Hydrogen production from glycerol by Escherichia coli and other bacteria: An overview and perspectives

Applied Energy 156 (2015) 174–184

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Applied Energy journal homepage: www.elsevier.com/locate/apenergy

Hydrogen production from glycerol by Escherichia coli and other bacteria: An overview and perspectives Karen Trchounian a,b, Armen Trchounian a,⇑ a b

Department of Microbiology, Plants and Microbes Biotechnology, Yerevan State University, 1 A. Manoukian Str., 0025 Yerevan, Armenia Department of Biophysics, Faculty of Biology, Yerevan State University, 1 A. Manoukian Str., 0025 Yerevan, Armenia

h i g h l i g h t s  Glycerol is a perspective carbon containing source to produce H2 by Escherichia coli.  Metabolic pathways, responsible hydrogenases and dependence of H2 production on external factors are summarized.  H2 production can be improved by glycerol added to glucose containing medium.  H2 production biotechnology would be further developed using glycerol as a feedstock.

a r t i c l e

i n f o

Article history: Received 20 March 2015 Received in revised form 25 June 2015 Accepted 4 July 2015

Keywords: Biohydrogen production Glycerol Dark fermentation Hydrogenases E. coli and other bacteria

a b s t r a c t Hydrogen (H2) is a clean, effective and renewable fuel which can be produced by different methods including biological ones, namely fermentation and biophotolysis. To improve fermentative H2 production the strategies, implicating use of by-products, utilization of carbon containing organic wastes and optimization of biotechnology process conditions, are developed. Glycerol, a biodiesel by-product, can serve as a cheap carbon containing source to produce H2 by Escherichia coli. Recent data on metabolic pathways, responsible hydrogenases and dependence of H2 production on external factors during glycerol fermentation are summarized. The strains are constructed to enhance H2 yield. The mixed carbon sources (glycerol and glucose) fermentation is a novel approach: glycerol added to glucose containing medium increases H2 production; different carbon sources comprising wastes can be used. H2 production from glycerol by different bacteria is overviewed; cultures types, new technologies and optimal conditions, purification of H2 and developing bioreactors are highlighted. All of these are significant for further developing H2 production biotechnology from glycerol and perspective for applied energy systems. Ó 2015 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9.

Hydrogen as a perspective fuel and estimation of its global market . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biological methods in global hydrogen production and dark fermentation by Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Novel discovery of glycerol fermentation by E. coli and biohydrogen production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glycerol fermentation by E. coli: pathways, different from sugar fermentation, and bioenergetic advantage . . . . . . . . . . . . . . . . . . Hydrogenases for hydrogen production by E. coli during glycerol fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Proton ATPase, proton-motive force and hydrogenases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Metabolic engineering of E. coli to improve hydrogen production from glycerol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrogen production by E. coli during mixed carbon sources (glycerol and glucose) fermentation . . . . . . . . . . . . . . . . . . . . . . . . . Glycerol fermentation and hydrogen production by different bacteria; cultures types, new technologies and optimal conditions

⇑ Corresponding author. Tel.: +374 60710520. E-mail address: [email protected] (A. Trchounian). http://dx.doi.org/10.1016/j.apenergy.2015.07.009 0306-2619/Ó 2015 Elsevier Ltd. All rights reserved.

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10. 11.

Purification of biohydrogen produced from glycerol and developing bioreactors . . . . . . . . . . . . Concluding remarks and perspectives for developing effective systems for energy production . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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181 181 181 181

1. Hydrogen as a perspective fuel and estimation of its global market

2. Biological methods in global hydrogen production and dark fermentation by Escherichia coli

Molecular hydrogen (H2) is a valuable gas as an effective, clean and renewable energy source and as feedstock for chemical, food, pharmaceutical and some other industries, metallurgy, production of electronic devices. H2 is also used in space exploration, especially in space shuttles. Importantly, high energy (142 kJ g1; 3.5 time higher than oil) is released and only water (H2O) but not carbon dioxide is formed during H2 combustion [1,2]:

Different methods have been developed for H2 production. Electrolysis of water, steam reforming of methane or hydrocarbons, gasification of coal, oxidation of oil and natural gas, and auto-thermal processes are well-known and largely used methods for global H2 production, but not cost-effective due to high temperature requirements (>700 °C), non-friendly method for environment (greenhouse gas emission) and non-renewable one (except electrolysis of water) (Fig. 1) [1,2]. Biological production of H2 (biohydrogen) includes dark and photo-fermentation of carbon containing substrates, biophotolysis of water by bacteria and microalgae (see Fig. 1). The theoretical maximum yields for H2 production by these methods can be different according to the well-known reactions for:

2H2 þ O2 ! 2H2 O: H2 has low radiation level and relatively safe: the explosion limits by volume for H2 in air of 18.3–59% are much higher than those for gasoline (<3.3%) and natural gas (<14%) [3]. Moreover, H2 is not available in nature but it can be produced from unlimited sources like water or renewable ones like biomass and carbon containing wastes. Therefore, demand on H2 production has increased considerably in recent years and H2 can ultimately replace oil and natural gas. H2 is produced in both small-scale and large-scale volumes. World production of H2 is more than 50 million tons [4] and according to the International Energy Agency estimated to be of 65 million tons by production capacities in China, USA, European and other countries [5]. H2 production is increased over the world rapidly (6–10%); it will decrease costs of H2 production which is competitive with oil and natural gas [3]. This can improve the efficiency and reliability of energy systems. H2 is already used in gas stations (>200 stations distributed in North and South America, Europe, Asia and Australia and 1000 stations will be constructed by 2025) for engines of different transport vehicles (cars, buses and ships are made by different companies like Daimler, Ford, Nissan, Mercedes, BMW etc) and in fuel cells to generate electric power [3,6]. As for growing economy, proposed global energy demand by 2050 will be tripled, and produced fossil fuels cannot satisfy energy market. Thus, very soon H2 and fuel cells can become one of the main components in global energy economy.

Fig. 1. Comparison of chemical and biological methods for H2 production with their advantages (high yield, low temperature, low cost) and disadvantages (low yield, high temperature, high cost).

dark fermentation of sugars ðglucose; C6 H12 O6 Þ  C6 H12 O6 þ 6H2 O ! 12H2 þ 6CO2 ; photo-fermentation of organic acids ðacetic acid; C2 H4 O2 Þ  C2 H4 O2 þ 2H2 O þ light ! 4H2 þ 2CO2 or ðsuccinic acid;C4 H6 O4 Þ  C4 H6 O4 þ 4H2 O þ light ! 7H2 þ 4CO2 ; biophotolysis of water  2H2 O þ light ! 2H2 þ 6O2 ðdirectÞ or

Fig. 2. Combined putative metabolic pathways of glycerol and glucose fermentation in E. coli. The pathways are adapted from [2,18,20,23–25]. Linear arrows indicate pathways only for glycerol fermentation, broken arrows indicate pathways only for glucose fermentation, and solid broken arrows indicate pathways for both glucose and glycerol fermentation. The end products are formatted as italics. 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate; AcCoA, acetyl-Coenzyme A; ADP, adenosine diphosphate; ATP, adenosine triphosphate; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; GAP glyceraldehyde-3-phosphate; NADH, dihydrodiphosphopyridine nucleotide; NAD+, diphosphopyridine nucleotide; PGP, 1,3-diphosphate glycerate; G6P, glucose 6 phosphate; F1,6P, fructose-1,6 diphosphate; AP, Acetyl -phosphate.

