Hydrogen Generation from Food Industry and Biodiesel Wastes

C H A P T E R

9 Hydrogen Generation from Food Industry and Biodiesel Wastes Naomichi Nishio and Yutaka Nakashimada

1. INTRODUCTION

Xred 1 H1 -Xox 1 12H2

Hydrogen gas is considered to be a clean energy gas because it is converted to water when it burns. Hydrogen can be produced from organic substrates via either photo-fermentation by photosynthetic microorganisms or dark fermentation by strict and facultative anaerobes (Table 9.1). Dark fermentation is capable of constantly producing hydrogen from organic compounds, whereas photo-fermentation only proceeds in the presence of light. Fermentative hydrogen production from food industry wastes is feasible because the wastes abundantly contain carbohydrates, which constitute the best substrate for such production. On the other hand, short-chain fatty acids, such as lactate or acetate, are also feasible for photobiological hydrogen fermentation. Furthermore, fermentative hydrogen production has some advantages over photosynthetic hydrogen production, such as (1) availability of conventional bioreactors, (2) compactness of process, (3) availability of various sugars, and (4) productivity at high rate. This chapter is focused on the advancement of fermentative hydrogen production with strict or facultative anaerobes, and some challenges experienced, when treating food industry wastes. Photo-fermentation of hydrogen is also briefly reviewed as one of the technologies for hydrogen production from by-products of dark fermentation.

where X represents electron carriers such as formate, ferredoxin, nicotine adenine dinuclotide (NAD), or chytocrome; red and ox in suffix represent reduced and oxidized form of the electron carrier. This means that an electron in the reduced carrier binds a proton to produce hydrogen and reoxidize the carrier. Since the proton is the one of the most abundant ions in water, any microorganism can always use it as an electron acceptor in the absence of any other electron acceptors such as oxygen, sulfate, or nitrate. Thus, hydrogen production is ubiquitous in the environment. Metabolic pathways for hydrogen production from organic matter are classified into two types: dark hydrogen production and photobiological production. Dark hydrogen production occurs under anoxic or anaerobic conditions, because there is no oxygen present as an electron acceptor. A wide variety of microorganisms can reduce protons to hydrogen and re-oxidize the reduced form of the electron carrier that results from primary metabolism. For example, when microorganisms grow on glucose, glucose is mainly catabolized to pyruvate via a glycolytic pathway, in which excess electrons are generated and used for reduction of a native electron carrier (usually NAD1 to NADH2): C6 H12 O6 1 2NAD1 1 2H1 -2CH3 COCOOH 1 2NADH2

2. BASIC PRINCIPLE OF DARK HYDROGEN FERMENTATION Microbial hydrogen production is a simple reaction. The final step for hydrogen formation is denoted with the following equation:

Food Industry Wastes. DOI: http://dx.doi.org/10.1016/B978-0-12-391921-2.00009-3

ð1Þ

ð2Þ

Reduced electron carriers must be reoxidized to maintain the metabolic activity. In aerobic conditions, oxygen is used for reoxidation of the electron carrier, with water and ATP formation (respiration). In anaerobic conditions, however, other compounds must

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9. HYDROGEN GENERATION FROM FOOD INDUSTRY AND BIODIESEL WASTES

act as electron acceptors, which include metabolic intermediates such as pyruvate, acetyl-CoA, endproducts such as fatty acids (acetate, butyrate), sulfate, nitrate, and protons. The electron acceptor used for reoxidation of a native electron carrier depends on the nature of the microorganism. For example, lactic acid bacteria use pyruvate as the electron acceptor to reoxidize NADH formed in the glycolytic pathway: 2CH3 COCOOH 1 2NADH2 -2CH3 CHðOHÞCOOH 1 2NAD1 ð3Þ A beer yeast, Saccharomyces cerevisiae, uses acetaldehyde formed via decarboxylation as the electron acceptor: 2CH3 COCOOH-2CH3 CHO 1 2CO2

ð4Þ

2CH3 CHO 1 2NADH2 -2CH3 CH2 OH

ð5Þ

In this context, hydrogen-producing microorganisms use a proton as the electron acceptor. Dark hydrogen-producing microorganisms can be further classified into two metabolic types: strict anaerobes or facultative anaerobes.

2.1 Hydrogen Production by Strict Anaerobes Strict anaerobes such as Clostridium and Ruminococcus spp. reduce a proton to hydrogen with reduced electron carrier NADH2 or reduced ferredoxin (Figure 9.1). If NADH2 is directly used as the electron donor, 2 mol of hydrogen is produced using excess electrons generated at the glycolytic pathway from 1 mol of glucose: 2NADH2 -2NAD1 1 2H2

ð6Þ

Furthermore, strict anaerobes generate excess electrons during decomposition of pyruvate to acetyl-CoA with pyruvate dehydrogenase and reduce ferredoxin: 2CH3 COCOOH 1 2HS-CoA 1 2Fdox -2CH3 2 CO 2 S 2 CoA 1 2CO2 1 2Fdred 1 2H1

where Fd represents ferredoxin; CoA, coenzyme A. Hydrogen can be formed from reduced ferredoxin with hydrogenase: 2Fdred 1 2H1 -2Fdox 1 2H2

ð8Þ

The formed acetyl-CoA can be further catabolized to acetate: 2CH3 2 CO 2 S 2 CoA 1 H2 O-CH3 COOH 1 HS 2 CoA

TABLE 9.1 Examples of Hydrogen-Producing Microorganisms Microorganism (genera) DARK H2 FERMENTATION Strict anaerobes Clostridium

Substrates for H2 Production

C6 H12 O6 -4H2 1 2CH3 COOH 1 2CO2 Glucose EMP pathway

Starch, pyruvate

Thermococcus Facultative anaerobes Escherichia

Various carbohydrates

2 Glutaraldehyde-3P 2 NAD+ 2 NADH Products

2 Pyruvate PFL

CO2

Enterobacter PHOTOBIOLOGICAL H2 FERMENTATION Purple non-sulfur bacteria Rhodospirillium Organic acids, amino acids

