Improving Biohydrogen Productivity by Microbial Dark- and Photo- MARK Fermentations: Novel Data and Future Approaches ⁎ Karen Trchouniana,B, R

Renewable and Sustainable Energy Reviews 80 (2017) 1201–1216

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Improving biohydrogen productivity by microbial dark- and photo- MARK fermentations: Novel data and future approaches ⁎ Karen Trchouniana,b, R. Gary Sawersc, Armen Trchouniana,b, a Research Institute of Biology, Faculty of Biology, Yerevan State University, Armenia b Department of Biochemistry, Microbiology and Biotechnology, Faculty of Biology, Yerevan State University, 0025 Yerevan, Armenia c Institute of Biology/Microbiology, Martin-Luther University of Halle-Wittenberg, Halle (Saale), Germany

ARTICLE INFO ABSTRACT

Keywords: Hydrogen (H2)isaneffective, environmentally friendly and renewable source of fuel that can be produced Hydrogen production during dark- and photo-fermentation by different facultative and obligate anaerobic and purple bacteria and Dark- and photo-fermentation microalgae. This product is known as biohydrogen. It has the advantage of variable yield at low temperature (for Bacteria and microalgae mesophiles growing best at moderate temperature) and relatively low production cost, if compared with Hydrogenases and nitrogenases thermochemical methods. To develop fermentative H production biotechnology using cheap carbonaceous by- Glycerol 2 products and utilization of organic wastes, the selection or construction of effective bacterial strains and Carbon-containing wastes Renewable and sustainable energy optimization of technology process conditions are required. Here we review recent new data that have been obtained with Escherichia coli, Clostridium beijerinskii, Rhodobacter sphaeroides and other bacteria. Activities of [Ni-Fe]-hydrogenases of dark-fermentative bacteria and [Mo]-nitrogenase and [Ni-Fe]-hydrogenase of photo-fermentative species have been examined after growth with different carbon sources, using pure cultures,

as well as co-culture and mixed-cultures technologies. Importantly, H2 production from cheap and readily available substrates like crude glycerol or diﬀerent industrial, agricultural and other carbon-based wastes by bacteria is a sustainable technology. Consequently further approaches and strain-improvement could increase

H2 production in a cost-effective way, and they will lead to both small- and large-scale H2 production. Moreover, they will provide significant economic and environmental benefits for renewable and sustainable energy supply in the near future.

1. Introduction [2] (see Fig. 1). Furthermore, dark-fermentation does not need sun- light, has a signiﬁcantly higher rate of production and has the added

Hydrogen (H2)isaneffective, environmentally clean and renewable advantage that H2 can be continuously produced when compared with fuel, because it has three-fold higher energy content (~ 142 kJ g−1) photo‐fermentation. than fossil fuel, such as oil, so its conversion efficiency is high; it The European Union's aim is to improve the contribution of H2 generates only water and no toxic by-products and it can be produced production to 27% of our renewable energy budget, and to reduce from biomass [1,2]. Development of a low-carbon economy is im- green-house gas emissions by > 40% by 2030 (compared to the 1990 portant to decrease green house gas emissions and to avoid climate level) [4]. These are important goals for the global energy strategy to change, which can be irreversible processes [3].H2 can be produced by establish a low-carbon economy and flexible and adaptive power various methods including dark- and photo-fermentation through the systems. To achieve these goals this requires further development of activities of different facultative and obligate anaerobic and purple- biohydrogen biotechnology (Fig. 2). Moreover, H2 is an important part sulfur bacteria, as well as by microalgae (Fig. 1). It can also be of current and future transport development with the aim to increase produced through biophotolysis of water by microalgae and algae as use of H2 vehicles and to build H2 filling stations. Starting in 2018 biocatalysts. H2 production by bacteria, microalgae and algae is known Toyota will produce > 3000H2-powered vehicles per year (e.g. the as biohydrogen and this source remains a highly promising one in Toyota Mirai is one of the first vehicles to be sold commercially that terms of global energy supply [1]. It has the advantages of high yields at uses exclusively a H2 fuel-cell). A ‘H2 highway’ system is planned for comparatively low temperature (typically between 20 and 45 °C) and European countries these will be served by > 1000H2-filling stations, has relatively low cost if compared with other methods of production which will be constructed by 2023, including > 400 stations in

⁎ Correspondence to: Department of Biochemistry, Microbiology & Biotechnology, Yerevan State University, 1 A. Manoogian Str, 0025 Yerevan, Armenia. E-mail address: [email protected] (A. Trchounian). http://dx.doi.org/10.1016/j.rser.2017.05.149 Received 12 August 2016; Received in revised form 7 March 2017; Accepted 19 May 2017 1364-0321/ © 2017 Elsevier Ltd. All rights reserved. K. Trchounian et al. Renewable and Sustainable Energy Reviews 80 (2017) 1201–1216

be located in the proximity of carbonaceous resources. This H2 can also provide security and reduce operating expenses for its transportation. Furthermore, the generation of various contaminants, including volatile fatty acids, can be drastically reduced or avoided if the production site is close to the power station.

H2 is also a signiﬁcant feedstock for the chemical, food, pharma- ceutical and some other industries, and is important in metallurgy and

the production of electronic devices [7]. These are the main ﬁelds of H2 application.

To develop fermentative H2 production biotechnology using carbonaceous by-products and organic wastes, studies to select eﬀective H2- producing heterotrophic bacteria or the genetic modiﬁcation of already characterized strains for the optimization of this technological process

have been initiated. These approaches could increase H2 production in inexpensive ways (Fig. 2). They will beneﬁt both small- and large-scale

H2 production capabilities globally and provide significant benefits for renewable and sustainable energy supply in the near future. Although there have been several excellent reviews on microbial Fig. 1. H2 production sources and methods of dark- and light-driven fermentations by biohydrogen production published within the last few years [1,2,7–19], bacteria as biocatalysts, highlighting their advantages. this is a rapidly-expanding field and there have been many exciting new recent findings in this subject, particularly from patents (Fig. 4) and, thus, an updated review is required. This review highlights new

findings in the field and developments in H2 production biotechnology (see Fig. 2) focusing on biohydrogen from organic by-products like glycerol, their mixtures with different organic compounds like sugars and organic acids contained in organic wastes and various carbon- based wastes like distillers grains (DG) and brewery wastes by bacterial fermentation processes. The findings are with Escherichia coli, Clostridium beijerinskii, Rhodobacter sphaeroides and other ferment- ing bacteria; pure, co- and mixed bacterial cultures are considered and compared; some novel ideas concerning basic and applied research on biohydrogen production like general physiology, genetics and biochemistry of responsible enzymes – hydrogenases (Hyd), interaction between these enzymes and hydrogen cycling through the membrane in the cells and the stimulatory and inhibitory effects of different heavy Fig. 2. Main approaches adopted to enhance H2 production by bacteria. metals and their mixtures are also presented and discussed. They will

be useful to draw novel approaches (see Fig. 2) for enhanced H2 production by bacteria.

2. Cheap organic substrates and advantages for H2 production

Future economically viable alternatives for the development of H2 production biotechnology in both the small- and large-scale require the

Fig. 3. H2-ﬁlling stations numbers biulded in 2015–2016 and planned for 2017–2032 [6].

Germany alone [5]. Generally a rapid increase in the number of H2- ﬁlling stations (Fig. 3) and their distribution worldwide is planned [6].

In addition, H2 will be explored as an alternative fuel source in airplanes and space shuttles. As with hybrid technology, which is now widely adopted, we see an even more rapid acceleration in the development and acceptance of H2 vehicles. There is also one important argument in favor of biohydrogen from Fig. 4. Number of articles published in international peer-reviewed journals and of carbon-containing by-products and wastes: it makes feasible to man- patents between 2006 and 2015 by Scopus. Titles on H2 production by bacteria were ufacture decentralized power stations, where the generating units can searched.

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H2 by E. coli [29] (Table 1). As mentioned above, crude glycerol is much cheaper than pure one (at least ~ 7 fold), so economic beneﬁtof

its using for H2 production is obvious. Moreover, there is no need to use pre-treatment or treatment of biomass when crude glycerol is used, as fermentation substrate by bacteria. Although the mechanism underlying this ﬁnding is unexplained, this is nevertheless an additional advantage in the use of glycerol (see Fig. 5). Then, it is of interest that glycerol added into the medium with glucose has been shown to

increase signiﬁcantly (2-fold and more) H2 production by E. coli [30,31]. However, in the case of E. coli H2 production is optimal at high pH and decreases when growth occurs at acidic pH [30,31].

Nevertheless, this presents an interesting new approach to H2 production because both glucose and glycerol are found in agricultural, kitchen and other organic wastes [27]. Because sugars such as glucose are fermented optimally at low pH by many bacteria, including E. coli, this allows a wide range of acidic and alkaline pHs to be exploited for

the production of H2 by bacteria: this ﬂexibility has obvious technical advantages for biohydrogen processing.

H2 can be produced from different substrates by bacteria during Fig. 5. Crude glycerol from biodiesel industry to produce biofuels and its applications in photo-fermentation [16,32–37]. Among these substrates are sugars other fields of industry and agriculture. Based on data from [2,7,22,29]. (glucose or sucrose), carboxylic acids (formic, acetic, succinic, malic and other acids) as carbon source [38–40] and amino acids (glycine, use of cheap organic growth substrates. One substrate that fits this glutamic acid, lysine, proline and others) [39,41] or from yeast extract requirement is glycerol (C3H8O3). Glycerol is produced during the when used as a nitrogen source [42]. degradation of biomass, vegetable oils and animal fat, and is also Various carbon-based organic wastes, which contain sugars, pro- generated in large quantities as the main by-product of the biodiesel teins, amino acids and lipids [43–45], can also be used for biohydrogen industry (1 kg glycerol per 9 kg of biodiesel [20,21]). Over 65% of production by bacteria. Moreover, glycerol content in different wastes glycerol is obtained as by-product of biodiesel industry, which is might provide effective option for H2 production [27]. Indeed, DG produced much in the European Union and the other countries [2]. derived from the wine and ethanol industry (the cost is 2 cents/kg) Importantly the biodiesel production has become one of the most fast [35,36,42], wastes from potato, rice and wheat straw, maize stalks [46– growing industries worldwide [2,21] making crude glycerol highly 48], brewery [49,50], food (kitchen) [51], municipal solid wastes [52], available substrate for applications (Fig. 5); its market value is cheese whey [53–55], oil mills [56] and dairy industry [57] waste- estimated to grow at 7.9% till 2022. waters and many other sources [11,17,58–61] can be fermented to

It has been discovered recently that glycerol can be fermented by produce of H2 by E. coli, Rhodobacter and Clostridia species and other many bacterial species at acidic [22] and alkaline [23–26] pHs and, bacteria and by their co- or mixed cultures including various lactococci therefore, serves as an excellent substrate to produce biohydrogen. The and enterobacteria (Table 2). Importantly, H2 production yield from discovery of glycerol fermentation by bacteria initially by Gonzales [22] different organic wastes is lower than from by-products (glycerol) and then by our group as a source of H2 production [23,24] has several (comp. Tables 1, 2) but it is reasonable if costs for wastes and glycerol implications. Firstly, although it is clear that the maximal H2 yield from are compared. Recently, Kumar et al. [62] published an update on the glycerol (C3H8O3 +3H2O->7H2 + 3CO2) is lower than that from production of lignocellulose-derived H2 by dark fermentation and ffi sugars (glucose, C6H12O6 +6H2O - > 12H2 + 6CO2; or xylose, C5H10O5 discussed pretreatment and hydrolysis methods to achieve e cient ff +5H2O - > 10H2 + 5CO2), nevertheless glycerol is very cheap (nowa- and cost-e ective processing. All this demonstrates high-potential of days cost is roughly 4–11 cents/kg for crude glycerol and 70–101 different carbon-based wastes as renewable feedstock to produce cents/kg for pure glycerol) and widely available [2]. Moreover, market bioenergy, especially H2, having a central role in biorefinery concept price of glycerol is decreasing since increasing biodiesel production [45,63]. While the search for cheap alternative potential waste as

[21,27]. This will lead to economic benefit lowering the costs for carbon sources for H2 production continues, and with obvious eco- substrate (glycerol is cheaper than glucose) and operating expenses for nomic advantages, the optimal conditions are very different for various its transportation in addition to that a comparatively low temperature crops, by-products and wastes. But, as mentioned above (see is applied. But glycerol is not only abundant and inexpensive but it has [13,43,60–62]), different wastes require pre-treatment and treatment, unique characteristics generating more reducing equivalents than which could increase expenses for H2 production even of cheap wastes. sugars (glucose or xylose); therefore, glycerol has quickly become a A further advantage of utilization of organic wastes for H2 produc- competitive raw material to produce different fuels and chemicals, tion by bacteria is that this process can also contribute to maintain a along with various other applications, compared with sugars [7,25,28]. clean environment and control climate change since different wastes Importantly, crude and pure glycerol proved to yield similar levels of are accumulated in large quantities and at critical level dangerous for

Table 1

H2 production from crude and pure glycerol in batch cultures of Escherichia coli. Data were from [29].

