of substrates such as wastewater can be metabolized. Because of the versatility of the microbial community, MECs are not confined to using fermentation metabolites as substrate but can also decompose glucose or glucose-containing substrates such as cellulose to hydrogen (Ditzig et al., 2007). Achieving satisfactory efficiency of MEC with nominal electrical inputs, however, is still a major challenge. Hydrogen production rates are still substantially lower than those derived from dark fermentations at relatively high voltages (800 mV). There remain substantial challenges to the practical implementation of this technology, including the replacement of expensive platinum electrode, high current densities, and reduction of the electrical input requirement.

Hybrid Bioreactors Hybrid two-stage systems are operated on the notion that conversion of substrate to hydrogen and organic acids takes place in a conventional reactor in a first stage of process and additional gaseous energy in the form of methane or hydrogen is extracted in the second stage (Koutrouli et al., 2009). In optimizing gas production, the second-stage reactor is operated under different conditions, such as higher pH and longer HRT (Hwang et al., 2011). Applications of photofermentation (Kapdan and Kargi, 2006) or fuel cell (Garcia-Pena et al., 2009) for maximizing the overall energy extraction in the second stage have been reported. Additional hydrogen was recovered from metabolites of the dark fermentation in the studies. In another approach, MECs were incorporated into the second stage, in which electricity applied to a microbial fuel cell provided the energy needed in converting organic acids to hydrogen (Call and Logan, 2008; Wang et al., 2011). In principle, a second-stage MEC could produce 12 H2/glucose with only a small input of energy. A hybrid two-stage photofermentation system in which the second stage was fed with effluent from a hydrogen-producing reactor has been investigated (Nath and Das, 2006). Hydrogen yields well below the hypothetical values were reported. Conversely, it was found that a combination of dark and photofermentation in a hybrid two-stage system enhanced the overall yield of hydrogen (Nath and Das, 2006). In the first stage, the biomass is fermented to acetate, carbon dioxide, and hydrogen in a thermophilic dark fermentation. In the secondstage photobioreactor, acetate is converted to hydrogen and carbon dioxide. Enhancement of the hybrid system was attributable to maximal utilization of the substrate with improved thermodynamics. The combination of dark and photofermentation has the potential to almost attaining the theoretical maximum production of 12 mol of hydrogen per mol glucose equivalent. Theoretically, complete conversion of organics into hydrogen via photofermentations is

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possible due to the fact that hydrogen production is driven by ATP-dependent nitrogenase. The required ATP is formed with energy derived from sunlight. While research work has so far indicated encouraging findings, the main technical barrier to practical application of photofermentation lies with low photosynthetic efficiencies with concomitant low hydrogen yields under moderate- to high-light intensities. The hybrid two-stage system has been scaled up to the pilot plant stage (Ueno et al., 2007). The pilot system offers several merits over conventional methanogenesis-enhanced substrate solubilization, and tolerance to high OLRs is among the merits reported. Successful operation of such pilot systems using actual wastes has also been documented. A pilot-scale fermentation of kitchen waste generated relatively high production rates of 5.4 m3 H2 m-3 day-1 and 6.1 m3 CH4 m-3 day-1 while achieving a COD removal of 80% (Ueno et al., 2007). The superior performance of this hybrid system is illustrated from the methane yields, which were twofold higher than a single-stage process (Ueno et al., 2007).

Multistage Bioreactors Multistage bioreactors involving three or even four stages have been reported in biohydrogen production (Show et al., 2011; Wang et al., 2011; U.S. DOE, 2007). Sunlight is first filtered through first-stage direct photolysis in which visible light is utilized by blue-green algae, and an unfiltered infrared ray is used by photosynthetic microbes in the second-stage photofermentative reactor. Effluent from the second-stage photofermentation, together with the biomass feedstock, is fed into a third-stage dark fermentation reactor where bacteria convert the substrate into hydrogen and organic acids. As the effluent is enriched with organic acids, a supply of external organic acids for the photofermentative process can be eliminated. The fourth stage involves use of a MEC to convert the organic acids generated from dark fermentation into hydrogen under the light-independent process. It thus can be operated during the night or low-light conditions. Integration of multiple biochemical conversion processes poses significant challenges for multistage reactor engineering, system design, process control, operation, and maintenance. Major challenges with the simultaneous production of hydrogen and oxygen from photolytic hydrogen production include respiration to photosynthetic capacity ratio, coculture balance, and concentration and processing of cell biomass (Holladay et al., 2009).