H2

FHL

PYDH NAD+

Formate

H2

Fdox NFOR

NADH Fdred

Main H2 producing pathway of facultative anaerobes

Rhodopseudomonas

2H+ H2ase

Main H2 producing pathway of strict anaerobes

Acetyl-CoA NADH

Water, starch, glycogen

NAD+ Products

Synechococcus

Chlorella

ð10Þ

Various carbohydrates

Caldicellulosiruptor Archaea Pyrococcus

Spirulina Green algae Chlamydomonas

ð9Þ

As the overall reaction, 4 mol of hydrogen are produced from 1 mol glucose:

Thermotoga

Rhodobacter Cyanobacteria Anabaena

ð7Þ

Water, starch, glycogen

FIGURE 9.1 Simplified metabolic pathways for H2 production by strict and facultative anaerobes from various kinds of carbohydrates. FHL, formate hydrogen lyase; PFL, pyruvate formate lyase; PYDH, pyruvate dehydrogenase; NFOR, NADH-ferredoxin oxidoreductase; H2ase, hydrogenase.

III. IMPROVED BIOCATALYSTS AND INNOVATIVE BIOREACTORS FOR ENHANCED BIOPROCESSING OF LIQUID FOOD WASTES

2. BASIC PRINCIPLE OF DARK HYDROGEN FERMENTATION

Although this is the theoretical maximum of hydrogen yield from glucose by strict anaerobes, actual hydrogen yield is usually much lower than the theoretical value because of the formation of more reduced by-products besides acetate and the use of reducing power for anabolism (cell growth). Strict anaerobes isolated and characterized for hydrogen production were mainly Clostridium spp. For example, Clostridium beijerinckii strain AM21B was isolated from termites (Taguchi et al., 1993). The strain AM21B produced 2.0 mol H2/mol glucose at uncontrolled pH and 37 C. Since strain AM21B produced amylase, it can produce hydrogen from starch at the yield of 1.7 mol H2/mol glucose at pH 6.0 (Taguchi et al., 1994). Strain AM21B also produced hydrogen from arabinose, cellobiose, fructose, galactose, lactose, sucrose, and xylose, which are abundantly contained in food wastes, with conversion efficiencies ranging from 15.7 to 19.0 mmol/g substrate for 24 h. Continuous fermentation of xylose and glucose to hydrogen using the other clostridia, Clostridium sp. strain No. 2, isolated from termites, was also carried out in a culture containing 0.3% substrate with the pH controlled at 6.0 (Taguchi et al., 1995). The maximal hydrogen production rates of 21.0 and 20.4 mmol/L/h were obtained from xylose and glucose with dilution rates of 0.96/h and 1.16/h, respectively. C. paraputrificum M-21, isolated from a soil sample collected from Mie University campus, utilized chitin and N-acetyl-D-glucosamine (GlcNAc), a constituent monosaccharide of chitin, to produce a large amount of gas along with acetic acid and propionic acid as major fermentation products (Evvyernie et al., 2000). The bacterium grew rapidly on GlcNAc, with a doubling time of around 30 min, and produced hydrogen yielding 1.9 mol H2/mol GlcNAc at initial medium pH 6.5 and 45 C. Strain M-21 produced 1.5 mol H2 from ball-milled chitin equivalent to 1 mol of GlcNAc at pH 6.0 (Evvyernie et al., 2001). In addition, strain M-21 efficiently degraded and fermented ball-milled raw shrimp and lobster shells to produce hydrogen: 11.4 mmol H2 from 2.6 g of the former and 7.8 mmol H2 from 1.5 g of the latter. Characteristics of continuous hydrogen production and fatty acid formation by Clostridium butyricum strain SC-El were examined under vacuum and non-vacuum culture systems (Kataoka et al., 1997). The cultures grown without vacuum showed 2.0 to 2.3 mol H2/mol glucose and 1.4 to 2.0 mol H2/mol glucose at 0.5% and 1.0% substrate concentration, respectively. The cultures conducted at 0.28 atm under vacuum gave 1.8 to 2.3 mol H2/mol glucose and 1.3 to 2.2 mol H2/mol glucose with the same substrate concentration, respectively. In addition, the total hydrogen production rate by a two-stage bioreactor consisting of a 1 L anaerobic

159

fermenter (HRT 10 h) and a 4 L photobioreactor (HRT 36 h), feeding at 2.4 L of 1.0% glucose per day, was estimated at 1.4 to 5.6 mol H2/mol glucose, which is 1247% of theoretical values. Fermentation processes with strict anaerobes grown under thermophilic (4560 C) and extreme thermophilic (.65 C) conditions can be also investigated for H2 production, because they possibly result in higher hydrogen yields due to favorable thermodynamics and lower variety of soluble by-products (van Groenestijn et al., 2002; Abreu et al., 2012). Actually, Thermotoga maritima grown at 80 C (Schro¨der et al., 1994) and Caldicellulosirupter saccharolyticus grown at 70 C (van Niel et al., 2002) produce H2 at 4 and 3.3 mol/mol from glucose, respectively. Also, it is attractive that higher hydrolysis rates of cellulosic material have been observed in studies performed under thermophilic conditions, with the concurrent formation of higher amounts of fermentable sugars (Lu et al., 2008).