−1 Type of glycerol Glycerol concentration, g L Culture medium composition pH Maximal H2 yield, production, mmol −1 H2 L

Crude (40–80% 10 MOPSa buﬀered minimal media; 10 g L−1 tryptone, pH 6.3 ~ 13 glycerol) 5gL−1 yeast extract Pure ( > 98% glycerol) 10 MOPS buffered minimal media; 10 g L−1 tryptone, pH 6.3 ~ 11.5 5gL−1 yeast extract

a 3-morpholinopropane-1-sulfonic acid.

1203 .Tcona tal. et Trchounian K.

Table 2 H2 production from diﬀerent organic wastes by diﬀerent bacteria during dark- and light-driven fermentation processes.

Organic wastes Bacteria, fermentation type Technology conditions Characteristics of H2 production References

Distillers grains (DG) with solubles Escherichia coli BW25113, Batch culture; pH 7.5, 37 °C H2 was produced during 24 h; 2–5 fold diluted DG enhanced 2 fold H2 yield; [35] from ethanol industry dark fermentation undiluted and 10 fold diluted DG decreased H2 yield in comparison with culture − grown on glucose (2 g L 1) DG with solubles from ethanol Rhodobacter sphaeroides MDC6521, light Batch culture; Ormerod medium, pH 7.0, H2 was produced after 24 h and continued till 96 h; 2 fold diluted DG enhanced 4.6 [35,36] 2 industry fermentation 30 °C; light intensity of approx. 36 W / m fold H2 yield and 5 fold diluted one enhanced 5.5 fold H2 yield; DG with solubles from ethanol Co-culture of E. coli BW25113 and R. Batch culture; Ormerod medium, pH 7.0, H2 was produced after 24 h and continued till 96 h; 2 fold diluted DG enhanced [36] 2 industry sphaeroides MDC6521 30 °C; light intensity of approx. 36 W / m 1.5–3 fold H2 yield for 72–96 h in comparison with appropriate pure cultures DG from wine industry Mixed culture of unidentified bacteria from cow Batch culture; pH 6.0, 50 °C; pre-treatment H2 was produced during 156 h; H2 production was dependent on DG concentration [43] dung at 95 °C and with 3 N HCl, −1 −1 Industrial municipal solid waste Mixed culture of Eubacteria and Archaea, Batch culture, crude glycerol added (1%), pH H2 production rate of 51 H2 (kg total volatile solid) and yield of 2.32 mol H2 L [52] containing mainly butyrate and dark fermentation 5.5 were doubled by glycerol added; H2 was cummulated during 7 days acetate Leaf-shaped vegetable wastes with Mixed culture of Enterobacteria-ceae, Strepto- Batch culture; H2 yield depended on glucose concentration; higher yield of 1.6–2.2 mol H2 / mol [47] −1 1204 potato pills coccaceae, Pectobacterium, Raoultella, glucose added, 28 °C; glucose at low concentration (1 g L ) but maximal production at a concentration of − Rahnella and 10 g L 1 Lactococcus −1 Raw corn stalk Clostridum butyricum FS3, dark fermentation Response surface methodology; Maximal H2 yield was from sucrose; maximal H2 yield (92.9 mL g ) from corn [48] − diﬀerent sugars stalk was at the concentration of 20 g L 1 −1 Brewery wastewater Bacillus sp., dark fermentation Batch culture; pH 6.0, 35 °C H2 production yield of 61 mL H2 (g volatile solids) [49] −1 Brewery wastewater R. sphaeroides O.U.001, Bioreactor; Biebl and Pfennig médium; pH The highest H2 production (2.2 L H2 L ) was achieved with ﬁltered and sterilized [50] photo fermentation 6.0; light intensity of 116 W / m2 wastewater at concentration of 10% nd Food (kitchen) wastes Rhodopseudo-monas palustris CGA009; Bioreactor; carbon-free complex medium; The highest H2 production rate was achieved on the 2 day, H2 was cummulated [51]

photo fermentation light was from 2 fluorescent lamps of 20 W during 7 days Renewable andSustainableEnergyReviews80(2017)1201–1216 −1 Cheese whey Clostridium sp. IODB−03, Batch culture; pH 8.5; 37 °C; H2 production yield of 6.35 mol H2 mol (mol lactose) [55] dark fermentation Cheese whey wastewater Rahnella aquatilis 9, Anaerobic respirometer; pH 7.1; 20 °C H2 was reached a high content ( > 65%) in exaust gas; the highest rate was 474 mL [54] − dark fermentation (g bacterial biomass) 1 per day Olive mill wastewater R. sphaeroides O.U.001, Bioreactor; pH 7.2, 32 °C; light intensity of H2 was produced after 15 h and continued till 54 h; H2 production rate was 1 mL [56] 2 −1 −1 photo fermentation 200 W / m H2 L h and increased by metal ions additions Palm oil, pulp and paper mills R. sphaeroides NCIMB8253, Batch culture; 30 °C; light intensity of The highest H2 yield was with the mixture of palm iol mill (25%) and pulp and [59,60] −1 −1 effluents photo fermentation 7000 lx paper mill (75%) effluents; maximal H2 production rate was 496 mL H2 L h ;H2 production could be increased 1.9 fold by ultrasonification K. Trchounian et al. Renewable and Sustainable Energy Reviews 80 (2017) 1201–1216

are diﬀerent carboxylic organic acids, ethanol and gaseous H2 from sugars (glucose) or glycerol [1,2,25,31,64,66–70] (see Fig. 6). CO2 from formic acid might be used by bacteria for cell growth (key carboxylation reactions) and regulation of intracellular pH, as has been suggested [71]. The ratio of the end products is variable and depends on the concentration of substrates, pH, redox potential and

other external factors [72]. Moreover, H2 has a negative impact on the formation of end products [22,73]. The absence of formation of lactic acid and reduced formation of acetic acid from glycerol, but not from glucose, are distinctive characteristics of mixed-acid fermentation of glycerol, as compared with sugars [25,67]. Moreover, unlike the glycolysis (for sugars), glycerol fermentation enters central metabolism via dihydroxyacetone phosphate and is converted to phosphoenolpyruvate (PEP) and then pyruvate (PYR) generating a loop pathway (see Fig. 6). The latter can lead to formation of succinic acid. These are important for regulating metabolic fluxes during glucose or glycerol fermentation. Furthermore, there is no net gain of ATP during glycerol fermentation (see Fig. 6). These together have various bioenergetic effects: a lower intracellular pH is maintained and changed (lower) proton-motive force is generated during glycerol fermentation compared to glucose: these have been reported Fig. 6. Combined putative metabolic pathways of glucose and glycerol fermentation in E. recently [74]. Thus, even with E. coli, our knowledge about glycerol coli. The scheme is adapted from [1,2,25,31,64,66–70]. Dashed arrows indicate path- fermentation and H2 metabolism in bacteria is still incomplete and ways only for glucose fermentation, solid arrows indicate pathways only for glycerol further study is required. The other area requiring further study is the fermentation, and solid + dashed arrows indicate pathways common to both glucose and links between the diverse H2-oxidizing and -producing pathways in glycerol fermentation. The end products are formatted as italics. 2PG, 2-phosphoglyce- different bacteria. A deeper understanding of fermentation and H2 rate; 3PG, 3-phosphoglycerate; AcCoA, acetyl-Coenzyme A; ADP, adenosine dipho- production and the underlying physico-chemical and molecular me- sphate; ATP, adenosine triphosphate; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate; GAP glyceraldehyde-3-phosphate; NADH, dihydrodiphosphopyridine nucleo- chanisms is essential for the development of high-quality biohydrogen tide; NAD+, diphosphopyridine nucleotide; PGP, 1,3-diphosphate glycerate; G6P, glucose biotechnology. 6 phosphate; F1,6 P, fructose-1,6 diphosphate; AP, Acetyl -phosphate.

4. Hydrogenases and H2 cycling the environment. This provides additional and significant advantage for H2 production even cheap substrates and cost-effective technology H2 metabolism in bacteria leading to its production depends on conditions are used. Hyd enzymes which are either soluble or membrane-associated en- + Thus, there are cheap, cost-effective and almost unlimited resources zymes catalyzing the reversible redox reaction of 2H to H2 [75]. like organic by-products (glycerol) and different wastes for biohydro- Different Hyd enzymes have been identified in many bacteria; among gen production, which should accelerate H2 production and change our the important characteristics of these enzymes is their metal content. future energy conservation strategy. Not only cheap substrates but also Hyd enzymes are divided into two main groups based on metal content the other approaches (see Fig. 2) would significantly decrease the cost in the active sites namely nickel (Ni) and iron (Fe): the [Ni-Fe]-Hyds of H2 produced; these would be reviewed hereafter. occur in a large number of bacteria and are evolutionarily more ancient that the [Fe-Fe]-Hyds or [Fe]-Hyds [76]. The latter sub-group of (Fe)-

3. Dark fermentation of organic substrates and H2 dependent Hyd enzymes is found in a specialized group of the archaea production [76]. E. coli encodes four membrane-associated [Ni-Fe]-Hyd enzymes Many bacteria under anaerobic conditions in the absence of [68,77]. These Hyd enzymes are genetically determined: Hyd-1 and external electron acceptors can perform the so-called dark fermenta- Hyd-2 are encoded by the hyaABCDEF and hybOABCDEFG operons, tion of organic substrates, i.e. without the need for light as an energy respectively [78–80], and consist of large and small subunits contain- source. A common fermentation is the mixed acid fermentation ing [Ni-Fe] active site and [Fe-S] clusters, respectively [81,82] (Fig. 7). performed by enterobacteria, resulting in the formation of lactic, In contrast to Hyd-1, Hyd-2 has a ferredoxin-like subunit [81,83], formic and different other carboxylic acids and ethanol. The further which might be important in electron-transfer to and from the quinone disproportionation of formic acid (HCOOH) forms H2 and carbon pool [84]. Hyd-1 and Hyd-2 oxidize H2 to transfer electrons to quinone dioxide (CO2) (HCOOH - > 2H + CO2 and 2H - > H2)(Fig. 6) [2,64]. pool and to provide the cell with additional energy during glucose Therefore, dark fermentations producing H2 usually produce a mixture fermentation but also under other conditions [85]. Hyd-3 is encoded by of other products. Why is H2 produced from formic acid, as the end the hycA-hycBCDEFGHI operon and consists of a total of six subunits product of fermentation? H2 reduces formic acid formation but [86] (see Fig. 7). Sequence identity between some Hyd-3 subunits and provides H+ and, thus, represents an important factor in regulating proton-translocating NADH: ubiquinone oxidoreductase complex of intracellular pH [65]. This has been considered as an important role (complex I) allows to propose that Hyd-3 might be coupled to for H2 in balancing proton gradients and off-setting acidic pH in generation of a proton motive force [77]. bacterial physiology and bioenergetics. The fourth hydrogenase in E. coli is Hyd-4, which is encoded by the E. coli is a well-studied facultative anaerobic bacterium carrying out hyfABCDEFGHIJ-hyfRfocB operon and consists of three more sub- under anaerobic conditions (in the absence of oxygen), mixed-acid units in addition to Hyd-3 [87] (see Fig. 7). Hyd-3 and Hyd-4 form the fermentation of carbohydrates (sugars), which are common and stored major parts of the large, multi-protein formate hydrogenlyase (FHL) in plant tissues as well as in other organic carbon sources, particularly complexes [87,88]. It was proposed that, unlike FHL-1, which com- in different wastes [2]. The regulation of fermentation pathways in E. prises Hyd-3, FHL-2 includes the Hyd-4 enzyme and may be an coli is complex [64]. They can be represented schematically as a set of energy-transducing complex since, like the proton-translocating biochemical redox reactions where the end products of fermentation NADH: ubiquinone oxidoreductase complex and unlike Hyd-3, the

1205 K. Trchounian et al. Renewable and Sustainable Energy Reviews 80 (2017) 1201–1216

Fig. 7. [Ni-Fe]-hydrogenase enzymes and their genes of E. coli. Structural components are given with their molecular mass predicted or determined. Proteins of Hyd-4 have not been isolated and characterized yet. Based on data from [82,86–88].