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12H2 O þ light ! 12H2 þ 6O2 ðindirectÞ:

12000

There is also the method of bioconversion of carbon oxide (CO) to H2 by bacteria when H2 is produced from water according to the reaction:

10000

Biohydrogen has significant advantages over chemical methods since it could be performed at relatively low temperatures (25–37 °C), atmospheric pressure and with relatively high rates (see Fig. 1). This can reduce the costs for H2 production. There is one important argument: biohydrogen production creates an opportunity to develop decentralized energy systems when energy production plants can be located not so far from carbon containing resources. Moreover, generation of different contaminants with high level, including volatile fatty acids, can be avoided. Therefore, biohydrogen production will be increased in upcoming years. Among biological methods dark fermentation with microbes, including bacteria, is the best promising one [2,7], and Escherichia coli is well-studied facultative anaerobic bacteria [8,9] performing mixed-acid fermentation of carbohydrates (sugars), which are common and largely present and stored in plant tissues, and of other organic carbon sources – different wastes. This bacterium is easy for manipulation in lab and large-scale conditions because of difficulty in maintaining strict anaerobic conditions. Fermentation pathways in E. coli are complex (Fig. 2) [10]. But due to known genome sequence and analysis (defined transcription and translation systems, broad spectrum of already constructed mutant strains), metabolic flux analysis, as well as the ability of fast growth on cheap substrates (wastes), they can be genetically and metabolically engineered for constructing highly effective strains with increased H2 production [8], compared to the theoretical maximum yield of 12 mol H2 per mole of glucose (see reaction above) [9,11,12]. However, sugars are expensive, and this maximum is hard to be reached by bacteria even of external energy supply; 4 mol H2 per mole of glucose is considered as nowadays reality. Moreover, some mechanical, thermal (<150 °C), alkaline or acidic, ultrasonic or other pretreatment of different resources and wastes is recommended; but this requires additional costs. Interestingly, dark fermentative production of H2 is also more efficient and rate is higher than H2 production by photosynthetic pathways; the cost of fermentative H2 production is 300-fold lower than of photosynthetic one [7]. There is also one important property of E. coli – this is a non-pathogenic (conditionally pathogenic) bacterium. And H2 production is stable for a relatively long period (several tens of days, depending on culture type). Thus, dark fermentation has advantages due to rapid H2 production, relatively high H2 yield, no solar light requirement, stable bacteria and low cost.

Biodiesel, milion L

CO þ H2 O ! CO2 þ H2 :

USA EU

8000

Brazil Indonesia

6000

China Thailand

4000

2000

0 2010

2011

2012

2013

2014

Fig. 4. Biodiesel production in developed countries (USA, EU, Brazil, Indonesia, China and Thailand) during 2010–2014. Data are from US Energy Information Administration, Form EIA-22 M ‘‘Monthly Biodiesel Production Report’’; USDA Foreign Agricultural Service Global Agricultural Information Network Reports # NL4025, # ID1420, #BR13005, #CH14038, TH4057.

A 2H++2e- ->H2

GLYCEROL

Hyd-4 FORMATE

In Hyd-2

GlpF

Hyd-1

Hyd-3

Out +

GLYCEROL

-

H2->2H +2e pH 7.5 2H++2e- ->H2

GLYCEROL

B Hyd-4 In

FORMATE Hyd-2

GlpF

Hyd-1

Hyd-3

Out GLYCEROL

H2->2H++2e-

pH 5.5 Fig. 5. H2 cycling and responsible Hyd-enzymes in E. coli cell membrane during glycerol fermentation. These schemes are adapted from [2,13,25,34]. The GlpF is glycerol transporter [19]. The direction of enzyme operation to produce and/or to oxidize H2 is shown (arrows) at pH 7.5 (A) and 5.5 (B).

Fig. 3. Strategies for main approaches to enhancing of H2 production by bacteria.

Different strategies are developed for the improvement of fermentative H2 production and lowering the costs of its production [2,9,11,12], including use of by-products, utilization of carbon containing organic wastes, construction of effective bacterial strains and optimization of biotechnology process conditions (Fig. 3). The most important problem is to find out a cheap substrate for

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fermentation and to construct effective strains with stable responsible enzymes with high activity of H2 production. 3. Novel discovery of glycerol fermentation by E. coli and biohydrogen production It has been recently discovered by Gonzalez’s group [13] that glycerol (C3H8O3) can be fermented by E. coli; and H2 is detected among the end products during glycerol fermentation at slightly acidic pH (pH 6.3). Furthermore, glycerol fermentation and H2 liberation have been also revealed by our group [14] at slightly alkaline pH (pH 7.5) in batch culture. This is very intriguing phenomenon applicable in clean and sustainable energy production because (1) glycerol, a biodiesel by-product (1 kg crude glycerol per 10 kg biodiesel), or product of biomass, vegetable oils and animal fats, is a very cheap (prices of crude glycerol is 3–10 cents/lb and reformed glycerol is 40–50 cents/lb); (2) glycerol is largely available (hundreds of plants in different countries worldwide in North and South America, Europe and Asia with large-scale biodiesel production (Fig. 4); average annual growth of biodiesel market is estimated to be of 42%; crude glycerol could be reformed into pure one) and (3) glycerol is effective renewable carbon containing source for obtaining biomass, bacterial fermentation and H2 production, compared to sugars and other organic carbon sources [1,2,15]. Crude glycerol from biodiesel as well as pure glycerol has rapidly become a competitive raw material to produce different fuels and chemicals, compared with sugars (glucose) [11–13]. The crude glycerol has an acidic (pH 2.4) to alkaline pH (pH 12) and contains 45–88% (v/v) glycerol; however, pure glycerol (>98%) has an acidic pH (pH 5.5). Glycerol is completely soluble in water (>500 g L1 at 20 °C) and stable. These properties of crude and pure glycerol are important for application to produce H2. The high degree of reduction of carbon atoms in glycerol confers the ability to produce H2 at higher yields when compared with glucose [15,16]. On the other hand, the theoretical yield is 7 mol H2 for every mole glycerol according to the reaction [17]:

activity and operation mechanisms during glycerol fermentation at different environmental conditions, especially pH, cultures types and new technologies based on glycerol would be of great importance. 4. Glycerol fermentation by E. coli: pathways, different from sugar fermentation, and bioenergetic advantage Glycerol can permeate into the cell of E. coli [19]. The glycerol fermentation pathways within the cell are relatively simple inter-linked biochemical reactions known from glycolysis [2,20–23]. But unlike glycolytic pathways they have a unique coupled reaction on the level of phosphoenolpyruvate (PEP) conversion into pyruvate (PYR) generating a cycle pathway (see Fig. 2). These pathways of glycerol fermentation can be presented as shown, at the stage of PEP some intermediates may be used for succinate formation, whereas all other end products, including formic acid (HCOOH), are formed from PYR [10,24,25] (see Fig. 2). The glycerol fermentation pathways are not clear yet although succinic, acetic and formic acids and ethanol as well as H2 are shown to be produced but lower acetic and less lactic acids could be ended from glycerol [18]. Especially, H2 can be produced by E. coli from formate [26–28]. Moreover, additional ATP could be obtained (see Fig. 2): glycerol fermentation has a bioenergetic advantage. These differences between the fermentations of glycerol and sugars by E. coli are important enough and could reveal novel pathways to regulate H2 production. Importantly, H2 generated from formate could be evolved whereas CO2 (see Fig. 2) might be used for cell growth [13,29]. No other gaseous products can be ended from dark fermentation. Interestingly, glycerol concentration of 10 g L1 has been shown to be optimal for E. coli cell growth and H2 production: a higher concentration of glycerol can decrease H2 production rate and yield [9,14,18,22]. This is likely to the effect of sugars (glucose) concentration on H2 production by E. coli when the highest yield was obtained with culture limited for glucose [9]. 5. Hydrogenases for hydrogen production by E. coli during glycerol fermentation

C3 H8 O3 þ 3H2 O ! 7H2 þ 3CO2 : It is difficult to suggest so high yield in the application of glycerol to produce H2 by bacteria, however it can be reached. But H2 produced by E. coli has negative impact on the cell growth and glycerol fermentation [18]. To avoid this effect, gaseous H2 removal and cumulating can be essential steps to improve H2 production and should be used in continuous culture. An updated overview of H2 production from glycerol by E. coli and other bacteria with perspectives for developing of effective systems of energy production is provided in the present paper. The glycerol fermentation pathways, the mechanisms of hydrogenase (Hyd) enzymes biosynthesis, determination of their specific

E. coli processes multiple membrane-associated [Ni–Fe]containing Hyd enzymes to produce H2 due to simple redox reaction [2,16,25,30,31]:

2Hþ þ 2e ! H2 : These enzymes are responsible for the stable H2 production. However, these enzymes not only produce but also oxidize H2 [11,16,25,32–35], and therefore H2 cycling is suggested (Fig. 5). The latter is a novel phenomenon in the bioenergetics of mixed-acid fermentation and can play a role in maintaining a

Table 1 H2 production by E. coli Hyd enzymes during glycerol fermentation in batch culture at different pHs. H2 productiona

Characteristics or properties of Hyd enzymes

H2 production rate in wild type, mmol H2 min Responsible Hyd enzymes Recycling H2 Inhibition by DCCD Requirement of the FOF1-ATPase Coupled generation of a proton-motive force Intracellular pH Sensitivity to osmotic stress a b

1

1

(g dry weight)

pH 7.5

pH 5.5

4.57 Hyd-2 is major, and Hyd-1 is less +b  + + 7.0 Hyd-3 and Hyd-4

6.79 Hyd-3 + + + + 5.7 Hyd-1, Hyd-3 and Hyd-4

For references, see the text. The signs ‘‘+’’ or ‘‘’’ represent the presence or the absence of the characteristics, respectively.