2.2 Hydrogen Production by Facultative Anaerobes Facultative anaerobes such as Escherichia coli and Bacillus spp. possess another type of metabolic pathway for hydrogen production. Although such microbes also catabolize glucose to pyruvate using the glycolytic pathway, resulting in formation of NADH2, they cannot reoxidize NADH2 using a proton as the electron acceptor, perhaps because of lack or poor activity of NADH-dependent hydrogenase. Instead, NADH2 is mainly reoxidized via formation of lactate or 2,3-butanediol from pyruvate, or ethanol from acetyl-CoA according to the nature of the microorganism. In the case of facultative anaerobes, hydrogen is produced by formate hydrogen lyase from formate that is formed in the process of conversion of pyruvate to acetyl-CoA with pyruvate formate lyase (Figure 9.1): 2CH3 COCOOH 1 2HS 2 CoA2CH3 CO 2 S 2 CoA 1 2HCOOH 2HCOOH-2H2 1 2CO2

ð11Þ ð12Þ

Since NADH2 has to be reoxidized by reduction of intermediary metabolites, the theoretical maximum yield of hydrogen from glucose is 2 mol/mol, when 1 mol ethanol and 1 mol acetate are produced from 1 mol glucose: C6 H12 O6 1 H2 O-2H2 1 CH3 CH2 OH 1 CH3 COOH 1 2CO2

ð13Þ

There is an interest in hydrogen production by facultative anaerobes, especially Enterobacter spp., because they have a high growth rate, rapidly consuming oxygen and thereby restoring anaerobic conditions immediately

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9. HYDROGEN GENERATION FROM FOOD INDUSTRY AND BIODIESEL WASTES

High Redox state C6H14O6 mannitol sorbitol

Low C6H10O7 glucuronate

C6H12O6 glucose fructose galactose

C3H8O3 glycerol

NAD+ NADH

NAD+ NADH

GAP NAD+ NADH CO2

H2

Lactate

Pyruvate NADH NAD+

Formate NADH

Acetyl-CoA

+

CO2

a-Acetolactate

NAD

CO2 Acetaldehyde

Acetyl-phosphate

Acetoin

NADH

NADH

NAD+

NAD+ Ethanol

Acetate

2,3-Butanediol

FIGURE 9.2 Simplified anaerobic catabolic pathway of various kinds of carbohydrates with different redox states by Enterobacter aerogenes. GAP, Glutaraldehyde 3P.

in the reactor, ability to utilize a wide range of carbon sources, and lack of an inhibitory effect on hydrogen generation under high hydrogen pressure. Strict anaerobes are very sensitive to oxygen and do not survive low oxygen concentration. Furthermore, strict anaerobes are also sensitive to high hydrogen pressure, and their growth and hydrogen production are severely inhibited. Tanisho et al. first reported hydrogen production by E. aerogenes E.82005 isolated from leaves of a plant (Tanisho et al., 1983). It is known that E. aerogenes produces mainly ethanol, 2,3-butanediol (BD), lactate, acetate, and formate besides hydrogen (Figure 9.2) (Johansen et al., 1975; Magee and Kosaric, 1987). They investigated the effects of pH and temperature (Tanisho et al., 1987), the usability of various substrates (Tanisho et al., 1989b), and the effect of CO2 removal (Tanisho et al., 1998) on hydrogen production. Continuous hydrogen production from molasses was also tested and yielded 20 mmol/L/h of hydrogen production rate at dilution rate 0.6/h and pH 6 (Tanisho and Ishiwata, 1994, 1995). However, the hydrogen yield from glucose was ca. 0.71.0 mol/mol, which was lower than the normal yield of Clostridia. This is due to the formation of by-products such as lactate and BD that are produced via pathways without participation in hydrogen formation.

In E. aerogenes, hydrogen is usually produced from formate, which is formed by the way of metabolism to acetyl-CoA from pyruvate via pyruvate formate-lyase. However, Tanisho et al. proposed that E. aerogenes possessed a hydrogen-producing pathway via NADH as the electron donor (Tanisho et al., 1989a). Provided that the hydrogen generation is derived from the pyruvate metabolism to ethanol and acetate formations in glucose fermentation, the maximum molar hydrogen yield should not exceed the sum of the ethanol and acetate produced. In the experiments by Rachman et al., however, even if acetate and ethanol production was reduced by mutation, hydrogen production was greatly enhanced (Rachman et al., 1997). This suggested that hydrogen should be generated from excess NADH. Furthermore, when in vitro enzymatic evolution of hydrogen from NADH was tested, both NADH and NADPH supported hydrogen formation on extract of E. aerogenes after a reaction time of 48 h. NAD(P)H-dependent hydrogenase was localized in the cell membrane (Nakashimada et al., 2002), like a membrane-bound hydrogenase previously reported for Klebsiella pneumoniae, a close relative of E. aerogenes (Steuber et al., 1999). If all NADH is converted to hydrogen, the theoretical maximum hydrogen yield is 4 mol/mol glucose, similar to that from clostridia. Breeding of such a mutant will make hydrogen production by E. aerogenes even more attractive. To improve the hydrogen yield of facultative anaerobes, we have tested a mutation of E. aerogenes HU-101, isolated from anaerobic methanogenic sludge, as a hydrogen producer with high growth rate and hydrogen production rate (Rachman et al., 1997). Since hydrogen is produced from formate, which is formed with formation of acetyl-CoA from pyruvate (Figure 9.2), it would be possible to produce mostly 2 mol H2/mol glucose by decreasing BD and lactate production that hampers hydrogen production. Such mutants can be screened by using the allyl alcohol (AA) method (Rachman et al., 1997). In this method, since AA is oxidized by ADH and/or BDDH to a toxic aldehyde (acrolein), mutants deficient in these enzymes can survive (Du¨rre et al., 1986). The hydrogen yield of AA-resistant mutant A-1 increased to 0.84 mol/mol glucose with less production of alcoholic metabolites but more production of acids, compared with the wild strain HU-101 (Table 9.2). To isolate a non- or low-lactic-acid-producing mutant, a proton suicide method can be used (Rachman et al., 1997). This method is based on lethal effects of bromine and bromite produced from a mixture of NaBr and NaBrO3 during production of acids such as lactate and acetate (Pablo and Mendez, 1990). The mutant HZ-3 screened by this method yielded hydrogen at 0.83 mol/mol glucose with less production of acids, but more alcoholic metabolite production compared with the wild strain HU-101 (Table 9.2). Double mutation by the AA

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3. EFFECT OF INTRACELLULAR AND EXTRACELLULAR REDOX STATES ON HYDROGEN PRODUCTION

TABLE 9.2 Yield of End-Products of Fermentation by E. aerogenes HU-101 and Mutants (mollmol glucose)