Table 3

H2 production by E. coli and characteristics of hydrogenase enzymes for batch-culture during glucose and glycerol fermentation at diﬀerent pHs.

Characteristics or properties H2 production from glucose H2 production from glycerol of Hyd enzymesa pH 7.5b pH 5.5b pH 7.5b pH 5.5b

−1 −1 H2 production rate in wild type, mmol H2 min (g dry weight) 8.43 4.57 6.79 Responsible Hyd enzymes Hyd−4 Hyd−3 Hyd−2 is major, and Hyd−1 is less Hyd−3 Sensitivity to osmotic stress Hyd−3 and Hyd−4NDc Hyd−3 and Hyd−4 Hyd−1, Hyd−3 and Hyd−4 d Recycling H2 + ++ + Inhibition by DCCD + –– +

Requirement of the FOF1-ATPase + – ++ Coupled generation of a proton-motive force + + + + Intracellular pH 7.5 6.1 7.0 5.7

a For references, see the text. b Initial pH, ﬁnal pH in buﬀered medium (20 g L−1 peptone) was 7.2 or 5.2 for initial pH 7.5 or 5.5, respectively. c Not determined. d The signs “+” or “–“ represent the presence or the absence of the characteristics, respectively.

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Hyd-4 complex possesses ND2/ND4/ND5-like subunits [87]. It was 3 activity. At pH 5.5 during glucose fermentation Hyd-1 was essential shown that during the fermentation of glucose, the hycB gene product for H2 metabolism, with Hyd-3 contributing little in the wild type to the is required for the activity of Hyd-4 at neutral and slightly alkaline pH total activity under these conditions [85]. Interestingly, Hyd-1 could

[89], and might, therefore, be a component of the functional complex become an effective H2 producer at pH below 4 [113], but bacteria such FHL-2. This may explain why E. coli mutants lacking Hyd-1, Hyd-2 as E. coli cannot grow under these extreme conditions. Hyd-3 had a and Hyd-3 do not produce H2 [86,90,91]. more significant contribution to total Hyd activity with glycerol at pH The expression of the hya or hyb genes was shown to be induced 5.5 [85]. These findings substantiate the important role of H2-oxidizing under anaerobic conditions upon fermentation of glucose at acidic or Hyd-1 during growth at acidic pH. It is also suggested that FHL alkaline pHs [92] and under reducing or oxidizing conditions, respec- (including of Hyd-3) can change its operation direction to synthesize tively [93–96], and by formate [80,97]; the hyc operon was also formic acid at low pH [114]. induced by formate [98] and regulated by the transcriptional regulator Moreover, some metabolic crosstalk between Hyd-1 and Hyd-2 is FhlA [99] and in response to pH [98,99]. [Ni-Fe]-Hyd enzymes suggested on both the gene expression and enzyme activity levels [115]. undergo a complex maturation, and some proteins, such as the Hyp This crosstalk was also offered when Hyd-2, but not Hyd-1, activity enzymes, are required for this process [82,100]; intracellular CO2 is increased at a low activity of Hyd-3 or when Hyd-1, but not Hyd-2, also important in this process [101]. activity was reduced when Hyd-3 activity was abolished [79]. The activity of E. coli Hyd enzymes and the direction of their Furthermore, Hyd-3 activity increased when the hyb but not hya operation have been shown to depend on fermentation substrates and operon was deleted [93]; however, this might be related to improved pH [68] (Table 3). Moreover, the concentration of substrates is maturation of Hyd-3. It is clear that the interaction between Hyd important: H2 production by Hyd-4 has been determined from glucose enzymes to oxidize and to produce H2 is important for H2-cycling; at low concentration and at slightly alkaline pH (pH 7.5) [102]; glucose however, further detailed study is required. at a higher concentration ( > 0.2%) can inhibit the expression of the It has been suggested that Hyd-1 of E. coli has H2-producing hyf operon [91] and Hyd-4 activity [102], thus affecting H2 production. capability in the presence of oxygen [97]. This is interesting as Hyd-1 is Different groups have attempted to determine Hyd-4 activity [78– active under oxidizing conditions [83,94] and oxygen can limit 81,83,86,97,100–102] but this work was hampered presumably be- biohydrogen production, which usually requires anaerobic conditions. cause of the high concentration of glucose used ( > 0.4%). It should be noted, however, that E. coli does not synthesize either

Furthermore, H2 production from glucose or glycerol is sensitive to Hyd-1 or Hyd-2 during aerobic growth but the Hyd-1 enzyme survives osmotic stress provided by sucrose (see Table 3): Hyd-3 and Hyd-4 are for at least short periods upon a shift back to aerobic conditions sensitive to osmotic stress but at different pHs, whereas Hyd-1 is [82,83]. osmosensitive at low pH [89,103]. The other important property of There is also an intriguing phenomenon during H2 production Hyd enzyme is their inhibition by diphenylene iodonium (Ph2I) whereby the Hyd enzymes in E. coli form a H2-cycle across the [31,103], which was shown to inhibit [Ni-Fe]-Hyd enzyme activity in cytoplasmic membrane when at least one Hyd enzyme operates in the several bacteria [104]. H2-oxidizing and the other one in H2-producing mode (Fig. 8) H2 production by E. coli has been established to be completely [1,2,24,66]. This is possibly one reason why E. coli has multiple Hyd absent when Ph2I is present at very low concentration (1 nM) [31,103]. enzymes. H2-cycling is suggested to occur in other bacteria, such as H2 production by Hyd enzymes can also be inhibited by N,N′- Salmonella typhimurium [116]. The other aspect of H2-cycling is its dicyclohexylcarbodiimide (DCCD) [23,24,89,105,106], which is an effect on proton cycling, which is a well-known phenomenon in + inhibitor of the H -translocating FOF1-ATPase and other proton bioenergetics: it results in generation and utilization of proton motive translocation pathways [107]. DCCD inhibition is different during force, having a key role in energy transformation in cells [117]. glucose or glycerol fermentations and depends on pH (see Table 3). During the fermentation of glucose or glycerol the H+-translocating

It has been established that H2 production was repressed at alkaline FOF1-ATPase is the main mechanism of bioenergetics relevance to (pH 7.5) but not acidic (pH 5.5) pH during glucose fermentation, proton motive force generation [8,118,119]. An interaction of H2- and whereas the reverse was observed during glycerol fermentation [24].It H+-cycles in the membrane has been suggested to maintain proton is likely that DCCD inhibits H2 production indirectly: it interacts with motive force and to regulate intracellular pH when bacteria consume the FOF1-ATPase, which might play a role in operation of Hyd enzymes. reduced substrates [10] (see Fig. 8). In this respect, two very different Indeed, DCCD inhibited the FOF1-ATPase activity during glucose and findings are important: firstly, proton motive force generation is glycerol fermentation but during glycerol fermentation the inhibition of changed in an E. coli hypF mutant lacking all Hyd enzymes [74] due the enzyme was absent in hypF mutant (where all four Hyd enzymes to the effects on the FOF1-ATPase [109]. Therefore, Hyd enzymes have are absent [108]) at pH 7.5; however, inhibition was still observed at a direct impact on the overall proton-motive force generation and H+- pH 5.5 [109]. Interestingly, during glucose fermentation Hyd-4 activity pumping by Hyd is therefore suggested. Second, the H2 production rate was shown to depend on K+ [105,110], which is presumably the reason determined in liquid and gaseous phases was markedly different during + why H2 production by Hyd-4 had not been observed in K -depleted glycerol but not during glucose fermentation by E. coli; significantly cells. (30-fold) more H2 was observed in liquid phase [120]. This suggests an Whole-cell Hyd enzyme activity and H2 production by E. coli wild increased importance of H2-cycling during glycerol fermentation. type and mutant strains during glucose and / or glycerol fermentation at different pHs (7.5, 6.5 or 5.5) was intensively investigated during the 5. Fermentation of mixed carbon sources and H2 production last few years. During glucose fermentation at pH 7.5, Hyd-2 (exhibits

H2-oxidation and proton reduction) and Hyd-3 form the basis of the An interesting aspect for enhancing H2 production is the fermenta- Hyd activity, with negligible contribution to overall activity made by tion of mixed carbon sources, especially glucose and glycerol together Hyd-1 [85,93,111]. In contrast, fermentation of glycerol at pH 7.5 or with other carboxylic acids – a novel approach developed over the demonstrated that Hyd-1 and Hyd-2 formed the main part of the last few years. Indeed, since glycerol is such a cheap substrate for dark overall Hyd activity [85,112]. Therefore, Hyd-2 is suggested to be a mixed-acid fermentation by different bacteria, therefore, sugars (glu- unique enzyme that can switch between H2-oxidizing and H2-produ- cose) remain of interest and work effectively in improving H2 yields, cing modes in response to the redox status of the quinone pool [77];H2 especially when mixed with glycerol or with carboxylic acids, the latter production from glycerol by Hyd-2 requires reverse electron transfer being components of different carbon-containing wastes. [84]. Hyd-3 had the highest activity after growth at pH 6.5 in the It is interesting that, in addition to only the fermentation by E. coli presence of either glucose or glycerol [85] so pH 6.5 is optimal for Hyd- and a few other bacteria [23,24,85], it has been shown that glycerol ( >

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Fig. 8. H2-cycling in bacteria during dark-fermentation. A- H2-cycle across the cytoplasmic membrane in bacteria formed by combined actions of H2-oxidation and H2-production via hydrogenase enzymes working in opposite directions. B- H2- and proton-cycles in E. coli during glucose fermentation at alkaline pH (pH 7.5). The scheme is adapted from [1,10,68]. The + directions of operation of the respective hydrogenases to produce and / or to consume H2 are shown (arrows). If suﬃcient ATP is available it can be hydrolyzed also to drive H + + translocation through the F0F1-ATPase, generating proton motive force. The latter drives a set of secondary H transporters (H -porters). (a-b) is for amount of H2 evolved by cell; (m-n) – H+ secreted from cell.

Table 4 fermentation [123]. Moreover, ATPase activity determination suggests

Hydrogenase enzymes responsible for H2 production by E. coli batch cultures during that Hyd-1 and Hyd-2 are required for the FOF1-ATPase [109]. These mixed-carbon fermentation at diﬀerent pHs. interesting and provocative ﬁndings clearly indicate that further

a research is required on this topic. Mixed carbon Responsible Hyd enzymes Approximately a decade ago it was shown that in addition to its

pH 7.5 pH 5.5 high activity at pH 6.5, Hyd-3 is also a major H2-producing enzyme of E. coli when the bacterium grows at pH 7.5 by glucose fermentation Glucose (2 g L−1) and Hyd−3 Hyd−3, Hyd−4 mainly, − and when the substrate for H formation is formate (30 mM) glycerol (10 g L 1) and Hyd−2 in some 2 extent [124,125]. Interestingly, during glucose fermentation by E. coli up to Glucose (2 g L−1) and Hyd−3 and Hyd−4 Hyd−3 one-third of this carbon source can be converted to formate reaching a formate (10 mM) maximal concentration of ~ 20 mM [126,127]. This formate can be Glycerol (10 g L−1) and Hyd−3, Hyd−4 mainly, Hyd−3 oxidized within the cell to produce H2, as noted before, or it can be formate (10 mM) and Hyd−2 in some extent exported. At the same time external formate can be re-imported into the cell through the formate channels FocA and FocB at diﬀerent pHs; a For references, see the text. moreover, external formate directs FocA to import the anion [128]. Formate added into the medium in the presence of glycerol stimulated −1 10 g L ) can be co-fermented in the presence of sugars (glucose) at E. coli growth and H2 production at diﬀerent pHs [129,130]. During slightly alkaline pH [30,121–123]. Moreover, H2 production can be glycerol fermentation formate might also be imported through FocB, inhibited by DCCD and total inhibition of H2 production by DCCD was whereas formate is exported preferentially through FocA at pH 7.5 observed in fhlA (encodes transcriptional activator FhlA of hyc operon [128]. Interestingly, during glycerol fermentation formate (10 mM)

[99]) mutant at pH 7.5 and pH 5.5 [123] suggesting that FhlA protein recovered H2 production in Hyd-2 (hybC) or Hyd-4 (hyfG) mutants, or more likely the Hyd-3 might directly interact with the FOF1-ATPase. depending on the pH value [129,130]. These results highlight the key Importantly, diﬀerent Hyd enzymes are responsible for H2 production role of Hyd-3 at both pH 6.5 and pH 7.5, as well as the role of Hyd-2 depending on the conditions (Table 4): the activities of mainly Hyd-4 and Hyd-4 at pH 7.5 for H2 production by E. coli during glycerol and and, to some extent, Hyd-2 to produce H2 have been revealed at low pH formate co-fermentation. Moreover, under these conditions H2 pro- [115,121–123]. When glucose and glycerol were used together, Hyd-3 duction depends on the formate channels. It is proposed that the was the main H2-producing enzyme either in slightly alkaline (pH 7.5) absence of formate channels might lead to increased H2 production or acidic (pH 6.5 and 5.5) medium. In addition, previously unknown [128], but this needs to be demonstrated experimentally.