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proton-motive force [31]. Interestingly, the standard redox potential of 2H/H2 couple is 414 mV: this value depends on H2 steady state concentration and can be lowered to 270 mV [16]. The range of redox potential is important in determining H2 production and oxidation mode of Hyd enzymes. It is also important that genes coding these Hyd enzymes and proteins participating in their maturation are known, mechanisms of their expression and regulation are complex and have not been understood well [10,32,36–42]. However, manipulating with these genes is an effective tool to regulate and especially to increase H2 production. The problem is to determine Hyd enzymes, which are responsible for H2 production during glycerol fermentation, and to reveal their dependence on external and other factors. The principal findings on Hyd enzymes and H2 production are clearly shown with E. coli during glycerol fermentation and summarized in Table 1. E. coli wild type and mutant strains with defects in different genes coding Hyd enzymes and their maturation have different Hyd enzymatic activity and H2 production rate depending on pH. Hyd-2 (hyb) mostly and Hyd-1 (hya) partially are responsible for H2 production at slightly alkaline pH (pH 7.5); Hyd-3 (hyc) and Hyd-4 (hyf) can work in H2 oxidizing mode (see Fig. 5) [2,14,34]. H2 producing Hyd-2 activity is confirmed by the other groups [43,44] and by the results with E. coli hya hyc or hyb hyc double mutants [45]. In the stationary growth phase and in the absence of Hyd-3, Hyd-2 can be forced to evolve H2 at pH 7.5 [45]. Thus, the major contribution of Hyd-2 to H2 production during glycerol fermentation at pH 7.5 resulted from changed metabolism and surprisingly influenced on proton reduction. At the same time, H2 production by E. coli using glycerol is 1.5-fold higher at pH 5.5 than at pH 6.5 and Hyd-3 is a major H2 producing enzyme during glycerol fermentation at acidic pH (see Fig. 3) [34]. The latter seems to be clear due to the result decade ago that in addition to pH 6.5 Hyd-3 becomes a major H2 producing enzyme also at pH 7.5 during glucose fermentation when formate (30 mM) is supplemented [26,27]. All these findings with Hyd enzymes, which are responsible for H2 production at different pHs, are significant for manipulation to produce substantial amount of H2 depending on external conditions. Otherwise it is possible to have incorrect conclusion on the absence of substantial H2 production by E. coli using different substrates when modified strain (hyaB hybC hycA) lacking Hyd enzymes was used and aerobic conditions were applied [46]. It is very interesting that H2 production rate determined in liquid and gaseous phases was markedly different during glycerol but not glucose fermentation by E. coli: a significantly (30-fold) more H2 is observed in liquid [46]. This suggests an increased role of H2 cycling upon glycerol fermentation.

Then, proton-motive force is generated at different pHs. The intracellular pH and, hence, proton-motive force were lower at pH 7.5 compared with those during glucose fermentation [50]. Proton motive force generation is changed in E. coli hypF mutant lacking all Hyd enzymes [50] due to the effects on the FOF1-ATPase too [52]. Therefore, Hyd impact on an overall proton-motive force generation is disclosed; H+ pumping by Hyd is suggested. H2 production is inhibited completely by 0.5 mM N-ethylmaleimide (NEM) [50,51], an inhibitor of glycerol kinase [53], which catalyzes the formation of GP and then DHAP (see Fig. 2). However, NEM had no effect on membrane potential [51] suggesting different mechanism for NEM action on glycerol metabolism and H2 production by E. coli. Moreover, H2 production is very sensitive to diphenylene iodonium (Ph2I), an inhibitor of [Ni–Fe]-Hyd enzymes in the other bacteria [54]: even 1 nM Ph2I inhibited completely H2 production [51]. These results indicate the absolute role of known Hyd enzymes in H2 production during glycerol fermentation. Besides, H2 production from glycerol is sensitive to osmotic stress provided by sucrose (see Table 1): Hyd-3 and Hyd-4 are sensitive to osmotic stress but at different pHs, whereas Hyd-1 is osmosensitive at low pH [55]. Importantly, these data have been mainly obtained by using electrochemical determination of H2 with a pair of oxidation– reduction (redox) titanium-silicate and platinum electrodes, as described elsewhere [14,22,30,35,47,56–58]. In contrast to platinum electrode, titanium-silicate electrode is not sensitive to H2 and oxygen; therefore this pair of redox electrodes allows detecting exclusively H2 in liquids during fermentation under anaerobic conditions (in the absence of oxygen). Various controls have ruled out interference by the other fermentation end products (see Fig. 2). This approach is close to the method with Clark-type electrode employed by different groups in worldwide labs [46,59–62]: a good correlation between redox potential measuring different redox electrodes readings difference and H2 production was shown in liquids. These redox electrodes have a high sensitivity to H2, a long life expectancy and can be applied to control H2 production in different systems. Moreover, the results obtained with redox electrodes have been confirmed by chemical (with using permanganate in sulfur acid [63,64]) and other (for instance, with Durham tubes [65]) methods. There is a problem with comparison of data on H2 yield since this parameter is given by different groups in moles of H2 per mole of substrate, dry weight of biomass or volume of culture.

6. Proton ATPase, proton-motive force and hydrogenases

7. Metabolic engineering of E. coli to improve hydrogen production from glycerol

E. coli wild type and atp mutant lacking the proton FOF1-ATPase also have different Hyd enzymatic activity and H2 production rate depending on pH [47]. Hyd activity was inferred by native PAGE electrophoresis to be dependent on the active FOF1-ATPase during glycerol as well as glucose fermentation, especially at extreme pHs [48]. This might be resulted from link between Hyd enzymes and the FOF1-ATPase, physiological role of which is in maintaining a proton-motive force, key intermediate in energy conversion in cells [31,49]. H2 production is inhibited by N,N0 -dicyclohexylcarbodiimide (DCCD), inhibitor for the FOF1-ATPase [49]: DCCD inhibited H2 production at acidic (pH 5.5) but not slightly alkaline (pH 7.5) medium (see Table 1). It should be noted that DCCD inhibition was reversed during glucose fermentation [34,49]. Moreover, under glycerol fermentation Hyd-2 activity could disappear by protonophore action indicating the requirement of a proton-motive force [50,51].

The progress with glycerol fermentation pathways and enhanced H2 production from glycerol has been achieved by metabolic engineering of E. coli [2,9,14,18,21,43]. Potential strategies for increasing H2 production by Hyd enzymes have been outlined and whole-cell systems and cell-free systems were compared recently by Wood’s group [66]. It is possible to redirect metabolic pathways, to induce the DHAP production pathway, to block lactic, acetic acids and ethanol production (see Fig. 2). Indeed, Hu and Wood [67] obtained an improved strain with H2 production of 0.68 mmol H2 L1 h1 in glycerol medium; this was 20-fold increased H2 production compared with precursor one. A new strain with 5-fold enhanced H2 yield has been created since the old one for H2 production from glucose was not suitable [68]. Moreover, Wood with co-workers [43,69] have identified uncharacterized genes (54% of E. coli genome is experimentally determined; the other part is uncharacterized or computationally predicted) whose inactivation

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K. Trchounian, A. Trchounian / Applied Energy 156 (2015) 174–184 Table 2 Comparison of H2 production by E. coli from crude and pure glycerol as substrates for dark fermentation. Type of glycerol Crude (80% glycerol) Pure (>98%) a

Culture medium composition and pH a

Characteristics of H2 production

1

1

MOPS minimal media; 10 g L tryptone, 5 g L yeast extract (pH 6.3) MOPS minimal media; 10 g L1 tryptone, 5 g L1 yeast extract (pH 6.3)

References 1

1

Maximal yield of 13 mmol H2 L at 10 g L glycerol Maximal yield of 11.5 mmol H2 L1 at 10 g L1 glycerol

[72] [72]

3-(N-morpholino) propane sulfonic acid.