TABLE 9.3 Yields of Products from Various Carbon Sources by E. aerogenes

Strain

Yield (mmol/g-substrate) †

HU-101*

A-1*

HZ-3*

AY-2*

VP-1

Substrate

Formula

Cave

H2

Ethanol

Acetate

BD

Lactate

H2

0.56

0.84

0.83

1.17

1.76

Gluconate

C6H12O7

3.67

1.44

0.86

2.69

1.59

1.35

Ethanol

0.49

0.32

0.54

0.34

0.70

Glucose

C6H12O6

4.00

1.97

2.59

0.81

2.66

1.99

BD

0.37

0

0.46

0.04

0.11

Fructose

C6H12O6

4.00

2.17

2.73

1.32

2.46

1.53

Formate

0.16

0.11

0.14

0.18

ND

Galactose

C6H12O6

4.00

1.90

2.65

1.02

2.61

1.28

Lactate

0.29

0.55

0.16

0.31

0.07

Sorbitol

C6H14O6

4.33

4.96

5.80

0.74

1.27

1.08

Acetate

0.17

0.48

0.13

0.14

1.02

Mannitol

C6H14O6

4.33

5.20

5.30

0.37

1.43

2.15

Pyruvate

0.02

0.08

0.09

0.14

ND

Glycerol

C3H8O3

4.67

6.69

7.05

0.17

0.15

1.95

CO2

1.08

0.88

1.07

1.22

1.07

Adapted from Nakashimada et al. (2002). BD, 2,3-butanediol; Cave 5 (Available electrons in 1 mol compound)/(Number of carbon atoms in 1 mol compound). Culture conditions: substrate, 10 g/L; culture time, 14 h.

*

Data from Rachman et al. (1997). † Data from Ito et al. (2004). BD, 2,3-butanediol; ND, not determined.

and proton suicide methods successfully blocked production of both alcoholic and acidic metabolites and increased hydrogen yield compared with single mutation (Rachman et al., 1997). The hydrogen yield of AY-2 obtained by double mutation reached 1.17 mol mol/glucose, which is 2.1-fold higher than that of HU-101 (Table 9.2). As the latest test, we screened a mutant strain VP-1 with decreased α-acetolactate synthase activity, which was isolated using the Voges-Proskauer (VP) test (Ito et al., 2004). Since the VP test detects acetoin that is the precursor of BD, a negative mutant for the VP test is expected to be a BD-deficient strain. In pH-uncontrolled batch culture with a complex medium, a screened mutant VP-1 showed the highest hydrogen yield of 1.76 mol/ mol glucose with a decrease not only of 2,3-butanediol but also of lactate. Since the theoretical maximum yield of hydrogen is 2 mol/mol glucose, this result demonstrates that hydrogen yield can be improved to near optimum by using conventional breeding methods with mutagenesis and adequate screening strategy. Although improvement of hydrogen yield by means of genetic engineering has been intensively investigated, the availability of a conventional method to enhance hydrogen production is useful, because the use of a genetically engineered microorganism is usually very restricted and it would be difficult to gain approval to use one to treat organic wastes discharged from the food industry.

3. EFFECT OF INTRACELLULAR AND EXTRACELLULAR REDOX STATES ON HYDROGEN PRODUCTION One of the important factors in determining the diversity of fermentation end products is the intracellular redox state. Intracellular electron carriers such as NADH

and NADPH especially play an important role in this state. Their actions in numerous anabolic and catabolic reactions range widely throughout the biological system (Foster and Moat, 1980). In the case of hydrogen production in clostridia under acidogenic conditions, NADH is reoxidized with H2 formation via NADH-ferredoxin oxidoreductase and hydrogenase (see Figure 9.1) (Girbal et al., 1995). The activity of the former enzyme is regulated by the ratios of NADH/NAD and acetyl-CoA/ CoA, resulting in a disposal of excess reducing power as hydrogen (Adams et al., 1980). Regarding the facultative anaerobes such as the genera Enterobacter, Klebsiella, and Bacillus, NADH is usually used as the reductant for the production of 2,3-butanediol, ethanol, and lactate from pyruvate, but it is not used for hydrogen production. For these bacteria, hydrogen is produced from formate generated by splitting pyruvate. The intracellular redox state is affected by various environmental factors such as substrates (Tanisho et al., 1989b), culture pH (Jones and Woods, 1986), and the nature of the electron acceptor (de Graef et al., 1999). To optimize microbial hydrogen production, it is important to know the cellular responses to such conditions. Although carbohydrates (sugars) are the preferred substrate for hydrogen production, it is difficult to compare hydrogen yield between different carbohydrates. As one example, end-product yields from various carbon sources by E. aerogenes are shown in Table 9.3. Considering the hydrogen yield based on weight, the yields on fructose and galactose are similar to that on glucose, while the H2 yields on mannitol and sorbitol were 2.5-fold higher than that on glucose. The H2 yield on glycerol was even 3.3-fold higher than that on glucose. On the other hand, H2 yield on gluconate was much lower than that on glucose. Ethanol yield gives similar trends to those of H2 yield, while that of acetate

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H2 yield (mol/mol-C)

0.25 0.20 0.15 0.10 0.05 0 3.5

3.8

4.1 Cave

4.4

4.7

FIGURE 9.3 Dependence of redox state of carbohydrates on yields of metabolites per mol carbon in substrate for E. aerogenes HU-101. Symbols: yield of (’), H2; (K), lactate; (&), acetate; (ƒ) 2,3-butanediol; (), ethanol. Adapted from Nakashimada et al. (2002).

yield contradicts the trends. Lactate yield is not affected by carbon sources, but butanediol yield decreases drastically on glycerol. This result can be explained by introducing a concept of redox state of carbohydrate. To clarify the relationship between H2 production and carbon source, the available electrons per one carbon atom for each carbon source (Cave) can be defined as follows: Cave 5

Available electrons in one mole of compound Number of carbon atoms in one mole of compound

where available electrons in one mole of compound are calculated as C 5 4, O 5 22, and H 5 1. Using this equation, for example, Cave of glucose 5 24/6 5 4 and Cave of mannitol 5 26/6 5 4.33. Since Cave implies the redox state of one carbon in each compound, it can be used as the parameter to compare each redox state of compounds even if conformation and numbers of carbon of carbohydrate were different. In Figure 9.3, the relationship between Cave and product yields per mol carbon is illustrated. It is denoted that Cave of glucose, fructose and galactose, or mannitol and sorbitol is the same value because the chemical formula is the same. The figure clearly demonstrates that H2 and ethanol yields increased linearly with Cave, while the acetate yield decreased with Cave. This indicates that the redox state of carbohydrate mainly affects hydrogen productivity by E. aerogenes.