Hyd-4 activity at pH 5.5 was demonstrated. Moreover, Hyd-1 and Hyd- H2 generation has been investigated using the mixture of glycerol −1 −1 2 formed H2-oxidizing activity at pH 7.5 and compared to this, Hyd- (10 g L ) and acetate (5 g L )atdiﬀerent pHs [131]. Note, acetic acid 1has no any H2-oxidizing activity at the same pH during glucose-only (C2H4O2) can produce H2 (C2H4O2 +2H2O->4H2 + 2CO2). The

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Fig. 9. Photo-fermentation to H2 production and H2-cycling in purple non-sulfur bacteria. The scheme is adapted from [1,32,33,142,143].A-H2 production by nitrogenase and H2- + - cycling under nitrogen-limited conditions. B- H2 production by Hyd enzyme under nitrogen excess conditions. aH translocated through the membrane via e transfer chain generates a - proton motive force, and the FOF1-ATPase makes ATP. e transfer paths from carbon source are shown (arrows).

−1 highest H2 yield of 5.16 mmol L was detected at the log growth phase bacteria – Eubacteria and Archaea during a dark fermentation [52]. of E. coli at pH 7.5. H2 production rate was increased at pH 6.5 when The mixed-carbon substrate fermentation under optimized conditions cells were grown with mixed carbon sources (glycerol and acetate) might be used for pre-cultivation of bacterial cells, regulation of Hyd

[131]. Interestingly, H2 generation was continuously detected at pH 7.5 enzymes activity towards enhancing H2 production and utilizing and pH 5.5 for 96 h of growth. There are currently no data available on glycerol and carbon-containing organic wastes. There are both eco- which Hyd enzymes activity and which is responsible for H2 produc- nomic (cheap wastes) and environmental (wastes utilization) benefits. tion. Moreover, with knowledge of the genome sequence (defined Interestingly, formic or acetic acids are known as weak acids having transcription and translation systems, broad spectrum of already uncoupling effects on bacteria [65,132]. However, these end products constructed mutant strains), metabolic flux analysis (see metabolic of fermentation are proposed to be re-assimilated back into the cell network of glucose and glycerol fermentations on Fig. 6), as well as the once glucose or glycerol is consumed [133], and the consumption of ability of fast growth on cheap substrates (wastes) and based on the acetate by bacteria has been shown in the presence of different sugars results already obtained, genetic and metabolic engineering it should

[133]. be possible to construct E. coli strains with increased H2 production. Recently, the conversion of glycerol and its mixture with glucose In this respect it is of great importance that Wood and his into H2 has been demonstrated with C. beijerinckii DSM791: a higher colleagues [135–137] have identified uncharacterized genes (54% of H2 production was at pH 7.5 [26]. But responsible Hyd enzymes and E. coli genome is determined experimentally; the other part is regulatory mechanisms are not clear. Interestingly light intensity has uncharacterized or predicted on the basis of computational analysis) been shown to have negative effect on H2 production by this bacterium whose inactivation is beneficial for H2 production from glycerol. Using [134]. This effect should be taken into consideration if co-culture of C. the recombinant DNA technology, they constructed the strains with beijerinckii with photosynthetic bacteria is formed and light is applied. ability for ~ 1.6-fold higher H2 production rate and ~ 2.1-fold higher Moreover, crude glycerol added could double the methane-free H2 H2 yield. Output of H2 has reached 95% of the theoretical maximum production rate and yield from municipal solid waste by the other but there is still some room for improvement. However, the conditions,

1209 K. Trchounian et al. Renewable and Sustainable Energy Reviews 80 (2017) 1201–1216 particularly substrates and pH, are important in the use of these strains enzyme as well. However, it has also been suggested that the Hyd for increasing H2 production to have maximal activity of H2-producing enzyme can be reversible and operate in H2-producing and H2- Hyd enzymes. In addition, a new model for metabolic engineering oxidizing modes (see Fig. 9). This seems to be similar to the situation based on an in vitro synthetic enzymatic pathway has also been with Hyd enzymes in E. coli [23,24]. In addition, the H2-producing developed to enhance H2 production from biomass in a distributed, enzyme can be involved in generation of proton motive force [156,157]. carbon-neutral and inexpensive way [138]. This led to a 67-fold Н2 production by these bacteria is essentially increased by supple- increase in the production of H2 by E. coli. This approach can be used mentation of different organic substrates, as organic acids or sugars as for effective H2 production by other bacteria using variable conditions carbon sources and as amino acids and yeast extract as nitrogen with substrates (by-products and wastes) and pH. sources [39,41,158]. Increased H2 production has been determined Moreover, a high-throughput methodology for the systematic with succinic acid as carbon source, and glutamic acid or yeast extract analysis of E. coli knockout strains for the study of glycerol consump- as nitrogen source [39,42]. R. palustris was shown to produce H2 from tion and H2 metabolism has been designed recently in order to identify crude and pure glycerol with high efficiency (conversion efficiency was novel phenotypes with enhanced H2 yield [139]. This methodology was about 90%) [159,160], but this finding needs additional more detailed useful for screening E. coli mutants, and novel effective strains were analysis. Moreover, H2 production was shown in medium containing found; it can facilitate an engineering strategy with other H2-producing salt ( < 2% sea salt) [140]. Interestingly, nitrogen-limited and nitro- bacteria. gen-excess conditions (with 2 g L−1 and 5 g L−1 (2.5 fold higher

amount) yeast extract, respectively) can significantly affect H2 produc- 6. Photo fermentation of organic substrates and responsible tion by R. sphaeroides:H2 production was higher in nitrogen-limited enzymes conditions [153]. It is likely that H2 can be produced as the final step following different metabolic routes (see Fig. 8): for example, in Fermentation of organic substrates in the presence of light resulting nitrogen-limited conditions nitrogenase catalyzes reduction of H+ to + – in the production of H2 (photo fermentation) has been found in purple H2 according to the reaction: 8H +8e + 16ATP - > 4H2 + 16 ADP+ non-sulfur photosynthetic bacteria and microalgae (see Fig. 1), parti- 16Pi (Pi is inorganic phosphate) and in nitrogen excess conditions cularly in R. sphaeroides (the main workhorse of these photosynthetic nitrogenase catalyzes the reduction of N2 to NH3 according to the + – genera), Rhodobacter capsulatus or Rhodopseudomonas palustris reaction: N2 +8H +8e + 16ATP - > 2NH3 +H2 + 16ADP + 16Pi (H2 [1,16,32–34,140]. could be produced by Hyd enzyme). Therefore, H2 can be produced at The photosynthetic apparatus of purple non-sulfur bacteria consists higher levels in nitrogen-limited conditions. That is why nitrogenase is of light-harvesting complexes and reaction centers and contains mainly considered as a main H2-producing enzyme in purple bacteria but it bacteriochlorophylls (BChl) and carotenoids [141]. Upon absorption of should be clarified and the Hyd enzyme cannot be excluded as a source light energy by the light-harvesting pigment-protein apparatus, elec- of H2. trons from carbon substrates can be transferred to nitrogenase via the The nitrogenase has also been suggested to function in a different respiratory chain of cytochromes and ferredoxins (Fd) and this results mode depending on nitrogen source limitation; additionally, in the + in the formation of H2; protons are pumped through the membrane to absence of nitrogen in the medium nitrogenase reduces H only [32]. generate a proton motive force [1,32,33,142,143] (Fig. 9). It is known Thus, nitrogen source in the medium is important to regulate the that H2-production by R. sphaeroides is coupled to [Mo-Fe]-nitrogen- substrate used by nitrogenase, but the exact range of nitrogen source ase, which not only participates in nitrogen fixation, but also catalyzes used needs to be defined more clearly. In this context it is interesting to + - the reduction of H to H2.H2-production by nitrogenase is an suggest that under nitrogen excess conditions e can bifurcate passing irreversible process, requiring a large amount of ATP, which is possibly from NADPH to Hyd enzyme and nitrogenase via Fd (see produced by the FOF1-ATPase, the main membrane-associated enzyme Fig. 9). This is similar to electron-bifurcation in energy conservation of bioenergetic relevance. R. sphaeroides also has a [Ni-Fe]-Hyd processes described for Fd and different enzyme systems in bacteria enzyme, which is responsible for H2 uptake under anaerobic photo- and archea [161]. fermentative conditions; unlike nitrogenase it does not require ATP. A role for the FOF1-ATPase in H2 production by R. sphaeroides is Another Hyd enzyme is also proposed for this bacterium [144]. crucial, that is why the inhibition of the FOF1-ATPase by DCCD has Interestingly, nitrogenase is sensitive to oxygen, ammonia (NH3) and been shown to suppress H2 production [38,157]. In contrast, over- to a high ratio of nitrogen to carbon [145–147]. The main problem for expression of the FOF1-ATPase complex in R. sphaeroides HY01 has H2-production biotechnology is to overcome the oxygen-sensitivity of been shown to improve ATP content, enhancing nitrogenase activity nitrogenase and Hyd enzyme [148]. and, significantly, increasing H2 production [162]. Genetic studies with R. sphaeroides have not achieved a significant Thus, H2 production by purple non-sulfur bacteria can result from progress; transcription and especially regulation of responsive genes interactions between different enzymes and factors, where nitrogenase, expression is complex. R. sphaeroides has been shown to have one Hyd enzyme and the FOF1-ATPase are the main proteins involved. It is [Mo-Fe]-nitrogenase, which is encoded by nifHDK genes [149,150]; likely that this interaction network is highly interconnected and the Hyd enzyme is encoded by hupSL genes [142]. Deletion of hupSL depends on the cellular nitrogen status. Moreover, nitrogenase and genes resulted in 1.2 fold increase in H2 production from malic but not Hyd enzyme operating in opposite directions may form a type of H2- from acetic acid [151]. As for other enzymes involved in H2 metabolism cycling (see Fig. 9), but this is not obligatory with these bacteria. in different bacteria, the Hyd enzyme consists of large and small subunits containing catalytic sites and [Fe-S] clusters, respectively. 7. Novel approaches with photo fermentation to produce H2 Additional nitrogenases could be found in R. capsulatus increasing H2 production [152], but their role in physiology of the bacterium is not Novel approaches for increased H2 production by photo-fermenta- clear. tion are constantly being developed over the last few years. They might

H2 production by R. sphaeroides MDC6521 and MDC6522 has have application in H2-production biotechnology using photosynthetic been shown to be inhibited by metronidazol (2-methyl-5-nitroimida- purple non-sulfur bacteria or their co-cultures with diﬀerent bacteria. zole-1-ethanol) ( < 50 μM) [143,153,154], a long-range electron Interesting results with R. sphaeroides MDC6521 batch cultures acceptor from Fd and inhibitor of nitrogenase [155], and Ph2I have been obtained recently where succinate (30 mM) was used as a (0.5 μM), an inhibitor of [Ni-Fe]-Hyd enzyme [104]. These eﬀects carbon source and yeast extract (2 g L−1) – as a limited nitrogen were concentration-dependent. The inhibitory studies performed con- source. Firstly, the alternation of the light-dark period and of the light