is beneficial for H2 production from glycerol. They found out the strains with ability for 1.6-fold higher H2 production and 2.1-fold higher H2 yield. The yield of H2 reached was 95% of the theoretical maximum but this was not perfection. However, the conditions, especially pH, are important in using these mutant strains for H2 production to have maximal activity of responsible Hyd enzymes (see Table 1). The results can be explained due to that the strains produce more formate and less ethanol and acetate than the wild type during glycerol fermentation (see Fig. 2), as shown [18,45], and the transcription of genes for Hyd enzymes might be increased as determined for hycE during glucose fermentation [70]. Interestingly, the mutant strains grow faster on glycerol than parent strains so they achieve a reasonable anaerobic growth rate [67,69] which is important for biomass synthesis in biotechnology. Interestingly, a new model for metabolic engineering based on an in vitro synthetic enzymatic pathway has also been developed to enhance H2 production from biomass in a distributed, carbon– neutral and low-cost manner [71]. The latter resulted in an increase of 67-fold in H2 production by E. coli. This approach might be applied for effective H2 production from glycerol. This is a good basis to consider glycerol as a perspective substrate to produce H2 with high rate and yield due to effective external factors and metabolic engineering as well. 8. Hydrogen production by E. coli during mixed carbon sources (glycerol and glucose) fermentation It is interesting that both crude and pure glycerol can result in a high H2 yield by E. coli; however, use of crude glycerol instead of pure glycerol as a carbon source did not change H2 production (Table 2) [72]. Given this finding, the useful approach to increase H2 production would be determining H2 production detected during the mixed carbon sources (pure glycerol and glucose) fermentation:  Glycerol could be fermented in the presence of sugars (glucose) at slightly alkaline pH, and DCCD inhibited H2 production in a pH-dependent manner; especially, total inhibition of H2 production was observed in fhlA (gene for transcriptional activator of hyc [73]) mutant at pH 7.5 and pH 5.5 suggesting that FhlA might directly relate to the FOF1-ATPase. Optimal concentration was 10 mM glycerol. In addition, Hyd-4 activity mainly and

Table 3 Changes in H2 production rates by E. coli wild type strain (BW25113) upon glycerol added into the peptone medium with glucose. H2 production by the cells grown on mixed carbon sources (glucose and glycerol) was assayed with either adding glucose (glucose assay) or glycerol (glycerol assay) in the same concentrations as during the growth [57]. For comparison, H2 production rates by E. coli grown on sole glucose or sole glycerol added into the peptone medium were determined. The standard errors were not more than 3%. pH

H2 production rate (mmol H2 min1 (g dry weight)-1) 0.1% glucose

7.5

8.43

0.5% glycerol

3.70

0.1% glucose + 0.5% glycerol Glucose assay

Glycerol assay

15.54

9.60

Hyd-2 activity to some extent to produce H2 have been revealed at low pH [42,74]. Moreover, Hyd-1 and Hyd-2 formed H2 oxidizing activity at pH 7.5.  Glycerol added into the medium with glucose increased significantly (2- and more fold) H2 production [57]; this was observed at slightly alkaline pH (Table 3). However, the decrease in H2 production was shown for acidic pH [57]. Thus, glucose and glycerol concentrations might have significant role in H2 production rate changing.  Formate added into the medium with glycerol stimulated E. coli growth and H2 production at different pHs [75,76]. Interestingly, formate (10 mM) recovered H2 production of Hyd-2 (hybC) or Hyd-4 (hyfG) mutants depending on pH [75,76]. These results highlight the key role of Hyd-3 at both pH 6.5 and pH 7.5, as well as the role of Hyd-2 and Hyd-4 at pH 7.5 for H2 production by E. coli during glycerol and formate co-fermentation. The mixed carbon sources fermentation is novel phenomenon, its pathways are complex but particularly clear as represented (see Fig. 2); a detailed study is required. However, this approach has been already employed with different fermentation substrates and different bacteria [2,11,12]. In addition to strategies already designed (see Fig. 3), the mixed carbon sources fermentation might be used in biotechnological applications for pre-cultivation of bacterial cells, regulation of Hyd enzymes activity towards enhancing H2 production and utilizing glycerol and other carbon sources containing industrial, agricultural and food (kitchen) wastes as well as wastewater. There are both economic (cheap wastes) and environmental (wastes utilization) benefits. 9. Glycerol fermentation and hydrogen production by different bacteria; cultures types, new technologies and optimal conditions Glycerol fermentation has been established in different cultures types - batch culture with changing conditions and continuous steady-state culture using different approaches with several bacteria like Klebsiella pneumoniae [77], Halanaerobium saccharolyticum [78], Clostridium sporogenes [46], Enterobacter sp. and Citrobacter freundii [79] and many others [46,80–89]; and H2 is among the end products of higher yield (Table 4). Probably, glycerol can be fermented by different bacteria due to specific enzymes namely glycerol dehydrogenase catalyzing the first steps of fermentation pathways to DHA (see Fig. 2). H2 production from glycerol was concentration dependent and depended on crude or pure glycerol as well as on medium composition, adaptation time and other conditions. But higher glycerol concentration has been shown to decline H2 yield enhancing by Bacillus thuringiensis [88]; this might be related to H2 negative impact on glycerol fermentation, as suggested with E. coli (see above). It was reported that the production of H2 decreased with an increase of the concentration of biodiesel wastes [90]. Therefore, dilution of glycerol or continuous culture might increase H2 production. Moreover, glycerol based on organic wastes has been evaluated for H2 production by Clostridium sp. or Enterobacter aerogenes using different – response surface methodology [83,84,86] (see Table 4). Recently, immobilization of E.

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Table 4 H2 production from glycerol by different bacteria during dark fermentation; culture types used.

a

Bacteria

Culture type; fermentation method

pHa

Characteristics and efficiency of H2 production from glycerol

References

Bacillus thuringiensis

Batch and continuous culture

7.0

[88]

Clostridium pasteurianum

Batch and continuous culture

7.0

Clostridium sporogenes

Batch culture

7.3

Enterobacter aerogenes

Batch and continuous aerobic culture, bioreactor

Enterobacter aerogenes

Response surface culture

5.0

Halanaerobium saccharolyticum

Batch culture

7.0–7.4

Klebsiella pneumoniae

Batch culture, bioreactor

Klebsiella sp.

Batch culture, response surface culture

Increased production yield of 0.646 and 0.748 mol H2/mol glycerol by 20 g L1 glycerol in minimal salt medium (2% ammonium nitrate and 1% sodium nitrate, respectively) and 2.2 mol H2/mol glycerol in complex media in batch culture with small volume (250 mL); 0.393 mol H2/mol glycerol in the immobilized culture on banana leaves (1% sodium nitrate) Improved production rate of 256 mL H2 L1 h1 and yield of 1.11 mol H2/mol glycerol by 10 g L1 crude glycerol in batch culture; production rate of 103 mL H2 L1 h1 and yield of 0.5 mol H2/mol glycerol by 10 g L1 pure glycerol or 166 mL H2 L1 h1 and yield of 0.77 mol H2/mol glycerol by 10 g L1 crude glycerol in continuous culture High production rate of 1.50 and 1.42 mmol H2 L1 h1 by 22 g L1 crude glycerol in liquid and in gaseous phases in flasks (200 mL) containing Luria– Bertani medium with phosphate buffered saline trace elements, respectively Increased H2 production yield by optimization of the medium composition (15 g L1 pure glycerol and different salt contents) in 500 mL bioreactor High productivity from pure glycerol of 9 mmol H2 L1 h1 and from crude glycerol of 6.2 mmol H2 L1 h1; stability and reusability by the immobilized cells Effective production of 0.6 mol H2/mol glycerol under 2.5 g L1 pure glycerol Increased H2 production yield by optimization of the medium composition (11 g L1 glycerol and different salt contents) Increased H2 production yield by optimization of the medium composition (20 g L1 crude glycerol and different salt contents for batch culture in 36 mL medium in 60 mL serum bottle or 11 g L1 crude glycerol and different salt contents for response surface culture)

8.0

[86]

[46,82]

[86,87]

[83,84]

[78,90,91] [77]

[85]

Temperature was 35–40 °C.

aerogenes has been optimized to develop biocatalyst for continuous H2 production from glycerol [83–85]. In addition, B. thuringiensis in continuous culture by immobilization on cheap and available plant waste has been shown to give H2 yield from glycerol to be suitable in large-scale H2 production [88]. The last methods are extensively developed for optimizing the biotechnology conditions and for long period H2 production due to biomass retention. Importantly, fermentation pathways in H. saccharolyticum have been reconstructed according to genome sequence analysis; multiple [Fe–Fe]-Hyd enzymes of different type were suggested; two of these Hyd enzymes were identified [89]. There are interesting findings for different bacteria on the dependence of H2 production