4. BIOREACTOR SYSTEM FOR HIGHRATE HYDROGEN PRODUCTION For hydrogen production from organic wastes, although the Continuously Stirred Tank Reactor (CSTR) can be used, the hydrogen production rate is as important as its overall yield for industrial use. This means that, for the practical operation of a bioreactor, a high cell density

is required. To achieve this end, a variety of reactor systems with immobilized cells using several microbial supports have been investigated. Several examples of such attempts are listed in Table 9.4. More detailed reviews about bioreactor design for dark H2 production are reported by Jung et al. (2011) and Ren et al. (2011). In an early study, the use of polyurethane foam for E. aerogenes E 82005 gave 13 mmol/L/h of hydrogen production rate using molasses as a substrate (Tanisho and Ishiwata, 1995). Agar gel or porous glass beads can be used for cell immobilization. When these supports were applied to aciduric E. aerogenes HO-39, the hydrogen production rate was 850 mL-H2/L/h from glucose (Yokoi et al., 1997b). It is also effective to use self-immobilized cells like “granules” in high-rate methane fermentation. Flocculation of E. aerogenes was reported by Tanisho and coworkers (Tanisho and Ishiwata, 1994). Additionally, Yokoi and coworkers reported that Enterobacter sp. BY-29 produced a new biopolymer flocculant consisting of polysaccharides, which caused flocculation of a suspension of kaolin, active carbon, cellulose, and yeast in the presence of Al31, Fe31, or Fe21 (Yokoi et al., 1997c). We also observed strong flocculation of E. aerogenes HU-101 in a cylindrical column reactor during continuous culture (Rachman et al., 1998). An upflow anaerobic packed-bed (UAPB) reactor with immobilized cells has several advantages over a stirred tank reactor (Figure 9.4) in terms of the lower energy demands, the high cell density per reactor volume, and the ease of scale-up due to the simple construction of the reactor. The use of flocculated cells is advantageous compared with the use of support material because the space utility in the reactor is maximized. In our study with a continuous culture, using glucose as the substrate in a UAPB reactor, cells from both strains of HU-101 and higher hydrogen-producing mutant AY-2 successfully flocculated and settled into the bottom of the reactor. Thereafter, the hydrogen production rate increased together with increase in the dilution rate in both strains. For the HU-101 strain, the hydrogen evolution rate was 30 mmol/L/h at a dilution rate 0.67/h, while for the AY-2 the hydrogen evolution rate reached 58 mmol/L/h at 0.67/h (Figure 9.5), giving a hydrogen yield of more than 1.1 at dilution rates from 0.13/h to 0.55/h, when the pH in the effluent was kept above 6.0. To increase biomass in the reactor, the concept of the upflow anaerobic sludge blanket (UASB) reactor, developed originally for high-rate methane fermentation (see Chapter 7), has also been investigated for high-rate H2 production. For example, Hu and Chen (2007) investigated H2 production with methanogenic granules as a seeding source after heat, acid, or chemical shock and observed that only chemical shock with chloroform caused irreversible damage to methanogens, while H2 productivity reached 0.48 L-H2/L/h without granular structure breakage.

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5. HYDROGEN PRODUCTION FROM INDUSTRIAL ORGANIC WASTES

TABLE 9.4 Hydrogen Production with Cell Immobilization Substrate Substrate Conc. (g/L)

H2 Production Rate (mmol/L/h)

H2 Yield (mol/molsubstrate)

Reference

Support Material

Bacteria

Porous glass beads

C. butyricum IFO13949 Glu

5

51

1.9

Yokoi et al. (1997a)

Porous glass beads

E. aerogenes HO-39

Glu

10

38

0.73

Yokoi et al. (1997b)

Urethane foam

E. aerogenes E.82005

Mol

20

13

1.8

Tanisho and Ishiwata (1995)

Self-flocculated cells

E. aerogenes AY-2

Glu

15

58

1.1

Rachman et al. (1998)

Self-flocculated cells

E. aerogenes HU-101

Gly

10

80

0.95

Ito et al. (2005)

Lignocellulose carrier

E. clocae IIT-BT 08

Glu

10

76

1.8

Kumar and Das (2001)

Activated carbon

Microflora from anaerobic sludge

Suc

18

59

1.3

Chang et al. (2002)

Glu

19

21

1.3

Hu and Chen (2007)

Pretreated methanogenic Microflora from granule granules

1.0

10

0.8

8

0.6

6

0.4

4

0.2

2

40 30 20 10 0

0.0

0 0

4

8

12

16

Residual glucose (mM)

16 50

Dilution rate (h-1)

(B)

H2 production rate (mmol/L/h)

60

(A)

12

8

4

0

Height of flocculated cells (cm)

Glu, glucose; Gly, glycerol; Mol, Molasses; Suc, sucrose.

20

Time (d)

FIGURE 9.5 Hydrogen production of E. aerogenes mutant AY-2 in an upflow anaerobic packed-bed reactor. (K), H2 production rate; (’), residual glucose; (¢) height of bed of flocculated cells from the bottom in the reactor; (&), dilution rate. Adapted from Rachman et al. (1998).

FIGURE 9.4 Photographs of (A) Upflow anaerobic packed-bed reactor for high-rate hydrogen production by E. aerogenes HU-101 and (B) self-flocculated cells in the bottom of the reactor.