ﬁrm that H2 production resulted from both nitrogenase and a Hyd intensity has led to changes in the production of H2 [163]. It was shown

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that bacteria did not grow well in dark conditions, but the growth and to enhanced H2 production by Hyd-4; this is in line with the suggested H2 production were restored under illumination. When the culture was dependence of Hyd-4 on the proton motive force [173]. This also + illuminated during 24 h after the dark period, the H2 yield remained at confirms with the proposed H -translocating activity of Hyd-4 [10,87]. a constant high level during 96–144 h. When the culture was illumi- Moreover, nitrogenase and hydrogenase genes from R. sphaeroides nated during 48 h and then dark conditions were used, H2 production have been shown to be heterologously co-expressed in E. coli: this was maintained for 120 h, while the control cells did not produce H2. manipulation also led to increased H2 production [174]. Interestingly, These changes might be explained by changes in the formation of the non-native [Ni-Fe]-Hyd enzyme encoded by hupSL genes of R. photosynthetic pigments especially BCl 875 (component of light palustris could improve H2 production by E. coli [175]. harvesting apparatus) and decrease in H+ conductance of the mem- These approaches are attractive for potential large-scale application branes analyzed [163]. This is similar to how changes in the light- in H2 production. With genetic and metabolic engineering of purple harvesting complexes affect H2 production, as was shown previously, bacteria [32,37,61,144] and optimizing conditions, an increased and but using slightly different conditions [32,164,165].H2 production by effective production of H2 could be achieved, but attention must be R. sphaeroides decreased in a pufBA mutant, which lacked BChl 875; paid to the substrates and conditions used. but it increased in a pucBA mutant, which lacked the other component of light harvesting apparatus, BChl 800–850 [164], showing that light 8. Organic wastes and co-cultures and mixed cultures is not limiting for H2 production. Moreover, it was suggested that the ATP required for light-driven Another approach for increased H2 production from organic wastes H2-production was obtained during photophosphorylation, and suffi- by bacteria is using co-cultures or mixed cultures (see Fig. 2), which cient intensity and exposure of light supplied more reducing power and may be stable and allow use of a wider choice of feedstock. Two ATP via membrane-associated photosynthetic electron transfer, which different types of cultures have been studied and applied in this was involved in photo-fermentative H2 production [166].Different technology so far: co-cultures of identified bacteria and mixed-cultures light-dark cycles have been shown to change cumulative H2 production from different natural sources. by the other photosynthetic bacteria - R. capsulatus compared to Recently, the approach has been developed for H2 production using continuous illumination [167]. Thus, manipulation of the light-dark co-culture of dark fermentative E. coli with purple photo-fermentative cycle, especially the period of illumination after a 24 h dark period, in R. sphaeroides where H2 production by R. sphaeroides and its comparison with continuous illumination can be considered as optimal regulation were studied and a requirement for carbon and nitrogen conditions for enhanced Н2 yields providing energy savings. sources was determined [36]: the co-culture could grow on the carbon- Reducing agents like DL-dithiotreitol (DTT) or dithionite have been containing waste (DG with solubles) at pH 7.5, and, in a one-stage shown to stimulate H2 production in a concentration-dependent process, more H2 yield (2-fold diluted DG with solubles yielded a 1.5–3 manner [153]. These reagents decreased the extra-cellular redox fold increase in H2 yield in comparison with pure cultures) was potential [168], and reducing conditions were required for H2 produc- obtained (see Table 2). The maximal rate of H2 production by co- −1 −1 tion. In addition, they increased DCCD-inhibited ATPase activity of culture was 5.16 mmol H2 L day . It should be noted that the −1 −1 membranes [153]; thus, the changes in ATPase, the activity of which is highest H2 production rate was 7.9 mmol L day , reported with an required for photo-fermentative H2-production, might explain the unidentified mixed-culture and from DG from the wine industry; the effects of the reducing agents. process involved a heat-treatment step and acidic pH [43]. DG with Recently, it has been reported for the first time that low-intensity solubles can alter the modes of bacterial metabolic pathways (see electromagnetic field of extremely high frequences (51.8 GHz and Fig. 6) and improve H2 production. The increased H2 production from 53 GHz) can affect H2 production by R. sphaeroides [169]: short DG with solubles in co-culture could be explained by the disproportion exposures (15 min) increased, and long exposures (60 min) decreased between rates of substrate uptake and fermentation end product

H2 production. Interestingly this electromagnetic field effect also formation with changing pH. This might change activity of the Hyd + increased H transfer and the FOF1-ATPase activity. It could be enzymes leading to improved H2 production, as has been suggested suggested that in addition to water (which can change its structure [176]. and properties), the FOF1-ATPase is a basic target for electromagnetic The processes underlying the biotechnology development using DG field as in many bacteria under anaerobic conditions [170] and mediate fermentation end products with E. coli, namely acetate and succinate, its effect on H2 production. Electromagnetic field of high frequences could also be applied to R. sphaeroides for continued H2 production; has been already applied for pre-treatment of microalgal biomass to the enhanced H2 yield might also be related to use of reducing power increase biogas production [171]. These results are important for and to improved levels of ATP synthesis, as also shown with dark-light possible future use of electromagnetic irradiation technology. cycle effects with R. sphaeroides [163]. As a novel approach, the use of mixed carbon sources (glucose, Co-culture of Clostridium butyricum and R. sphaeroides has been xylose and acetic acid) has been adapted for photo-fermentative H2 reported recently to produce H2 from starch; H2 production depended production by R. sphaeroides S10 [172], as originally developed for use on species ratio and the yeast extract concentration [177]. The other with E. coli [121,123]. The mixed carbon composition strongly co-culture of Clostridium acetobutylicum and R. sphaeroides was also influenced H2 production. Depending on the composition of the mixed effective for H2 production from starch: enhanced H2 production was substrate, the cumulative H2 yield varied significantly, reaching 2.33 L observed at pH 7.0 by both bacteria; however, a higher H2 production −1 H2 L . The optimal substrate concentration ratio was a mixture of was obtained when pH was increasing from 6.0 to 7.5 but dark and 5 mM glucose, 18 mM xylose and 7 mM acetic acid [172]. The H2 yield photo-fermentation by appropriate bacteria were separated in time was 3.56 mol H2/mol of mixed substrate and the substrate-specific [178]. −1 −1 production rate was 7.26 mL H2 (g mixed substrate) h . Mixed cultures (anaerobic microbiota) obtained from sewage Interestingly, these carbon sources were consumed by R. sphaeroides sludge, cow dung and soil have been used for dark-fermentative H2 with equal efficacy [172]. production because they contained a variety of H2-producing bacteria Recently, it has been reported that proteorhodopsin, a light- [43]. Mixed cultures of different consortia of bacteria have also been harvesting protein in bacteria, can increase H2 production when it shown to produce more H2 from dairy or distillery wastewater by both was expressed in recombinant E. coli [173]. This effect was observed dark- and photo-fermentative processes [179]. However, this process when HyfR, the transcriptional activator for the hyf operon encoding depends on different factors, such as pH, substrates, light and other Hyd-4 (see Fig. 5), was overexpressed. It was proposed that the factors, and often requires pre-treatment of substrates. It is of interest + proteorhodopsin could increase the H gradient and thus contribute that predictive and explicative models have been developed for H2

1211 K. Trchounian et al. Renewable and Sustainable Energy Reviews 80 (2017) 1201–1216 production from solid organic wastes containing different substrates production [187]. The effects were different depending on bacterial for fermentative H2 production by mixed cultures: it was shown that growth phase; they were mostly observed during glycerol but not sugars content and butyrate and lactate concentrations were correlated glucose fermentation and at acidic pH (pH < 6.5) [188]. Moreover, 2+ and had significant role in the variation of H2 yield [45,180]. Changes Fe (50 μM) had no effect on H2 production in a hyaB hybC mutant in metabolic pathways and bacterial community variations are sug- (lacking Hyd-1 and Hyd-2), confirming a role for Hyd-1 and Hyd-2 in gested to result different H2 production [180]. However, these models reducing H2 production [187,188]. Interestingly, various metal ions 2+ 2+ 6+ should be further developed to give more clear predictions and including Ni ,Fe and Mo have also been shown to increase H2 determined process results. production in other bacteria, especially C. butyricum [189], R. Therefore, selection and formation of stable co-cultures or mixed sphaeroides [56,190–192] or mixed-cultures [193]; but the concentra- cultures and optimization of technological conditions could be a tions of metals, as well as the substrates for fermentation and promising area for further development. Another interesting aspect technology conditions used were different, thus making direct compar- to be understood is with regard to the interactions between bacterial ison of conditions difficult (see Table 5). Indeed, the modification of the species in co-culture or mixed culture, which can significantly change medium with some heavy metals and their mixtures causing increased

H2 production yield [36,43,176–178] and the fermentation product H2 yield would be important for optimization of the technology spectrum [181]; this is complex phenomenon and requires further in- conditions in H2 production biotechnology, especially when waste- depth study. products used as carbón source are contaminated with heavy metals. 2+ 3+ Discrimination between Fe and Fe is important for H2 produc- 2+ 3+ 9. Optimizing conditions: heavy metal ions tion; some interaction of Ni with Fe can affect activity of H2- 6+ producing Hyd enzymes, and Mo is important for H2 production. For effective biohydrogen production by [Ni-Fe]-Hyd enzymes a On the other hand, copper ions (Cu2+) in low concentrations have low concentration of Ni and Fe is required. These metals are necessary been shown to inhibit some membrane-associated enzymes in E. coli, for biosynthesis and maturation of Hyd enzymes [82,182,183]. They including the FOF1-ATPase and Hyd enzymes [194], and Hyd enzymes + are proposed can participate in redox reactions, transferring electrons in archaea [195]. Low concentration of Cu ( > 100 μM) had a H2 through several [Fe-S] clusters (typically 3) to the active site of Hyd production-suppressing effect on E. coli [185,187,188] and R. sphaer- enzymes [184]. Furthermore, the redox potential of the metal in the oides [189] (see Table 5), while very low concentration of Cu+ but not + - 2+ active site can influence the equilibrium of 2H +2e -> H2 reaction Cu (5 μM) increased H2 production by R. sphaeroides [196] (see [76]. Therefore, metals are suggested to regulate the activity of Hyd Table 5). It is likely to that Cu+ at low concentration ( < 50 μM) is enzymes and optimization of their delivery can lead to improved H2 required by many bacteria but in relatively moderate concentrations it + 2+ production. In addition, H2 production by the FHL complex requires is toxic. The difference between Cu and Cu effects may be coupled molybdenum (Mo) for the formate dehydrogenase component with reducing and oxidizing properties of copper and their efficiency of

[88,185,186]; Mo might be also important to increase H2 production. uptake. 2+ 3+ 2+ H2 yield from glycerol by E. coli in peptone medium has been Besides, some heavy metals (Fe ,Fe ,Ni ) and their mixtures 2+ 3+ 2+ 2+ 2+ shown to be stimulated 1.7–3 fold by the mixtures of Ni +Fe ,Fe (Fe ,Ni ) have simulatory effects on H2 production by C. beijerinckii +Mo6+,Fe3+ +Mo6+ and Ni2+ +Fe3+ +Mo6+, but not by the individual [26] (see Table 5) although different Hyd enzymes might be operated. 3+ 6 metals. The maximal increase was obtained by a mixture of Fe +Mo All this might be useful in optimization of technology conditions for H2 [187,188] (Table 5). It was shown that Ni2+ and Fe2+ at higher production by different bacteria. concentrations (200 μM and 100 μM, respectively) stimulated H2

Table 5

Effects of different metals on H2 production by diﬀerent bacteria during dark- and light-driven fermentations.