Fig. 6. Two stage technology for enhanced H2 production by bacteria using dark and photo-fermentation. Dashed lines present structure (selective membrane) to split cultures between stages.

from concentration of glycerol, composition of culture medium, pH, temperature and other factors [46,74,86,91,92]. All these conditions would be useful to apply for enhanced H2 production. Interestingly, the efficiency for photo-fermentative conversion of crude glycerol into H2 by Rhodopseudomonas palustris is nearly 90% of the theoretical maximum [80,81]. This would be studied in order to integrate dark and photo-fermentation into a two-stage process and to use mixed cultures with E. coli to have a high H2 yield and more effective H2 production; light and strict conditions are required (Fig. 6). Fermentative H2 producing bacteria can be combined with photosynthetic producers to evolve additional H2 at the second stage using organic acids of fermentation (see Fig. 2) from the first stage (see Fig. 6). For this technology it would be important to employ light and dark alternations, which can change H2 production during photo-fermentation [93]. Moreover, Mangahil et al. [94] have reported about H2 production from crude glycerol (pH 12; 45% glycerol) using enriched microbial community with dominated Clostridium sp.; optimal conditions (pH 6.5, 37 °C and 1 g L1 glycerol in small volume of batch culture (50 mL in 120 mL serum bottle) were established. Interestingly, H2 was suggested to be end product together with acetate and less butyrate; and methanol contained in crude glycerol was not utilized [94]. H2 production rate from crude and pure glycerol was similar each with other [94], as in E. coli (see Table 2). New technology for H2 production from glycerol containing dairy wastes has been evaluated with syntrophic consortium in single chamber microbial electrolysis cell [95].

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To have reliable and comparable data, a new index of maximum specific H2 producing activity by bacteria has been proposed recently for mixed cultures [96]. This will be beneficial for the developing of systems for energy production. It is important to use renewable resources for dark fermentation, and carbons containing organic wastes of different kind for obtaining bacterial biomass are valuable sources. Glucose, sucrose, lactose or starch mixtures [97,98], rice and wheat straws, corn stalks [99,100], cheese whey [101], and many other sources [2,9,11,12,102,103] have been shown to be useful for H2 production by E. coli and other bacteria and their mixtures during fermentation. Recently Kumar et al. [104] have published updated information about lignocellulose H2 production by dark fermentation discussing pretreatment and hydrolysis methods to achieve efficient process. But glycerol added to these sources and different bacterial mixtures would further enhance H2 production. Searching for cheap potential waste materials is being continued. The economic advantage is obvious; but biotechnology conditions should be optimized for different wastes and cultures. Besides, different groups [105–107] have reported H2 production from glycerol in batch and continuous cultures of hyperthermophilic bacteria Thermotoga neapolitana and T. maritima (>75 °C). Interestingly, H2 production from pre-treated glycerol was higher than that from untreated one [103]; pure glycerol was effective [106]. Again, H2 production decrease was with an increase in glycerol concentration [107]. However, H2 production by these bacteria at high temperatures has low rate, and expensive nitrogen substrates (yeast extract) are required for obtaining biomass. Probably H2 by these bacteria is produced from formate via hydrogenase as suggested with hyperthermophile Pyrococcus furiosus (>80 °C) [108]. This should be further studied. Thus, H2 production from glycerol by different bacteria has been reported with different culture types and wastes, under new technologies and optimal conditions. A progress in H2 production biotechnology by different bacteria using glycerol as a feedstock during dark and photo-fermentation can be achieved with comparison and critical analysis of the findings for E. coli. This would be of significance to design large-scale energy producing systems. 10. Purification of biohydrogen produced from glycerol and developing bioreactors There is an important problem in H2 production biotechnology: it is its purification. The H2 gas purification operational systems and different separation membranes have been reviewed recently by Bakonyi et al. [109]. For separating H2 gas from gaseous mixture of CO2/H2 (see Fig. 3 and Section 3) in batch cultures different membranes (inorganic, organic and mixed-matrix, non-porous and porous) have been applied [110,111]. Particularly, amorphous or crystal metallic membranes mainly based on palladium are extremely selective to H2 but they have disadvantages (fragility, hard operation, high cost) so they are less useful [112,113]. It has been shown that polymeric dimethyl siloxane membranes have high separation selectivity of CO2/H2 mixture [111]. Membrane technologies are compact, effective and ecologically friendly, they have simple operation mode and the further technological improvements of different materials such as modified polymers will increase the separation selectivity of H2 and thus will be employed in H2 economy; effects of fermentation technology process conditions should be addressed [109,114]. These membranes are key parts of different bioreactors for H2 production. The other problem is to develop bioreactors suitable for H2 production from glycerol. Serum bottles of small volume or lab-scale fermentors are used for batch cultures; packed-bed or upflow and other bioreactors (see Table 4) are employed for continuous cultures [109,115]. In addition, large-scale bioreactors are already

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used. However, it should be required to take into account H2 production dependence on substrates (wastes) concentration; using of immobilized cells; protection of bacteria from impurities in crude glycerol and from contamination by other bacteria; agitation, maintaining strict anaerobic conditions and long-term stability of biotechnology processes. Interestingly, lactic acid bacteria have been found in bioreactors and these bacteria as contaminants can inhibit H2 production by the other bacteria [116,117]. Therefore, it is required to protect pure or mixed cultures from contamination during production process. All these would be important for further optimization of technological process conditions for better H2 production. 11. Concluding remarks and perspectives for developing effective systems for energy production The important remark is in that [Ni–Fe]-Hyd enzymes in E. coli are reversible depending on bacterial growth phase, pH and fermentation substrates and possibly on metabolic pathways [2,14,33,34,43,69,74,118]. Therefore by optimizing these conditions it would be probable to enhance H2 production from cheap substrate – glycerol as well as from carbohydrates (sugars) and carbon containing organic wastes [11,12]. The economic value is advantaged. The other remark is with some link or cross-talk between Hyd enzymes forming H2 cycling suggested but the mechanisms underlying how this is controlled are still not clearly understood [31,55]. There are many unclear problems with glycerol fermentation by E. coli, metabolic pathways and end products, Hyd enzymes activity and reversibility and their dependence on pH, osmotic stress and other external factors. Besides, H+ cycle and H2 cycling are important to regulate H2 production by E. coli [31]. A little is known with thermodynamic and kinetic analysis of H2 production. Additional efforts are required to develop large-scale technology to produce H2 by E. coli and other bacteria using glycerol. In this respect, it is of interest that lab-scale two-stage anaerobic digestion should be more productive than one-stage process for H2 production (see Fig. 6) and will recover more energy from biomass [119]. In summary, H2 is stated to be a promising clean fuel, and bioconversion of glycerol to H2 has additional environmental benefit reducing greenhouse gas emission [120]. All the findings together are of significance for further development of effective H2 production biotechnology using glycerol and its mixtures as a carbon source for fermentation, applied energy production. Acknowledgements This study was supported by Research Grants from the State Committee of Science, Ministry of Education and Science of Armenia, to AT (13-1F002), Armenian National Science and Education fund (ANSEF, USA) to KT (Biotech-3460), German Academic Exchange Service (DAAD, Germany) scholarships to AT (A/13/03789) and KT (A/11/79636). References [1] Khanna S, Goyal A, Moholkar VS. Microbial conversion of glycerol: present status and future prospects. Crit Rev Biotechnol 2012;32:232–65. [2] Trchounian A. Mechanisms for hydrogen production by different bacteria during mixed-acid and photo-fermentation and perspectives of hydrogen production biotechnology. Crit Rev Biotechnol 2015;35:103–13. [3] Alazemi J, Ansdrews J. Automotive hydrogen fuelling stations: an international review. Renew Sustain Energy Rev 2015;48:483–99. [4] Winter CJ. Into the hydrogen energy economy – milestones. Int J Hydrogen Energy 2005;30:681–5.