5. HYDROGEN PRODUCTION FROM INDUSTRIAL ORGANIC WASTES 5.1 Carbohydrates As mentioned earlier, there are two types of hydrogen production: by strict anaerobes or facultative anaerobes. The advantage of strict anaerobes is higher hydrogen yield than that of facultative anaerobes, but they are very sensitive to oxygen and do not survive a

low oxygen concentration. On the other hand, facultative anaerobes rapidly consume oxygen, thereby restoring anaerobic conditions immediately in the reactor, although hydrogen yield is low. Thus, hydrogen production with a coculture of strict and facultative anaerobes is an attractive strategy to compensate for each disadvantage and treat actual industrial food wastes. Yokoi et al. (1998) reported that a continuous mixed culture of C. butyricum and E. aerogenes removed oxygen in a reactor and produced hydrogen from starch with a yield of more than 2 mol H2/mol glucose without any reducing agents in the medium. The repeated batch culture using the same coculture produced hydrogen with a yield of 2.4 mol H2/mol glucose under a controlled culture at pH 5.25 in a medium consisting of sweet potato starch residue and 0.1% polypepton without addition of any reducing agents (Yokoi et al., 2001).

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The use of mixed culture acclimated for hydrogen production has been investigated by several researchers. Ueno et al. (1995) found that anaerobic microflora acclimated from anaerobic sludge compost produced hydrogen from cellulose with a high efficiency of 2.4 mol/mol hexose in batch experiments at 60 C. Furthermore, Ueno et al. (1996) reported stable hydrogen production for 190 days from industrial water from a sugar factory by the same microbial flora at 0.53 days of HRT in a chemostat culture, yielding 8.8 mmol/L/h hydrogen production rate and 2.5 mol H2/mol hexose at 3 days of HRT. For continuous hydrogen production from a high-strength rice winery wastewater, Yu et al. (2002) applied microbial flora acclimated with glucose from the sludge derived from the secondary settling tank in a local municipal wastewater treatment plant. When the experiment was conducted in a 3.0 L UAPB reactor, an optimum specific hydrogen production rate of 9.33 L H2/g-VSS/day and 7.1 mmol/L/h of volumetric rate were achieved at an

HRT of 2 h, COD of 34 g/L, and pH 5.5 and 55 C, giving a hydrogen yield of 1.372.14 mol/mol hexose. We have also investigated hydrogen production from industrial wastewater from a brewery (Mitani et al., 2005) and solid bread wastes (Oki and Mitani, 2008), using the same mixed microbial flora acclimated from anaerobic sludge mainly composed of Thermoanaerobacterium thermosaccharolyticum PHE9. Regarding the hydrogen production from solid bread wastes, stable hydrogen formation for 300 days was possible in the 900 L reactor at 60 C, giving a hydrogen yield of ca. 3.2 mol H2/mol hexose and production rate of 1,600 m3 H2/day at the loading rate of 12.5 g wet weight per day of bread wastes (Box 9.1). In the effluent after hydrogen fermentation, although 5.7 g/L of hexose, 4 g/L of acetate, and 8 g/L of butyrate remained, they can be used as the influent for methane production (see Chapter 7 on methane fermentation) or photobiological hydrogen fermentation.

BOX 9.1

M AT E R I A L F L O W O F P I L O T S C A L E H Y D R O G E N F E R M E N TAT I O N O F B R E A D WA S T E S

500 L water

Solid wastes from bread factory 12.5 kg/day 111 MJ/day

Waste contents protein: 6% lipids: 4.5% carbohydrates: 38% ash: 1.5% water: 50%

Biogas flow H2: 1600 L/day CO2: 1300 L/day

Hydrogen fermentation (60oC, 500 L)

PSA H2 yield: 90%

Fuel cells Efficiency: 40%

Electrical energy 1450 Wh/day

Effluent concentration hexose: 5.7 g/L acetate: 4 g/L butyrate: 8 g/L

Methane fermentation Photobiological H2 fermentation

This is an example of material flow of hydrogen fermentation of bread wastes in a pilot scale plant carried out by Oki and Mitani (2008). Solid wastes from the bread factory are diluted with water and treated with a hydrogen fermenter, resulting in average hydrogen production of 1600 L (max. 2,500 L) from 12.5 kg wet weight of wastes per day. If hydrogen is used for electricity generation with a fuel cell system (assuming 40% of generating efficiency) after concentration of hydrogen with a pressure swing adsorption (PSA) system, 1450 Wh/day of electricity can be generated. In the effluent from the hydrogen fermenter, carbohydrates and fatty acids still remain. Thus, these can be used for further recovery of energy or material, such as methane production or hydrogen production. Source: Reprinted, with permission, from Oki and Mitani (2008).

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6. TREATMENT OF EFFLUENT AFTER DARK HYDROGEN FERMENTATION

E. aerogenes

Impure substance food oil CH2-OCOR1 | CH-OCOR2 water | CH2-OCOR3

Glycerol Ester exchange reaction

CH2-OH | CH-OH | CH2-OH

Ethanol + H2

TABLE 9.5 Microbial Production of Useful Chemicals from Glycerol Products

Microorganism

Reference

MATERIAL RESOURCES

BDF

1,3-propanediol

Klebsiella pneumoniae (NG) Clostridium butyricum (NG)

Moon et al. (2010) Hirschmann et al. (2005) Gonzalez-Pajuelo et al. (2004)

Succinate

Anaerobiospirillum succiniciproducens (NG)

Lee et al. (2001)

Lactate

Escherichia coli (NG)

Hong et al. (2009)

RnC=O

Deacidification

OMe

Impure substance Methanol/KOH

FIGURE 9.6 Schematic representation of production of biodiesel, via ester exchange reaction by alkali catalyst and H2/ethanol with facultative anaerobe, from food oil.