Bacteria, fermentation Technology conditions Eﬀect of metals on H2 production References type

2+ −1 2+ −1 Clostridium butyricum, Bioreactor; glucose fed continuous tank, glucose Ni (25 mg L )orFe (50 mg L ), increased 1.7 fold H2 yield; [188] −1 −1 2+ −1 3+ −1 dark fermentation (9 g L ) and yeast extract (2 g L ), pH 5.0 Ni (50 mg L )+Fe (50 mg L ) increased 1.4 fold H2 yield; further increase of Ni2+ and Fe2+ concentration had no additional

eﬀects on H2 yield 3+ 2+ Clostridium beijerinckii Batch culture; glucose (40 mM) or glycerol (110 mM) Fe (0.1 mM) increased 1.3 fold H2 yield upon glucose; Fe [26] 3+ 2+ DSM791, and formate (10 mM), pH7.5 (0.01 mM) Fe (0.5 mM) or Ni (0.001 mM) increased 1.3 fold H2 dark fermentation yield upon glycerol and formate Escherichia coli BW25113, Batch culture; peptone medium with glycerol Ni2+ (50 μM) + Fe3+ (50 μM), Fe2+ (50 μM) + Mo6+ (20 μM), Fe3+ + [186,188] −1 6+ 2+ 3+ 6+ dark fermentation (10 g L ), pH 6.5, 37 °C Mo or Ni +Fe +Mo increased 1.7–3 fold H2 yield at the late stationary growth phase; maximal increase was by Fe3+ +Mo6+ 2+ 2+ Escherichia coli BW25113, Batch culture; peptone medium with glycerol Ni (200 μM) stimulated 1.5 fold H2 production at pH 7.5; Fe (> [187,188] −1 2+ dark fermentation (10 g L ), 37 °C 50 μM) increased H2 production and Cu (> 50μM) inhibited H2 production at pH 6.5 and pH 7.5 3+ Rhodobacter sphaeroides Bioreactor; olive mills wastewater (2%), pH 7.2, 32 °C; Fe (100 μM) increased 1.6 fold H2 production duration, 2 fold and [56] 2 6+ O.U.001, light intensity of 200 W / m 3 fold H2 production rate and yield, respectively; Mo (16.5 μM)

photo fermentation decreased 2 fold H2 production duration but increased 3 fold and 1.6

fold H2 production rate and yield, respectively; in comparison with 10 μMFe3+ and 7 μMMo6+ 2+ 2+ Rhodobacter sphaeroides Batch culture; Ormerod medium with succinate Ni ( < 5 µM) enhanced 2.7 fold H2 yield; 80 µM Fe and 16 µM [190,191] –1 −1 6+ 2+ MDC6521, (3.54 g L ) and yeast extract (2 g L ), pH 7.0, 30 °C; Mo increased 2 fold H2 yield in comparison with 40 µM Fe and photo fermentation light intensity of approx. 36 W / m2 3µMMo6+. + + 2+ Rhodobacter sphaeroides, Batch culture; Ormerod medium with succinate Cu at low concentration ( < 5 μM) increased H2 yield; Cu and Cu [196] –1 −1 photo fermentation (3.54 g L ) and yeast extract (2 g L ), pH 7.0, 30 °C; at moderate concentrations ( > 50 μM) inhibited H2 yield light intensity of approx. 36 W / m2 −1 2+ −1 2+ Mixed culture from cow dung; Batch culture; salt medium with sucrose (15 g L )Ni(0.6 mg L ) enhanced 2 fold cumulative H2 production; Ni [193]

dark fermentation higher concentration decreased H2 production

1212 K. Trchounian et al. Renewable and Sustainable Energy Reviews 80 (2017) 1201–1216

10. Concluding remarks and future progress References

Dark- and photo-fermentation by facultative and obligate anaerobic [1] Trchounian A. Mechanisms for hydrogen production by different bacteria during and purple non-sulfur bacteria and microalgae is one of the main mixed-acid and photo-fermentation and perspectives of hydrogen production – fi biotechnology. Crit Rev Biotechnol 2015;35:103 13. methods employed for biological production of H2 [1]. It has signi cant [2] Trchounian K, Trchounian A. Hydrogen production from glycerol by Escherichia advantages when used both in small-scale and in large-scale H2- coli and other bacteria: an overview and perspectives. Appl Energy production processes. Therefore, future biotechnological developments 2015;156:174–84. ffi [3] Solomon S, Plattner GK, Knutti R, Friedlingstein P. Irreversible climate change in H2 production should aim to increase e ciency and reduce costs [2]. due to carbon dioxide emissions. Proc Natl Acad Sci USA 2009;106:1704–9. To develop fermentative H2 production biotechnology using of [4] Hof AF, den Elzen MGJ, Beltran AM. The EU 40% greenhouse gas emission carbon-based industrial by-products and recycling of organic wastes, reduction target by 2030 in perspective. Int Environ Agreem-P 2016;16:375–92. 〈 〉 selection of effective bacteria, construction of efficient strains and the [5] http://ec.europa.eu/energy/en/topics/energy-strategy/2030-energy-strategy . [6] 〈http://www.cio.com/article/3159680/car-tech/hydrogen-refueling-stations-for- technological optimization of the process conditions have been studied cars-to-reach-5000-by-2032.html〉. by many groups: important new data have been obtained with dark [7] Anitha M, Kamarudin SK, Kofli NT. The potential of glycerol as a value-added – fermentative bacteria like E. coli, and photo-fermentative like R. commodity. Chem Eng J 2016;295:119 30. [8] Azwar MY, Hussain MA, Abdul-Wahab AK. Development of biohydrogen pro- sphaeroides, mainly during the last two years. duction by photobiological, fermentation and electrochemical processes: a review. Activity of [Ni-Fe]-hydrogenases in dark fermentative bacteria Renew Sustain Energy Rev 2014;31:158–73. [68,76,77,88] and [Mo]-nitrogenase and [Ni-Fe]-hydrogenase of [9] Basak N, Jana AK, Das D, Saikia D. Photofermentative molecular biohydrogen fi production by purple-non-sulfur (PNS) bacteria in various modes: the present photo-fermentative ones [76,144] have been de ned for various carbon progress and future perspective. Int J Hydrogen Energy 2014;39:6853–71. sources, and for pure, co-cultures and mixed cultures, as well as the [10] Trchounian A, Sawers RG. Novel insights into the bioenergetics of mixed-acid technology conditions. It was shown that the activity of enzymes and fermentation: can hydrogen and proton cycles combine to help maintain a proton motive force?. IUBMB Life 2014;66:1–7. their role in the total H2 production is dependent on fermentation [11] Arimi MM, Knodel J, Kiprop A, Namango SS, Zhang Y, Geißen SU. Strategies for substrate, its concentration, pH and some other factors improvement of biohydrogen production from organic-rich wastewater: a review. [68,102,149,150,153]. Among carbon sources, cheap and affordable Biomass Bioenergy 2015;75:101–18. [12] Chandrasekhar K, Lee Y-J, Dong-Woo Lee D-W. Biohydrogen production: by-products, in particular glycerol [2,7,13], especially crude glycerol strategies to improve process efficiency through microbial routes. Int J Mol Sci [29,160] (see Fig. 5) and a variety of industrial, agricultural, food and 2015;16:8266–93. other organic wastes [11,13,57,61] have been studied for increased H2 [13] Ghimire A, Frunzo L, Pirozzi F, Trably E, Escudie R, Lens PNL, Esposito G. A production. Together, these findings point to biohydrogen production review on dark fermentative biohydrogen production from organic biomass: process parameters and use of by-products. Appl Energy 2015;144:73–95. as a being a suitable sustainable technology for the future. It is [14] Rosales-Colunga LM, Rodriguez ADL. Escherichia coli and its application to – important that conversion of by-products and wastes to H2 by bacteria biohydrogen production. Rev Environ Sci Biotechnol 2015;14:123 35. has environmental benefit especially 1 kg of crude glycerol converted [15] Bundhoo MZ, Mohee R. Inhibition of dark fermentative bio-hydrogen production: a review. Int J Hydrogen Energy 2016;41:6713–33. reduced 7.66 kg of greenhouse gas [21]. Of course, this research area is [16] Hallenbeck PC, Liu Y. Recent advances in hydrogen production by photosynthetic not without its problems, particularly because many different bacterial bacteria. Int J Hydrogen Energy 2016;41:4446–54. strains and conditions have been explored by different groups, and [17] Ren NQ, Zhao L, Chen C, Guo WQ, Cao GL. A review on bioconversion of ffi lignocellulosic biomass to H2: key challenges and new insights. Bioresour Technol comparison of the results obtained becomes di cult. Nevertheless, new 2016;215:92–9. approaches are being actively been developed, and particularly the use [18] Ross IL, Oey M, Stephens E, Hankamer B. Prospects for photobiological hydrogen of mixed carbon fermentation using sugars, glycerol and / or carboxylic as a renewable energy. Curr Biotechnol 2016;5:1–9. – – [19] Sivagurunathan P, Kumar G, Bakonyi P, Kim SH, Kobayashi T, Xu KQ, Lakner G, acids looks very promising [30,31,121 123,129 131,171]. Tóth G, Nemestóthy N, Bélafi-Bakó K. A critical review on issues and overcoming Furthermore, analysis of inhibitory compounds and redox regulation strategies for the enhancement of dark fermentative hydrogen production in of [Ni-Fe]-Hyd enzymes activity [24,31,38,103,104,143,153, continuous systems. Int J Hydrogen Energy 2016;41:3820–36. [20] Dasari MA, Kiatsimkul PP, Sutterlin WR, Suppes GJ. Low-pressure hydrogeno- 154,185,187,190,194], genetic and metabolic engineering of the bac- lysis of glycerol to propylene glycol. Appl Cat Gen 2005;281:225–31. teria [32,37,97,135–138,173,174], systematic analysis of mutants [21] He Q, McNutt J, Yang J. Utilization of the residual glycerol from biodiesel [139], co- and mixed cultures of dark- and photo-fermentative bacteria production for renewable energy generation. Renew Sustain Energy Rev – are all contributing significantly to the field [33,36,47,179,181].To 2017;71:63 76. [22] Dharmadi Y, Murarka A, Gonzalez R. Anaerobic fermentation of licerol by ensure optimal activity of the enzymes responsible for H2 production, Escherichia coli: a new platform for metabolic engineering. Biotechnol Bioeng suitable metal ions in different combinations are being employed 2006;94:821–9. [26,56,187–193,196]. Significant progress has been achieved; how- [23] Trchounian K, Trchounian A. Hydrogenase 2 is most and hydrogenase 1 is less responsbile for H2 production by Escherichia coli under glycerol fermentation at ever, for details of mechanisms further study is still required. neutral and slightly alkaline pH. Int J Hydrogen Energy 2009;34:8839–45. These approaches promise to make H2 production more efficient [24] Trchounian K, Sanchez-Torres V, Wood TK, Trchounian A. Escherichia coli and cost-effective. They will hopefully provide significant long-term hydrogenase activity and H2 production under glycerol fermentation at a low pH. – fi Int J Hydrogen Energy 2011;36:4323 31. economic and environmental bene ts for renewable and sustainable [25] Mattam AJ, Clomburg JM, Gonzalez R, Yazdani SS. Fermentation of glycerol and energy supply in the future. production of valuable chemical and biofuel molecules. Biotechnol Lett 2013;35:831–42. [26] Trchounian K, Müller N, Schink B, Trchounian A. Glycerol and mixture of carbon sources conversion to hydrogen by Clostridium beijerinckii DSM791 and effects of fl various heavy metals on hydrogenase activity. Int J Hydrogen Energy 2017. Con ict of interests http://dx.doi.org/10.1016/j.ijhydene.2017.01.011. [27] Pachapur VL, Sarma SJ, Brar SK, Bihan YL, Buelna G, Verma M. Energy balance The authors declare they have no conflict of interests. of hydrogen production from wastes of biodiesel production. Biofuels 2016. http://dx.doi.org/10.1080/17597269.2016.1153361. [28] de Oliveira Faber M, Ferreira-Leitão VS. Optimization of biohydrogen yield produced by bacterial consortia using residual glycerin from biodiesel production. Bioresour Technol 2016;219:365–70. Acknowledgements [29] Chaudhary N, Ngadi MO, Simpson B. Comparison of glucose, glycerol and crude glycerol fermentation by Escherichia coli K12. J Bioprocess Biotech 2012;S1:001. The study was supported by Research Grant from State Committee [30] Trchounian K, Sargsyan H, Trchounian A. Hydrogen production by Escherichia coli depends on glucose concentration and its combination with glycerol at of Science, Ministry of Education and Science of Armenia, to AT (15T- different pHs. Int J Hydrogen Energy 2014;39:6419–23. 1F123), Armenian National Science & Education Fund (USA) to KT [31] Trchounian K, Trchounian A. Clean energy technology development: hydrogen (Biotech-4184), German Academic Exchange Service (DAAD) production by Escherichia coli during glycerol fermentation. Progress Clean Energy 2015;2:539–49, Springer Int Publ. Scholarship to AT (#91611632).