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[5] Shubber K. Clean hydrogen fuel created with sunlight and water in new method. [accessed 05.08.13]. [6] Godula-Jopek A. Introduction. In: Godula-Jopek A editor. Hydrogen Production by Electrolysis. Wiley-C-VCH Verlag GmbH & Co. 2015. p. 1–33. [7] Hallenbeck PC. Fermentative hydrogen production: principles, progress, and prognosis. Int J Hydrogen Energy 2009;34:7379–89. [8] Maeda T, Sanchez-Torres V, Wood TK. Metabolic engineering to enhance bacterial hydrogen production. Microb Biotechnol 2008;1:30–9. [9] Rosales-Colunga LM, Rodriguez ADL. Escherichia coli and its application to biohydrogen production. Rev Environ Sci Biotechnol 2015;14:123–35. [10] Bock A, Sawers G. Fermentation. In: Neidhardt, FG, editor-in-Chief. Escherichia coli and Salmonella. Cellular and Molecular Biology. ASM Press: Washington DC, 2006. . [11] Ghimire A, Frunzo L, Pirozzi F, Trably E, Escudie R, Lens PNL, et al. A review on dark fermentative biohydrogen production from organic biomass: process parameters and use of by-products. Appl Energy 2015;144:73–95. [12] Arimi MM, Knodel J, Kiprop A, Namango SS, Zhang Y, Geiben S-U. Strategies for improvement of biohydrogen production from organic-rich wastewater: a review. Biomass Bioenergy 2015;75:101–18. [13] Dharmadi Y, Murarka A, Gonzalez R. Anaerobic fermentation of glycerol by Escherichia coli: a new platform for metabolic engineering. Biotechnol Bioengineer 2006;94:821–9. [14] Trchounian K, Trchounian A. Hydrogenase 2 is most and hydrogenase 1 is less responsible for H2 production by Escherichia coli under glycerol fermentation at neutral and slightly alkaline pH. Int J Hydrogen Energy 2009;34:8839–45. [15] Clomburg JM, Gonzalez R. Anaerobic fermentation of glycerol: a platform for renewable fuels and chemicals. Trends Biotechnol 2013;31:20–8. [16] Thauer RK, Kaster AK, Goenrich M. Energy conservation in chemotropic anaerobic bacteria. Bacteriol Rev 1977;41:100–80. [17] Nahar G, Dupont V. Hydrogen via steam reforming of liquid biofeedstock. Biofuels 2012;3:167–91. [18] Murarka A, Dharmadi Y, Yazdani SS, Gonzalez R. Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals. Appl Environ Microbiol 2008;74:1124–35. [19] Stroud RM, Miercke LJW, O’Connell J, Khademi S, Lee JK, Remis J, et al. Glycerol facilitator GlpF and the associated aquaporin family of channels. Curr Opin Struct Biol 2003;13:424–31. [20] Cintolesi A, Comburg JM, Rigou V, Zygourakis K, Gonzalez R. Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli. Biotechnol Bioengineer 2011;109:187–98. [21] Ganesh I, Ravikumar S, Hong SH. Metabolically engineered Escherichia coli as a tool for the production of bioenergy and biochemicals from glycerol. Biotechnol Bioproc Engineer 2012;17:671–8. [22] Poladyan A, Avagyan A, Vassilian A, Trchounian A. Oxidative and reductive routes of glycerol and glucose fermentation by Escherichia coli batch cultures and their regulation by oxidizing and reducing reagents at different pHs. Curr Microbiol 2013;66:49–55. [23] Kim K, Kim SK, Park YC, Seo JH. Enhanced production of 3-hydroxy-propionic acid from glycerol by modulation of glycerol metabolism in recombinant Escherichia coli. Bioresour Technol 2014;156:170–5. [24] Booth IR. Glycerol and methylglyoxal metabolism. In: Neidhardt, FG, editorin-Chief, EcoSal – Escherichia coli and Salmonella. Cellular and Molecular Biology. ASM Press: Washington DC, 2006. . [25] Trchounian K, Poladyan A, Vassilian A, Trchounian A. Multiple and reversible hydrogenases for hydrogen production by Escherichia coli: dependence on fermentation substrate, pH and FOF1-ATPase. Crit Rev Biochem Mol Biol 2012;47:236–49. [26] Mnatsakanyan N, Vassilian A, Navasardyan L, Bagramyan K, Trchounian A. Regulation of Escherichia coli formate hydrogenlyase activity by formate at alkaline pH. Curr Microbiol 2002;45:281–6. [27] Mnatsakanyan N, Bagramyan K, Trchounian A. Hydrogenase 3 but not hydrogenase 4 is major in hydrogen gas production by Escherichia coli formate hydrogenlyase at acidic pH and in the presence of external formate. Cell Biochem Biophys 2004;41:357–65. [28] Bakonyi P, Nemestóthy N, Lövitusz É, Bélafi-Bakó K. Application of PlackettBurman experimental design to optimize biohydrogen fermentation by E. coli (XL1-BLUE). Int J Hydrogen Energy 2011;36:13949–54. [29] Futatsugi L, Saito H, Kakegawa T, Kobayashi H. Growth of an Escherichia coli mutant deficient in respiration. FEMS Microbiol Lett 1997;156:141–5. [30] Bagramyan K, Trchounian A. Structure and functioning of formate hydrogen lyase, key enzyme of mixed-acid fermentation. Biochemistry (Moscow) 2003;68:1159–70. [31] Trchounian A, Sawers RG. Novel insights into the bioenergetics of mixed-acid fermentation: can hydrogen and proton cycles combine to help maintain a proton motive force? IUBMB Life 2014;66:1–7. [32] Redwood MD, Mikheenko IP, Sargent F, Macaskie LE. Dissecting the roles of Escherichia coli hydrogenases in biohydrogen production. FEMS Microbiol Lett 2008;278:48–55. [33] Lukey MJ, Parkin A, Roessler MM, Murphy BJ, Harmer J, Palmer T, et al. How Escherichia coli is equipped to oxidize hydrogen under different redox conditions. J Biol Chem 2010;285:3928–38. [34] Trchounian K, Sanchez-Torres V, Wood TK, Trchounian A. Escherichia coli hydrogenase activity and H2 production under glycerol fermentation at a low pH. Int J Hydrogen Energy 2011;36:4323–31.