5.2 Food Oil (Glycerol-Rich Residue Discharged after Biodiesel Manufacturing) Biodiesel fuel (BDF) is defined as long chain fatty acid methyl or ethyl esters produced by the transesterification of vegetable oils or animal fats used as food oils (Figure 9.6). BDF has various advantages as an alternative to petroleum-based fuel: renewable, lower harmful emissions, and nontoxic. BDF can be used as fuel in diesel engines or heating systems (Eggersdorfer et al., 1992; Chowdhury and Fouhy, 1993). Although BDF is produced chemically (alkali catalyst) or enzymatically (lipase), glycerol is essentially generated as a by-product (Du et al., 2003; Vicente et al., 2004). The glycerol generated is presently applied, for example, as a resource for cosmetics, but a further increase in the production of BDF will raise the problem of how to treat efficiently the wastes containing glycerol. Microbial conversion of glycerol to more useful compounds has been investigated recently, with particular focus on the production of 1,3-propanediol, which can be applied as a basic ingredient of polyesters (Gunzel et al., 1991; Biebl et al., 1992; Petitdemange et al., 1995). As mentioned, E. aerogenes HU-101 can convert crude glycerol to H2 and ethanol with a slight production of other by-products (Table 9.5). Thus, biological production of H2 and ethanol from crude glycerol is also an attractive technology for complete energy recovery from food oil wastes (Figure 9.7). Ito et al. (2005) reported efficient production of H2 and ethanol by E. aerogenes HU-101 from crude glycerol discharged after a BDF manufacturing process. They reported that yields of H2 and ethanol decreased with the increase of concentrations of biodiesel wastes, and the production rates of H2 and ethanol in biodiesel wastes were lower than those for the same concentration of pure glycerol, partially because of high salt content in the biodiesel wastes. In continuous culture with a packed-bed reactor using self-immobilized cells, the maximum production rate of H2 from pure glycerol was 80 mmol/L/h,

Poly-β-hydroxyalkanoates Burkholderia cepacia (NG) Cupriavidus necator (NG)

Zhu et al. (2010) Cavalheiro et al. (2009)

ENERGY RESOURCES H2/Ethanol

Escherichia coli (GE) Hu and Wood (2010) Enterobacter Ito et al. (2005) aerogenes (NG)

Butanol

Clostridium pasteurianum (NG)

Biebl (2001)

Methane

Methanogenic sludge

Lopez et al. (2009)

Propionic acid

Propionibacterium acidipropionici (GE)

Zhang and Yang (2009)

Succinate

Yarrowia lipolytica (NG)

Imandi et al. (2007)

OTHERS

GE, genetically engineered strain; NG, non-genetically engineered strain.

yielding 0.8 mol/mol of ethanol from 10 g/L glycerol, while hydrogen production rate from biodiesel wastes was only 30 mmol/L/h, due to low biomass retention in the reactor. The use of a porous ceramic as a support material was effective for fixing cells in the reactor, resulting in an increase in the maximum H2 production rate from crude glycerol to 63 mmol/L/h, giving an ethanol yield of 0.85 mol/mol glycerol (Figure 9.7). This means 930 L of BDF, 145 m3 of hydrogen, and 50 L ethanol can be produced from 1000 L of food oil.

6. TREATMENT OF EFFLUENT AFTER DARK HYDROGEN FERMENTATION Dark hydrogen fermentation is an incomplete oxidation process. This means that organic matter is not completely oxidized to CO2 but to intermediate

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166 80 A 60 40 20 0 120

B

C

60

100

50

80

40

60

30

40

20

20

10

0

Glycerol in effluent (mM)

End-products in effluent (mM)

H2 production rate (mmol/L/h)

9. HYDROGEN GENERATION FROM FOOD INDUSTRY AND BIODIESEL WASTES

0 0

0.5

1.0

Dilution rate (h–1)

1.5

0

0.2 0.4 0.6 0.8 1.0 0 Dilution rate (h–1)

1.0 1.5 0.5 Dilution rate (h–1)

FIGURE 9.7 Continuous production of hydrogen and ethanol from pure glycerol with self-flocculated cells of E. aerogenes HU-101 in an upflow anaerobic packed-bed reactor, (A); crude glycerol discharged after BDF manufacturing process with self-flocculated cells (B); cells supported by a porous ceramic (C) (K), hydrogen; (&), ethanol; (¢), formate; (ƒ), 1,3-propanediol; (’), lactate; (), acetate. Adapted from Ito et al. (2005).

compounds, like short-chain fatty acids—such as acetate, propionate, butyrate, and lactate—and alcohols such as ethanol and butanol. Even if a strict anaerobe ideally catabolizes glucose for hydrogen formation, acetate is produced as the by-product (see Eq (10)). Furthermore, since the actual industrial organic wastes are a mixture of several compounds with different characteristics, such as carbohydrates, lipids, and proteins, the composition of by-products is complicated. Although it is ideal to oxidize these intermediate compounds to H2 and CO2, further oxidation of these compounds is very unfavorable in the dark condition, since these reactions are endergonic under ordinary temperatures and pressures. However, since these compounds still retain chemical energy, the recovery of the energy will contribute to effective energy or material production from organic matter.

6.1 Methane Fermentation As described in Chapter 7, the above-mentioned intermediate compounds can be further metabolized to methane, using a hydrogen and methane two-stage process. Briefly, fatty acids and alcohols can be converted to methane even if reactions are endergonic under ordinary temperatures and pressures—in methane fermentation the significant decrease of H2 partial pressure by coupling with hydrogenotrophic methane formation enables further oxidation of these compounds to hydrogen and CO2. Since methane fermentation is well established for practical use, the combination of hydrogen fermentation and methane fermentation is a realistic choice as an energy recovery process from organic wastes.