1213 K. Trchounian et al. Renewable and Sustainable Energy Reviews 80 (2017) 1201–1216

[32] Ryu MH, Hull NC, Gomelsky M. Metabolic engineering of Rhodobacter sphaer- production. Energy Convers Manag 2016;119:142–50. oides for improved hydrogen production. Int J Hydrogen Energy [61] Ghosh S, Dairkee UK, Chowdhury R, Bhattacharya P. Hydrogen from food 2014;39:6384–90. processing wastes via photofermentation using purple non-sulfur bacteria (PNSB) [33] Tsygankov AA, Khusnutdinova AN. Hydrogen in metabolism of purple bacteria – a review. Energy Convers Manag 2016. http://dx.doi.org/10.1016/j.encon- and prospects of practical application. Microbiology 2015;84:1–22. man.2016.09.001. [34] Kumar GR, Chowdhary N. Biotechnological and bioinformatics approaches for [62] Kumar G, Bakonyi P, Periyasamy S, Kim SH, Nemestóthy N, Bélafi-Bakó K. augmentation of biohydrogen production: a review. Renew Sustain Energy Rev Lignocellulose biohydrogen: practical challenges and recent progress. Renew 2016;56:1194–206. Sustain Energy Rev 2015;44:728–37. [35] Sargsyan H, Trchounian K, Gabrielyan L, Trchounian A. Novel approach of [63] Mohan SV, Nikhil GN, Chiranjeevi P, Reddy CN, Rohit MV, Kumar AN, Sarkar O. ethanol waste utilization: biohydrogen production by mixed cultures of dark- and Waste biorefinery models towards sustainable circular bioeconomy: critical review photo-fermentative bacteria using distillers grains. Int J Hydrogen Energy and future perspectives. Bioresour Technol 2016;215:2–12. 2016;41:2377–82. [64] Bock A, Sawers G. Fermentation. In: Neidhardt FG, editor. Escherichia coli and [36] Sargsyan H, Gabrielyan L, Trchounian A. The distillers grains with solubles as a Salmonella. Cellular and molecular biology. Washington DC: ASM Press; 2006 perspective substrate for obtaining biomass and producing bio-hydrogen by 〈http://www.ecosal.org〉. Rhodobacter sphaeroides. Biomass- Bioenergy 2016;90:90–4. [65] Slonszewski JL, Fugisawa M, Dopson M, Krulwich TA. Cytoplasmic pH mea- [37] Wang Y, Zhou P, Tong J, Gao R. Advances in the genetic modification of surements and homeostatis in bacteria and archaea. Adv Microb Physiol Rhodobacter sphaeroides to improve hydrogen production. Renew Sustain Energy 2009;55:1–79. Rev 2016;60:1312–8. [66] Murarka A, Dharmadi Y, Yazdani SS, Gonzales R. Fermentative utilization of [38] Gabrielyan L, Trchounian A. Relationship between molecular hydrogen produc- glycerol by Escherichia coli and its implications for the production of fuels and

tion, proton transport and the FOF1-ATPase activity in Rhodobacter sphaeroides chemicals. Appl Environ Microbiol 2008;74:1124–35. strains from mineral springs. Int J Hydrogen Energy 2009;34:2567–72. [67] Cintolesi A, Comburg JM, Rigou V, Zygourakis K, Gonzalez R. Quantitative [39] Gabrielyan L, Torgomyan H, Trchounian A. Growth characteristics and hydrogen analysis of the fermentative metabolism of glycerol in Escherichia coli. Biotechnol production by Rhodobacter sphaeroides using various amino acids as nitrogen Bioeng 2011;109:187–98. sources and their combinations with carbon sources. Int J Hydrogen Energy [68] Trchounian K, Poladyan A, Vassilian A, Trchounian A. Multiple and reversible 2010;35:12201–7. hydrogenases for hydrogen production by Escherichia coli: dependence on

[40] Muzziotti D, Adessi A, Faraloni C, Torzillo G, De Philippis R. H2 production in fermentation substrate, pH and FOF1-ATPase. Crit Rev Biochem Mol Biol Rhodopseudomonas palustris as a way to cope with high light intensities. Res 2012;47:236–49. Microbiol 2016;167:350–6. [69] Poladyan A, Avagyan A, Vassilian A, Trchounian A. Oxidative and reductive routes [41] Gabrielyan L, Trchounian A. Concentration dependent glycine effect on the of glycerol and glucose fermentation by Escherichia coli batch cultures and their photosynthetic growth and bio-hydrogen production by Rhodobacter sphaeroides regulation by oxidizing and reducing reagents at different pHs. Curr Microbiol from mineral springs. Biomass Bioenergy 2012;36:333–8. 2013;66:49–55. [42] Hakobyan L, Gabrielyan L, Trchounian A. Yeast extract as an effective nitrogen [70] Kim K, Kim SK, Park YC, Seo JH. Enhanced production of 3-hydroxy-propionic source stimulating cell growth and enhancing hydrogen photoproduction by acid from glycerol by modulation of glycerol metabolism in recombinant Rhodobacter sphaeroides strains from mineral springs. Int J Hydrogen Energy Escherichia coli. Bioresour Technol 2014;156:170–5. 2012;39:6519–27. [71] Futatsugi L, Saito H, Kakegawa T, Kobayashi H. Growth of an Escherichia coli [43] Chuang Y-S, Ch-Yu Huang, Ch-H Lay, Ch-Ch Chen, Sen B, Ch-Y Lin. Fermentative mutant deficient in respiration. FEMS Microbiol Lett 1997;156:141–5. bioenergy production from distillers grains using mixed microflora. Int J [72] Riondet C, Cachon R, Wache Y, Alcaraz G, Divies C. Extracellular oxidoreduction Hydrogen Energy 2012;37:15547–55. potential modifies carbon and electron flow in Escherichia coli. J Bacteriol [44] Ch Wood, Rosentrater KA, Muthukumarappan K, Zh Gu. Quantification of 2000;182:620–6. physical and chemical properties, and identification of potentially valuable [73] Gonzalez R, Murarka A, Dharmadi Y, Yasdani SS. A new model for the anaerobic components from fuel ethanol process streams. Cereal Chem 2013;90:70–9. fermentation of glycerol in enteric bacteria: trunk and auxiliary pathways in [45] Alibardi L, Cossu R. Effects of carbohydrate, protein and lipid content of organic Escherichia coli. Metab Eng 2008;10:234–45. waste on hydrogen production and fermentation products. Waste Manag [74] Trchounian K, Blbulyan S, Trchounian A. Hydrogenase activity and proton-motive 2016;47:69–77. force generation by Escherichia coli during glycerol fermentation. J Bioenerg [46] Mirza SS, Qazi JI, Zhao Q, Chen S. Photo-biohydrogen production potential of Biomembr 2013;45:253–60. Rhodobacter capsulatus-PK from wheat straw. Biotechnol Biofuels 2013;6:1. [75] Sawers RG, Blokesch M, Böck A. Anaerobic formate and hydrogen metabolism. In: [47] Marone A, Izzo G, Mentuccia L, Massini G, Paganin P, Rosa S, Varrone C, Curtiss R, IIIeditor. EcoSal—Escherichia coli and Salmonella: cellular and Signorini A. Vegetable waste as substrate and source of suitable microflora for bio- molecular biology. Washington, D.C.: ASM Press; 2004, [Online.] 〈http://www. hydrogen production. Renew Energy 2014;68:6–13. ecosal.org〉. [48] Song ZX, Li XH, Li WW, Bai YX, Fan YT, Hou HW. Direct bioconversion of raw [76] Peters JW, Schut GJ, Boyd ES, Mulder DW, Shepard EM, Broderick JB, King PW, corn stalk to hydrogen by a new strain Clostridium sp. FS3. Bioresour Technol Adams MW. [FeFe]-and [NiFe]-hydrogenase diversity, mechanism, and matura- 2014;157:91–7. tion. Biochim Biophys Acta (BBA) – Mol Cell Res 2015;1853:1350–69. [49] Shah AT, Favaro L, Alibardi L, Cagnin L, Sandon A, Cossu R, Casella S, Basaglia [77] Sargent F. The model [NiFe]-hydrogenases of Escherichia coli. Adv Microb M. Bacillus sp. strains to produce bio-hydrogen from the organic fraction of Physiol 2016;68:433–507. municipal solid waste. Appl Energy 2016;176:116–24. [78] Menon NK, Robbins J, Wendt JC, Shanmugan KT, Przybyla AE. Mutational [50] Seifert K, Waligorska M, Laniecki M. Brewery wastewaters in photobiological analysis and characterization of the Escherichia coli hya operon, which encodes hydrogen generation in presence of Rhodobacter sphaeroides OU 001. Int J [NiFe] hydrogenase 1. J Bacteriol 1991;173:4851–61. Hydrogen Energy 2010;35:4085–91. [79] Menon NK, Chatelus CY, Dervartanian M, Wendt JC, Shanmugam KT, Peck HD, [51] Mekjinda N, Ritchie RJ. Breakdown of food waste by anaerobic fermentation and Przybyla AE. Cloning, sequencing, and mutational analysis of the hyb operon non-oxygen producing photosynthesis using a photosynthetic bacterium. Waste encoding Escherichia coli hydrogenase 2. J Bacteriol 1994;176:4416–23. Manag 2015;35:199–206. [80] Richard DJ, Sawers G, Sargent F, McWalter L, Boxer DH. Transcriptional [52] Zahedi S, Solera R, García-Morales JL, Sales D. Effect of the addition of glycerol regulation in response to oxygen and nitrate of the operons encoding the [NiFe] on hydrogen production from industrial municipal solid waste. Fuel hydrogenases 1 and 2 of Escherichia coli. Microbiology 1999;145:2903–12. 2016;180:343–7. [81] Dubini A, Pye RL, Jack RL, Palmer T, Sargent F. How bacteria get energy from [53] Rai Pankaj K, Singh SP, Asthana RK. Biohydrogen production from cheese whey hydrogen: a genetic analysis of periplasmic hydrogen oxidation in Escherichia coli. wastewater in a two-step anaerobic process. Appl Biochem Biotechnol Int J Hydrogen Energy 2002;27:1413–20. 2012;167:1540–9. [82] Forzi L, Sawers RG. Maturation of [NiFe]-hydrogenases in Escherichia coli. [54] Dębowski M, Korzeniewska E, Filipkowska Z, Zieliński M, Kwiatkowski R. BioMetals 2007;20:565–78. Possibility of hydrogen production during cheese whey fermentation process by [83] Pinske C, McDowall JS, Sargent F, Sawers RG. Analysis of hydrogenase 1 levels different strains of psychrophilic bacteria. Int J Hydrogen Energy reveals an intimate link between carbon and hydrogen metabolism in Escherichia 2014;39:1972–8. coli K-12. Microbiology 2012;158:856–68. [55] Patel AK, Vaisnav N, Mathur A, Gupta R, Tuli DK. Whey waste as potential [84] Pinske C, Jaroschinsky M, Linek S, Kelly CL, Sargent F, Sawers RG. Physiology

feedstock for biohydrogen production. Renew Energy 2016;98:221–5. and bioenergetics of [NiFe]-hydrogenase 2-catalyzed H2-consuming and H2- [56] Eroglu E, Gündüz U, Yücel M, Eroglu I. Effect of iron and molybdenum addition producing reactions in Escherichia coli. J Bacteriol 2015;197:296–306. on photofermentative hydrogen production from olive mill wastewater. Int J [85] Trchounian K, Pinske C, Sawers RG, Trchounian A. Characterization of Hydrogen Energy 2011;36:5895–903. Escherichia coli [NiFe]-hydrogenase distribution during fermentative growth at [57] Karadag D, Köroğlu OE, Ozkaya B, Cakmakci M, Heaven S, Banks C. A review on different pHs. Cell Biochem Biophys 2012;62:433–40. fermentative hydrogen production from dairy industry wastewater. J Chem [86] Sauter M, Böhm R, Böck A. Mutational analysis of the operon (hyc) determining Technol Biotechnol 2014;89:1627–36. hydrogenase 3 formation in Escherichia coli. Mol Microbiol 1992;6:1523–32. [58] Choi J, Ahn Y. Characteristics of biohydrogen fermentation from various [87] Andrews SC, Berks BC, Mcclay J, Ambler A, Quail MA, Golby P, Guest JR. A 12- substrates. Int J Hydrogen Energy 2013;39:3152–9. cistron Escherichia coli operon (hyf) encoding a putative proton-translocating [59] Budiman PM, Wu TY, Ramanan RN, Md Jahim J. Improvement of biohydrogen formate hydrogen lyase system. Microbiology 1997;143:3633–47. production through combined reuses of palm oil effluent together with pulp and [88] McDowall JS, Murphy BJ, Haumann M, Palmer T, Armstrong AF, Sargent F. paper effluent in photofermentation. Energy Fuels 2015;29:5816–24. Bacterial formate hydrogenlyase complex. Proc Nat Acad Sci USA [60] Budiman PM, Wu TY. Ultrasonication pre-treatment of combined effluents from 2014;111:E3948–E3956. palm oil, pulp and paper mills for improving photofermentative biohydrogen [89] Bagramyan K, Mnatsakanyan N, Poladyan A, Vassilian A, Trchounian A. The roles