[35] Poladyan A, Trchounian K, Sawers G, Trchounian A. Hydrogen-oxidizing hydrogenases 1 and 2 of Escherichia coli regulate the onset of hydrogen evolution and ATPase activity, respectively, during glucose fermentation at alkaline pH. FEMS Microbiol Lett 2013;348:143–8. [36] Menon NK, Robbins J, Wendt JC, Shanmugan KT, Przybyla AE. Mutational analysis and characterization of the Escherichia coli hya operon, which encodes [NiFe] hydrogenase 1. J Bacteriol 1991;173:4851–61. [37] Menon NK, Chatelus CY, Dervartanian M, Wendt JC, Shanmugam KT, Peck HD, et al. Cloning, sequencing, and mutational analysis of the hyb operon encoding Escherichia coli hydrogenase 2. J Bacteriol 1994;176:4416–23. [38] Sauter M, Bohm R, Bock A. Mutational analysis of the operon (hyc) determing hydrogenase 3 formation in Escherichia coli. Mol Microbiol 1992;6:1523–32. [39] Andrews SC, Berks BC, Mcclay J, Ambler A, Quail MA, Golby P, et al. A 12cistron Escherichia coli operon (hyf) encoding a putative proton-translocating formate hydrogenlyase system. Microbiology 1997;143:3633–47. [40] Richard DJ, Sawers G, Sargent F, McWalter L, Boxer DH. Transcriptional regulation in response to oxygen and nitrate of the operons encoding the [Ni– Fe] hydrogenases 1 and 2 of Escherichia coli. Microbiology 1999;145:2903–12. [41] Hube M, Blokesch M, Bock A. Network of hydrogenase maturation in Escherichia coli: role of accessory proteins HypA and HybF. J Bacteriol 2002;184:3879–85. [42] Trchounian K. Transcriptional control of hydrogen production during mixed carbon fermentation by hydrogenases 4 (hyf) and 3 (hyc) in Escherichia coli. Gene 2012;506:156–60. [43] Sanchez-Torres V, Yusoff MYM, Nakano C, Maeda M, Ogawa HI, Wood TK. Influence of Escherichia coli hydrogenases on hydrogen fermentation from glycerol. Int J Hydrogen Energy 2013;38:3905–12. [44] Pinske C, Jaroschinsky M, Linek S, Kelly CL, Sargent F, Sawers RG. Physiology and bioenergetics of [NiFe]-hydrogenase 2-catalyzed H2-consuming and H2producing reactions in Escherichia coli. J Bacteriol 2015;197:296–306. [45] Trchounian K, Trchounian A. Escherichia coli hydrogenase 4 (hyf) and hydrogenase 2 (hyb) contribution in H2 production during mixed carbon (glucose and glycerol) fermentation at pH 7.5 and pH 5.5. Int J Hydrogen Energy 2013;38:3919–27. [46] Dimanta I, Grunduls A, Nikolaeva V, Kleperis J, Muiznieks I. Crude glycerol as a perspective substrate for bio-hydrogen production in Latvia. Int Sci J Alt Energy Ecology 2012;9:28–31. [47] Blbulyan S, Avagyan A, Poladyan A, Trchounian A. Role of Escherichia coli different hydrogenases in H+ efflux and the FOF1-ATPase activity during glycerol fermentation at different pH. Biosci Rep 2011;31:179–84. [48] Trchounian K, Pinske C, Sawers RG, Trchounian A. Dependence on the F0F1ATP synthase for the activities of the hydrogen-oxidizing hydrogenases 1 and 2 during glucose and glycerol fermentation at high and low pH in Escherichia coli. J Bioenerg Biomembr 2011;43:645–50. [49] Trchounian A. Escherichia coli proton-translocating F0F1-ATP synthase and its association with solute secondary transporters and/or enzymes of anaerobic oxidation-reduction under fermentation. Biochem Biophys Res Comm 2004;315:1051–7. [50] Trchounian K, Blbulyan S, Trchounian A. Hydrogenase activity and protonmotive force generation by Escherichia coli during glycerol fermentation. J Bioenerg Biomembr 2013;45:253–60. [51] Trchounian K, Trchounian A. Clean energy technology development: hydrogen production by Escherichia coli during glycerol fermentation. In: Dincer I, Colpan CO, Kizilkan O, et al., editor. Proceedings of the 13th International Conference on Clean Energy, Istanbul (Turkey), 2014. p. 1322–8. [52] Blbulyan S, Trchounian A. Impact of membrane-associated hydrogenases on the FoF1-ATPase in Escherichia coli during glycerol and mixed carbon fermentation: atpase activity and its inhibition by N, N’dicyclohexylcarbodiimide in the mutants lacking hydrogenases. Arch Biochem Biophys 2015;579:67–72. [53] Pettigrew DW. Inactivation of Escherichia coli glycerol kinase by 5,5’dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide: evidence for nucleotide regulatory binding sites. Biochemistry 1986;25:4711–8. [54] Hakobyan L, Gabrielyan L, Trchounian A. Relationship of proton motive force and the FOF1-ATPase with bio-hydrogen production activity of Rhodobacter sphaeroides: effects of diphenylene iodonium, hydrogenase inhibitor, and its solvent dimethylsulphoxide. J Bioenerg Biomembr 2012;44:495–502. [55] Trchounian K, Trchounian A. Escherichia coli multiple [Ni–Fe]-hydrogenases are sensitive to osmotic stress during glycerol fermentation but at different pHs. FEBS Lett 2013;587:3562–6. [56] Bagramyan K, Mnatsakanyan N, Poladyan A, Vassilian A, Trchounian A. The roles of hydrogenases 3 and 4, and the F0F1-ATPase, in H2 production by Escherichia coli at alkaline and acidic pH. FEBS Lett 2002;516:172–8. [57] Trchounian K, Sargsyan H, Trchounian A. Hydrogen production by Escherichia coli depends on glucose concentration and its combination with glycerol at different pHs. Int J Hydrogen Energy 2014;39:6419–23. [58] Gabrielyan L, Sargsyan H, Hakobyan L, Trchounian A. Regulation of hydrogen photoproduction in Rhodobacter sphaeroides batch culture by external oxidizers and reducers. Appl Energy 2014;131:20–5. [59] Fernandez VM. An electrochemical cell for reduction of biochemical: its application to the study of the effect pf pH and redox potential on the activity of hydrogenases. Anal Biochem 1983;130:54–9. [60] Eltsova ZA, Vasilieva LG, Tsygankov AA. Hydrogen production by recombinant strains of Rhodobacter sphaeroides using a modified photosynthetic apparatus. Appl Biochem Microbiol 2010;46:487–91.

K. Trchounian, A. Trchounian / Applied Energy 156 (2015) 174–184 [61] Noguchi K, Riggins DP, Eldahan KC, Kitko RD, Slonczewski JL. Hydrogenase-3 contributes to anaerobic acid resistance of Escherichia coli. PLoS ONE 2010;5:e10132. [62] Piskarev IM, Ushkanov VA, Aristova NA, Likhachev PP, Myslivets TS. Establishment of the redox potential of water saturated with hydrogen. Biophysics 2010;55:13–7. [63] Bagramyan KA, Martirosov SM. Formation of an ion transport supercomplex in Escherichia coli. An experimental model of direct transduction of energy. FEBS Lett 1989;249:149–52. [64] Maeda T, Wood TK. Formate detection by potassium permanganate for enhanced hydrogen production in Escherichia coli. Int J Hydrogen Energy 2008;33:2409–12. [65] Barrett EL, Kwan HS, Macy J. Anaerobiosis, formate, nitrate, and pyrA are involved in the regulation of formate hydrogenlyase in Salmonella typhimurium. J Bacteriol 1984;158:972–7. [66] Maeda T, Sanchez-Torres V, Wood TK. Hydrogen production by recombinantEscherichia coli strains. Microb Biotechnol 2012;5:214–25. [67] Hu H, Wood TK. An evolved Escherichia coli strain for producing hydrogen and ethanol from glycerol. Biochem Biophys Res Comm 2010;391:1033–8. [68] Tran KT, Maeda M, Wood TK. Metabolic engineering of Escherichia coli to enhance hydrogen production from glycerol. Appl Microbiol Biotechnol 2014;98:4757–70. [69] Tran KT, Maeda T, Sanchez-Torres V, Wood TK. Beneficial knockouts in Escherichia coli for producing hydrogen from glycerol. Appl Microbiol Biotechnol 2015;99:2573–81. [70] Yoshida A, Nishimura T, Kawagushi H, Inui M, Yukawa H. Enhanced hydrogen production from formic acid by formate hydrogen lyase-overexpressing Escherichia coli. Appl Env Microbiol 2005;71:6762–8. [71] Rollin JA, del Campo JM, Myung S, Sun F, You C, Bakovic A, et al. High-yield hydrogen production from biomass by in vitro metabolic engineering: mixed sugars coutilization and kinetic modeling. Proc Nat Acad Sci USA 2015;112:4964–9. [72] Chaudhary N, Ngadi MO, Simpson B. Comparison of glucose, glycerol and crude glycerol fermentation by Escherichia coli K12. J Bioprocess Biotecniq 2012;S1:001. [73] Schlensog V, Bock A. Identification and sequence analysis of the gene encoding the transcriptional activator of the formate hydrogenlyase system of Escherichia coli. Mol Microbiol 1990;4:1319–27. [74] Trchounian K, Soboh B, Sawers RG, Trchounian A. Contribution of hydrogenase 2 to stationary phase H2 production by Escherichia coli during fermentation of glycerol. Cell Biochem Biophys 2013;66:103–8. [75] Trchounian K, Trchounian A. Escherichia coli hydrogen gas production from glycerol: effects of external formate. Renew Energy 2015;83:345–51. [76] Trchounian K, Abrahamyan V, Poladyan A, Trchounian A. Escherichia coli growth and hydrogen production in batch culture upon formate alone and with glycerol co-fermentation at different pHs. Int J Hydrogen Energy 2015. http://dx.doi.org/10.1016/j.ijhydene.2015.06.087. [77] Liu F, Fang B. Optimization of bio-hydrogen production from biodiesel wastes by Klebsiella pneumoniae. Biotechnol J 2007;2:374–80. [78] Kivisto A, Santala V, Karp M. Hydrogen production from glycerol using halophilic fermentative bacteria. Bioresour Technol 2010;101:8671–7. [79] Maru BT, Constanti M, Stchigel AM, Medina F, Sueiras JE. Biohydrogen production by dark fermentation of glycerol using Enterobacter and Citrobacter sp. Biotechnol Prog 2013;29:31–8. [80] Ghosh D, Tourigny A, Hallenbeck PC. Near stoichiometric reforming of biodiesel derived crude glycerol to hydrogen by photofermentation. Int J Hydrogen Energy 2012;37:2273–7. [81] Pott RWM, Howe CJ, Dennis JS. Photofermentation of crude glycerol from biodiesel using Rhodopseudomonas palustris: comparison with organic acids and the identification of inhibitory compounds. Bioresour Technol 2013;130:725–30. [82] Dimanta I, Nikolaeva Y, Grunduls A, Muiznieks I, Kleperis J. Assessment of bio-hydrogen production from glycerol and glucose by fermentative bacteria. Energetika 2013;59:124–8. [83] Han J, Lee D, Cho J, Lee J, Kim S. Hydrogen production from biodiesel byproduct by immobilized Enterobacter aerogenes. Bioproc Biosyst Engineer 2012;35:151–7. [84] Reungsang A, Sittijunda S, O-thong S. Bio-hydrogen production from glycerol by immobilized Enterobacter aerogenes ATCC 13048 on heat-treated UASB granules as affected by organic loading rate. Int J Hydrogen Energy 2013;38:6970–9. [85] Chookaew T, O-Thong S, Prasertsan P. Statistical optimization of medium components affecting simultaneous fermentative hydrogen and ethanol production from crude glycerol by thermotolerant Klebsiella sp. TR17. Int J Hydrogen Energy 2014;39:751–60. [86] Lo YC, Chen XJ, Yuan Y, Chang JS. Dark fermentative hydrogen production with crude glycerol from biodiesel industry using indigenous hydrogenproducing bacteria. Int J Hydrogen Energy 2013;38:15815–28. [87] Sarma SJ, Brar SK, Le Bihan Y, Buelna G, Soccol SR. Hydrogen production from meat processing and restaurant waste derived crude glycerol by anaerobic fermentation and utilization of the spent broth. J Chem Technol Biotechnol 2013;88:2264–71. [88] Kumar P, Sharma R, Ray S, Mehariya S, Patel SK, Lee JK, et al. Dark fermentative bioconversion of glycerol to hydrogen by Bacillus thuringiensis. Bioresour Technol 2015;182:383–8.