CHALLENGES AND THE WAY FORWARD Low hydrogen yields and production rates are two major challenges for practical application of biohydrogen production. Major efforts are underway to increase the hydrogen yield substantially, considering that less than 15% of the energy from the organic source can typically be obtained in the form of hydrogen (Logan, 2004). The U.S. DOE (2007) program goal for hydrogen fermentation technology is to realize yields of 4 and 6 mol hydrogen per mole of glucose by 2013 and 2018, respectively, as well as to achieve 3 and 6 months of continuous operation for the same years. On this perspective, some integrated strategies have been investigated, such as the two-step fermentation process (acidogenic þ photobiological or acidogenic þmethanogenic processes) or the use of modified microbial fuel cells (de Vrije and

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Claassen, 2003; Ueno et al., 2007). Through these coupled processes, more hydrogen or energy per mole of substrate can be achieved in the second stage. Enhancement in hydrogen yield may be possible using a suitable microbial strain, process modification, efficient bioreactor design, and genetic and molecular engineering technique to redirect the metabolic pathway. Applying genetic and metabolic engineering techniques to improve the hydrogen yields of those microbial cultures with higher hydrogen production rates may also be another feasible option (Chittibabu et al., 2006; Nath and Das, 2006). Metabolic flux analysis (MFA) may serve as an important tool to guide a priori most suitable genetic modifications oriented to a hydrogen yield increase. MFA or flux balance analysis has been used to provide valuable information for the optimization and design of the fermentative hydrogen production process (Cai et al., 2010; Kim et al., 2009; Manish et al., 2007; Navarro et al., 2009; Niu et al., 2011; Oh et al., 2008; Pasika et al., 2011). It should be noted that the improvement of hydrogen production by gene manipulation or modification is focusing mainly on the disruption of endogenous genes and not introducing new activities in the microorganisms. New pathways need to be discovered to directly take full advantage of the 12 mol of hydrogen available in a mole of hexose. Another challenge of hydrogen fermentation is maintaining stable production. Unstable hydrogen production is possibly attributed to the metabolic shift of hydrogen-producing bacteria, which could be minimized by an in-depth microbial study. A major technical prerequisite for stable and efficient hydrogen fermentations is the maintenance of low hydrogen partial pressures through continuous removal of hydrogen from the fermentation broth. An in-depth microbial study is also needed to shed light on the metabolic shift of hydrogen-producing bacteria causing unstable biochemical fermentation. At the current technology development, biohydrogen production is more expensive than other fuel options. Biohydrogen would play a major role in the economy in the long run if technology improvements succeed in bringing down the costs. Cost of the feedstock is another main barrier for economically viable fermentation technology. Biohydrogen production employing a renewable biomass may be a potential answer to overcome some of the economics. There is scope to use sugarcane juice, molasses, or distillery effluent as substrates, as they contain sugar in significant quantities. Apart from that more easily degradable substance, green wastes will, most probably, to a large extent be the targeted feedstocks for hydrogen fermentation because of their abundance. Lignocellulose is the most abundant renewable natural resource and substrate available for conversion to fuels. There is great potential in the use of green waste biomass as a renewable source of energy via microbial breakdown to sugars that can then be converted to biohydrogen (Lo et al., 2008, 2009). With technology advancements, biohydrogen production may offer a sustainable alternative energy resource in the future. Rigorous technoeconomic analyses, however, are necessary to draw a cost-effective comparison between biologically produced hydrogen and the various other conventional fossil fuels.

CONCLUSIONS Extensive work has indicated a promising prospect of biohydrogen production with substantial improvement in both the yield and the volumetric production rates of hydrogen fermentations. For realistic applications that make economic sense, hydrogen yields and

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production rates must be raised to a significant level, surpassing the current attainment. A technological breakthrough is needed to harvest most of the hydrogen available in the substrate. An intensified effort in scaling up of the process to full-scale systems would further refine the technology for practical application and commercialization. In order to accelerate and intensify technological advancement and to generate critical mass for development of a hydrogen-based economy, international knowledge exchange and cooperation need to be strengthened.

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