6.2 Photobiological Hydrogen Fermentation In methane fermentation, the significant decrease of H2 partial pressure by coupling with hydrogenotrophic methane formation enables further oxidation of these compounds to hydrogen and CO2. The other way to shift the endergonic reaction to an exergonic reaction is to provide energy from outside. Photoheterotrophic bacteria such as purple non-sulfur bacteria or cyanobacteria can oxidize the intermediate products in hydrogen fermentation to hydrogen and CO2 in the presence of light (Dasgupta et al., 2010). For purple non-sulfur bacteria, since fatty acids are preferred substrates rather than carbohydrates such as sugar, a two-stage process consisting of dark fermentation followed by photobiological hydrogen production can be used. These bacteria fix N2 with nitrogenase. The nitrogenase also catalyses the production of hydrogen, particularly in the absence of N2. The overall reaction is: N2 18H1 18e2 116ATP-2NH3 1H2 116ADP116Pi ð14Þ In the case that the organic substrate is converted to hydrogen, energy obtained from light is needed. When acetate is theoretically converted to hydrogen, CH3 COOH12H2 O1‘‘light energy’’-2CO2 14H2 ð15Þ Theoretical hydrogen yield from acetate, lactate, and butyrate, which are major fatty acids in the effluent from dark hydrogen fermentation, can be calculated to be 4, 6, and 10. A study with immobilized cells of Rhodopseudomonas sp. RV demonstrated that hydrogen

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6. TREATMENT OF EFFLUENT AFTER DARK HYDROGEN FERMENTATION

yields for acetate, lactate, and butyrate in the effluent from dark hydrogen fermentation were 1.6, 2.7, and 7.5 mol/mol substrate (Miyake et al., 1984). Maximum hydrogen production rate was reported to be 8 mmol/L/h (Dasgupta et al., 2010), which is much lower than that in dark fermentation (.60 mmol/L/h). Similar to hydrogenase, nitrogenase is also highly sensitive to oxygen and inhibited by ammonia ions. Thus, for photoheterotrophic hydrogen production from organic substrates, the bioreactor must usually operate under anaerobic and light conditions with illumination and limiting concentrations of nitrogen sources. Additionally, since photoheterotrophic hydrogen production essentially needs light energy, the reactor must be designed to allow penetration of light sufficient for efficient production, resulting in a two-dimensional reactor such as a flat or tubular type. This requires a huge place for extremely large-scale production, compared with dark fermentation. Thus, although a combination of dark and photobiological fermentation for sole hydrogen production from organic wastes is very attractive for practical use, dramatic enhancement of hydrogen productivity would be needed by means of breeding of photoheterotrophic bacteria and optimization of reactor design and culture condition. Several photobioreactors have been proposed for hydrogen production and recently reviewed by Dasgupta et al. (2010). Although the photobioreactors can be broadly classified into open system (i.e., raceway pond, lakes, etc.) and closed systems, the closed systems are used for hydrogen production because open systems cannot provide the anaerobic conditions needed and offer poor possibility for the collection of produced gas. Since light energy is essentially needed for H2 production in a closed photobioreactor, the Biogas

167

fundamental design factor of photobioreactors is transparency, which allows maximum penetration of light and a large surface area. Thus, the thicknesses of the reactors are usually small to avoid a shading effect by the growing cells, and a major issue limiting the largescale production of hydrogen is the restricted light penetration into the deeper regions of the reactor. Several types of bioreactor have been designed to improve hydrogen production (Figure 9.8). Among them, vertical-column (Figure 9.8A), tubular (Figure 9.8B), and flat-panel (Figure 9.8C) types of photobioreactor are widely used for hydrogen production. Vertical-column photobioreactors may be airlift or bubble column reactors. They consist of vertical transparent tubes in which agitation is achieved with the help of bubbling at the bottom. The illumination area is small, but still they are widely used for photosynthetic bacteria, owing to the compactness, low cost, ease of operation, and low shear stress in airlift and bubble columns (Asada and Miyake, 1999). Tubular photobioreactors consist of long transparent tubes with diameters ranging from 3 to 6 cm, and lengths ranging from 10 to 100 m. The culture liquid is pumped through these tubes by means of a mechanical or airlift pump. The tube can be positioned in many different ways such as in horizonal, vertical, or helical planes, although the shape of the light gradient in the tubes is similar in most designs. Compared with the vertical-column type, a large illumination area can be achieved in the tubular type (Molina et al., 2001). The length of the tubes is limited because accumulation of produced hydrogen gas inhibits hydrogenase activity, although this might not be so important for a nitrogenase-based process. Flat-panel reactors consist of a rectangular transparent box with a depth of only 1 to 5 cm. The reactors are

Biogas

Biogas Biogas

Biogas recycle

Inert gas (Argon, N2)

Biogas recycle

Inert gas

Inert gas Bubble column

Biogas recycle

Biogas recycle

Inert gas

Airlift reactor

A. Vertical-column reactor

B. Tubular reactor (Horizontal type)

C. Flat-panel reactor (Vertical type)

FIGURE 9.8 Schematic drawings of various kinds of photobioreactor for H2 production.

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9. HYDROGEN GENERATION FROM FOOD INDUSTRY AND BIODIESEL WASTES

mixed with gas introduced via a perforated tube at the bottom of the reactor. Usually the panels are illuminated from one side by direct sunlight, and the panels are placed vertically or inclined towards the sun. High photosynthetic efficiencies and effective control of gas pressure can be achieved in flat-panel photobioreactors, and they have been found to be more economical than other bioreactors (Lehr and Posten, 2009).

7. CONCLUDING REMARKS Hydrogen has become a focus of attention as the energy of the future. Therefore, dark hydrogen fermentation from biomass wastes has been investigated using various types of microorganisms and bioreactors. The technology will make a large contribution to the improvement of the global environment, because biologically produced hydrogen from biomass is renewable and clean. Indeed, the development, construction, operation, and regulation of proper processes is still challenging, and further research is needed before practical application of biological hydrogen production is possible at a large scale. Especially, efficient use of soluble by-products of hydrogen fermentation, such as alcohols or fatty acids, is important. In this context, crude glycerol discharged after biodiesel production from vegetable oils is an ideal feedstock for hydrogen production, because the main by-product is ethanol that can be directly used as a liquid fuel. However, the usual by-products from other organic wastes are a combined mixture of alcohols and acids. For efficient use of such a mixture, a hydrogen production process combined with methane production might be a candidate because of improvement of the overall energy yield from feedstocks. Combination of dark fermentation with photofermentation is another candidate, because it will achieve maximum overall hydrogen yield, although drastic improvement of the hydrogen productivity of photofermentation is needed. Overall, dark hydrogen fermentation production is a promising process for sustainable supply of renewable hydrogen for economic and commercial applications in the future.

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