183

[89] Mangayil R, Aho T, Karp M, Santala V. Improved bioconversion of crude glycerol to hydrogen by statistical optimization of media components. Renew Energy 2015;75:583–9. [90] Ito T, Nakashimada Y, Senba K, Matsui T, Nishio N. Hydrogen and ethanol production from glycerol-containing wastes discharged after biodiesel manufacturing process. J Biosci Bioeng 2005;100:260–5. [91] Kivisto A, Largo A, Ciranna A, Sanrala V, Roos C, Karp M. Genome sequence of Halanaerobium saccharolyticum subsp. saccharolyticum strain DSM 6643T, a halophilic hydrogen-producing bacterium. Genome Announcements 2013;1. e00187-13. [92] Jitrwung R, Verrett J, Yargeau V. Optimization of selected salts concentration for improved biohydrogen production from biodiesel-based glycerol using Enterobacter aerogenes. Renew Energy 2013;50:222–36. [93] Sargsyan H, Gabrielyan L, Hakobyan L, Trchounian A. Light-dark duration alternation effects on Rhodobacter sphaeroides growth, membrane properties and bio-hydrogen production in batch culture. Int J Hydrogen Energy 2015;40:4084–91. [94] Mangahil R, Karp M, Santala V. Bioconversion of crude glycerol from biodiesel production to hydrogen. Int J Hydrogen Energy 2012;37: 12198–204. [95] Montpart N, Rago L, Baeza JA, Guisasola A. Hydrogen production in single chamber microbial electrolysis cells with different complex substrates. Water Res 2015;68:601–5. [96] Mu Y, Yang H-Y, Wang Y-Z, He C-H, Zhao Q-B, Wang Y, et al. The maximum specific hydrogen producing activity of anaerobic mixed cultures: definition and determination. Sci Rep 2014;4:5239. [97] Gupta M, Velayutham P, Elbeshbishy E, Hafez H, Khafipour E, Derakhshani H, et al. Co-fermentation of glucose, starch, and cellulose for mesophilic biohydrogen production. Int J Hydrogen Energy 2014;39:20958–67. [98] Vendruscolo F. Starch: a potential substrate for biohydrogen production. Int J Energy Res 2014;39:293–302. [99] Marone A, Izzo G, Mentuccia L, Massini G, Paganin P, Rosa S, et al. Vegetable waste as substrate and source of suitable microflora for bio-hydrogen production. Renew Energy 2014;68:6–13. [100] Song ZX, Li XH, Li WW, Bai YX, Fan YT, Hou HW. Direct bioconversion of raw corn stalk to hydrogen by a new strain Clostridium sp. FS3. Bioresour Technol 2014;157:91–7. [101] De˛bowski M, Korzeniewska E, Filipkowska Z, Zielin´ski M, Kwiatkowski R. Possibility of hydrogen production during cheese whey fermentation process by different strains of psychrophilic bacteria. Int J Hydrogen Energy 2014;39:1972–8. [102] Kapdan IK, Kargi F. Biohydrogen production from waste materials. Enzyme Microb Technol 2006;38:569–82. [103] Choi J, Ahn Y. Characteristics of biohydrogen fermentation from various substrates. Int J Hydrogen Energy 2013;39:3152–9. [104] Kumar G, Bakonyi P, Periyasamy S, Kim SH, Nemestóthy N, Bélafi-Bakó K. Lignocellulose biohydrogen: Practical challenges and recent progress. Renew Sustain Energy Rev 2015;44:728–37. [105] Ngo TA, Kim MS, Sim SJ. High-yield biohydrogen production from biodiesel manufacturing waste by Thermotoga neapolitana. Int J Hydrogen Energy 2011;36:5836–42. [106] Ngo TA, Sim SJ. Dark fermentation of hydrogen from waste glycerol using hyperthermophilic eubacterium Thermotoga neapolitana. Environ Prog Sustain Energy 2012;31:466–73. [107] Maru BT, Bielen AAM, Constanti M, Medina F, Kengen SWM. Glycerol fermentation to hydrogen by Thermotoga maritima: proposed pathway and bioenergetic considerations. Int J Hydrogen Energy 2013;38:5563–72. [108] Sapra R, Bagramyan K, Adams MW. A simple energy-conserving system: proton reduction coupled to proton translocation. Proc Natl Acad Sci USA 2003;100:7545–50. [109] Bakonyi P, Nemestóthy N, Bélafi-Bakó K. Biohydrogen purification by membranes: an overview on the operational conditions affecting the performance of non-porous, polymeric and ionic liquid based gas separation membranes. Int J Hydrogen Energy 2013;38:9673–87. [110] Bakonyi P, Nemestóthy N, Lankó J, Rivera I, Buitrón G, Bélafi-Bakó K. Simultaneous biohydrogen production and purification in a doublemembrane bioreactor system. Int J Hydrogen Energy 2015;40:1690–7. [111] Ramírez-Morales JE, Tapia-Venegas E, Nemestóthy N, Bakonyi P, Bélafi-Bakó K, Ruiz-Filippi G. Evaluation of two gas membrane modules for fermentative hydrogen separation. Int J Hydrogen Energy 2013;38:14042–52. [112] Lai T, Yin H, Lind ML. The hydrogen permeability of Cu–Zr binary amorphous metallic membranes and the importance of thermal stability. J Membr Sci 2015;489:264–9. [113] Al-Mufachi NA, Rees NV, Steinberger-Wilkens R. Hydrogen selective membranes: a review of palladium-based dense metal membranes. Renew Sustain Energy Rev 2015;47:540–51. [114] Shao L, Low BT, Chung TS, Greenberg AR. Polymeric membranes for the hydrogen economy: contemporary approaches and prospects for the future. J Membr Sci 2009;327:18–31. [115] Barca C, Soric A, Ranava D, Giudici-Orticoni MT, Ferrasse JH. Anaerobic biofilm reactors for dark fermentative hydrogen production from wastewater: a review. Bioresour Technol 2015;185:386–98. [116] Noike T, Takabatake H, Mizuno O, Ohba M. Inhibition of hydrogen fermentation of organicwastes by lacticacid bacteria. Int J Hydrogen Energy 2002;27:1367–71.

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K. Trchounian, A. Trchounian / Applied Energy 156 (2015) 174–184

[117] Baghchehsaraee B, Nakhla G, Karamanev D, Margaritis A. Revivability of fermentative hydrogen producing bioreactor. Int J Hydrogen Energy 2011;34:2573–9. [118] Maeda T, Sanchez-Torres V, Wood TK. Escherichia coli hydrogenase 3 is a reversible enzyme possessing hydrogen uptake and synthesis activities. Appl Microbiol Biotechnol 2008;76:1036–42.

[119] Schievano A, Tenca A, Lonati S, Manzini E, Adani F. Can two-stage instead of one-stage anaerobic digestion really increase energy recovery from biomass? Appl Energy 2014;124:335–42. [120] Sarma SJ, Brar SK, Le Bihan Y, Buelna G. Bio-hydrogen production by biodiesel-derived crude glycerol bioconversion: a techno-economic evaluation. Bioproc Biosyst Eng 2013;36:1–10.