1214 K. Trchounian et al. Renewable and Sustainable Energy Reviews 80 (2017) 1201–1216

of hydrogenases 3 and 4, and the FOF1-ATPase, in H2 production by Escherichia Microbiol 2009;75:1456–9. coli at alkaline and acidic pH. FEBS Lett 2002;516:172–8. [117] Akopyan K, Trchounian A. Proton cycles through membranes in bacteria: [90] Skibinski DAG, Golby P, Chang Y-S, Sargent F, Hoﬀman R, Harper R, Guest JR, relationship between proton passive and active ﬂuxes and their dependence on Attwood MM, Berks BC, Andrews SC. Regulation of the hydrogenase-4 operon of some external physico-chemical factors under fermentation. Biophysics Escherichia coli by the σ54-dependent transcriptional activators FhlA and HyfR. J 2013;58:624–39. Bacteriol 2002;184:6642–53. [118] Konings WN, Poolman B, van Veen HW. Solute transport and energy transduction [91] Self WT, Hasona A, Shanmugam KT. Expression and regulation of a silent operon, in bacteria. Antonie Van Leeuwenhoek 1994;65:369–80.

hyf, coding for hydrogenase 4 isoenzyme in Escherichia coli. J Bacteriol [119] Trchounian A. Escherichia coli proton-translocating F0F1-ATP synthase and its 2004;186:580–7. association with solute secondary transporters and/or enzymes of anaerobic [92] King PW, Przybyla AE. Response of hya expression to external pH in Escherichia oxidation-reduction under fermentation. Biochem Biophys Res Comm coli. J Bacteriol 1999;181:5250–6. 2004;315:1051–7. [93] Redwood MD, Mikheenko IP, Sargent F, Macaskie LE. Dissecting the roles of [120] Dimanta I, Grunduls A, Nikolaeva V, Kleperis J, Muiznieks I. Crude glycerol as a Escherichia coli hydrogenases in biohydrogen production. FEMS Microbiol Lett perspective substrate for bio-hydrogen production in Latvia. Int Sci J Alt Energy 2008;278:48–55. Ecol 2012;9:28–31. [94] Lukey MJ, Parkin A, Roessler MM, Murphy BJ, Harmer J, Palmer T, Sargent F, [121] Trchounian K, Trchounian A. H2 producing activity by Escherichia coli during Armstrong FA. How Escherichia coli is equipped to oxidize hydrogen under mixed carbon fermentation at slightly alkaline and acidic phs: novel functions of diﬀerent redox conditions. J Biol Chem 2010;285:3928–38. hydrogenase 4 (hyf) and hydrogenase 2 (hyb). In Black Sea energy resource [95] Pinske C, Jaroschinsky M, Sargent F, Sawers RG. Zymographic diﬀerentiation of development and hydrogen energy problems, Springer Netherlands; 2013. p. [Ni-Fe]-hydrogenases 1, 2 and 3 of Escherichia coli K-12. BMC Microbiology 137–51. 2012;12:134. [122] Trchounian K. Transcriptional control of hydrogen production during mixed [96] Poladyan A, Trchounian K, Sawers G, Trchounian A. Hydrogen-oxidizing hydro- carbon fermentation by hydrogenases 4 (hyf) and 3 (hyc) in Escherichia coli. Gene genases 1 and 2 of Escherichia coli regulate the onset of hydrogen evolution and 2012;506:156–60. ATPase activity, respectively, during glucose fermentation at alkaline pH. FEMS [123] Trchounian K, Trchounian A. Escherichia coli hydrogenase 4 (hyf) and hydro-

Microbiol Lett 2013;348:143–8. genase 2 (hyb) contribution in H2 production mixed carbon (glucose and glycerol) [97] Kim JYH, Jo BH, Cha HJ. Production of biohydrogen by recombinant expression fermentation at pH 7.5 and pH 5.5. Int J Hydrogen Energy 2013;38:3921–9. of [NiFe]-hydrogenase 1 in Escherichia coli. Microb Cell Fact 2010;9:54. [124] Mnatsakanyan N, Vassilian A, Navasardyan L, Bagramyan K, Trchounian A. [98] Sawers RG, Ballantine SP, Boxer DH. Differential expression of hydrogenase Regulation of Escherichia coli formate hydrogenlyase activity by formate at isoenzymes in Escherichia coli K-12: evidence for a third isoenzyme. J Bacteriol alkaline pH. Curr Microbiol 2002;45:281–6. 1985;164:1324–31. [125] Mnatsakanyan N, Bagramyan K, Trchounian A. Hydrogenase 3 but not hydro- [99] Schlensog V, Bock A. Identification and sequence analysis of the gene encoding the genase 4 is major in hydrogen gas production by Escherichia coli formate transcriptional activator of the formate hydrogenlyase system of Escherichia coli. hydrogen lyase at acidic pH and in the presence of external formate. Cell Biochem Mol Microbiol 1990;4:1319–27. Biophys 2004;41:357–66. [100] Lutz S, Jacobi A, Schlensog V, Bohm R, Sawers G, Bock A. Molecular character- [126] Leonhartsberger S, Korsa I, Bock A. The molecular biology of formate metabolism ization of an operon (hyp) necessary for the activity of the three hydrogenase in enterobacteria. J Mol Microbiol Biotechnol 2002;4:269–76. isoenzymes in Escherichia coli. Mol Microbiol 1991;5:123–35. [127] Sawers RG. Formate and its role in hydrogen production in Escherichia coli. [101] Soboh B, Stripp ST, Bielak C, Lindenstrauss U, Braussemann M, Javaid M, Biochem Soc Trans 2005;33:42–6. Hallensleben M, Granich C, Herzberg M, HeberleJ , Sawers RG. The [NiFe]- [128] Trchounian K, Trchounian A. Different role of focA and focB encoding formate hydrogenase accessory chaperones HypC and HybG of Escherichia coli are iron- channels for hydrogen production by Escherichia coli during glucose or glycerol and carbon dioxide-binding proteins. FEBS Lett 2013;587:2512–6. fermentation. Int J Hydrogen Energy 2014;39:20987–91. [102] Trchounian K, Trchounian A. Hydrogen producing activity by Escherichia coli [129] Trchounian K, Abrahamyan V, Poladyan A, Trchounian A. Escherichia coli growth hydrogenase 4 (hyf) depends on glucose concentration. Int J Hydrogen Energy and hydrogen production in batch culture upon formate alone and with glycerol 2014;39:16914–8. co-fermentation at different pHs. Int J Hydrogen Energy 2015;40:9935–41. [103] Trchounian K, Trchounian A. Escherichia coli multiple [Ni-Fe]-hydrogenases are [130] Trchounian K, Trchounian A. Escherichia coli hydrogen gas production from sensitive to osmotic stress during glycerol fermentation but at different pHs. FEBS glycerol: effects of external formate. Renew Energy 2015;83:345–51.

Lett 2013;587:3562–6. [131] Trchounian K, Sargsyan H, Trchounian A. H2 production by Escherichia coli batch [104] Magnani P, Doussiere J, Lissolo T. Diphenyleneiodonium as an inhibitor for the cultures during utilization of acetate and mixture of glycerol and acetate. Int J hydrogenase complex of Rhodobacter capsulatus. Evidence for two distinct Hydrogen Energy 2015;40:12187–92. electron donor sites. Biochim Biophys Acta 2000;1459:169–78. [132] Kirkpatrick C, Maurer LM, Oyelakin NE, Yoncheva YN, Maurer R, Slonczewski JL. [105] Trchounian A, Bagramyan K, Poladian A. Formate hydrogenlyase is needed for Acetate and formate stress: opposite responses in the proteome of Escherichia

proton-potassium exchange through the FOF1-ATPase and the TrkA system in coli. J Bacteriol 2001;183:6466–77. anaerobically grown Escherichia coli. Curr Microbiol 1997;35:201–6. [133] Lakshmanaswamy A, Rajaraman E, Eiteman MA, Altman E. Microbial removal of [106] Mnatsakanyan N, Bagramyan K, Vassilian A, Nakamoto RK, Trchounian AFO. acetate selectively from sugar mixtures. J Ind Microb Biotech 2011;38:1477–84. cysteine, bCys21, in the Escherichia coli ATP synthase is involved in regulation of [134] Zagrodnik R, Laniecki M. An unexpected negative influence of light intensity on potassium uptake and molecular hydrogen production in anaerobic conditions. hydrogen production by dark fermentative bacteria Clostridium beijerinckii. Biosci Rep 2002;22:421–30. Bioresour Technol 2016;200:1039–43. [107] Azzi A, Casey RP, Nalecz KA. The effect of N,N′-dicyclohexylcarbodiimide on [135] Sanchez-Torres V, Yusoff MYM, Nakano C, Maeda M, Ogawa HI, Wood TK. enzymes of bioenergetic relevance. Biochim Biophys Acta – Bioenerg Influence of Escherichia coli hydrogenases on hydrogen fermentation from 1984;768:209–26. glycerol. Int J Hydrogen Energy 2013;38:3905–12. [108] Paschos A, Bauer A, Zimmermann A, Zehelein E, Böck A. HypF, a carbamoyl [136] Tran KT, Maeda M, Wood TK. Metabolic engineering of Escherichia coli to phosphate-converting enzyme involved in [NiFe] hydrogenase maturation. J Biol enhance hydrogen production from glycerol. Appl Microbiol Biotechnol Chem 2002;277:49945–52. 2014;98:4757–70. [109] Blbulyan S, Trchounian A. Impact of membrane-associated hydrogenases on the [137] Tran KT, Maeda T, Sanchez-Torres V, Wood TK. Beneficial knockouts in

FOF1-ATPase in Escherichia coli during glycerol and mixed carbon fermentation: Escherichia coli for producing hydrogen from glycerol. Appl Microbiol Biotechnol atpase activity and its inhibition by N,N′-dicyclohexylcarbodiimide in the mutants 2015;99:2573–81. lacking hydrogenases. Arch Biochem Biophys 2015;579:67–72. [138] Rollin JA, del Campo JM, Myung S, Sun F, You C, Bakovic A, Chandrayan SK, Wu [110] Trchounian A, Ohanjanyan Y, Bagramyan K, Vardanian V, Zakharyan E, Vassilian C-H, Adams MWW, Zhang YHP. High-yield hydrogen production from biomass A, Davtian M. Relationship of the Escherichia coli TrkA system of potassium ion by in vitro metabolic engineering: mixed sugars co-utilization and kinetic uptake with the F0F1-ATPase under growth conditions without anaerobic or modeling. Proc Nat Acad Sci USA 2015;112:4964–9. aerobic respiration. Biosci Rep 1998;18:143–54. [139] Valle A, Cabrera G, Cantero D, Bolivar J. Identification of enhanced hydrogen and [111] Maeda T, Sanchez‐Torres V, Wood TK. Metabolic engineering to enhance bacterial ethanol Escherichia coli producer strains in a glycerol-based medium by screening hydrogen production. Microb Biotechnol 2008;1:30–9. in single-knock out mutant collections. Microb Cell Fact 2015;14:1. [112] Abrahamyan V, Poladyan A, Vassilian A, Trchounian A. Hydrogen production by [140] Adessi A, Concato M, Sanchini A, Rossi F, De Philippis R. Hydrogen production Escherichia coli during glucose fermentation: effects of oxidative and reductive under salt stress conditions by a freshwater Rhodopseudomonas palustris strain. routes used by the strain lacking hydrogen oxidizing hydrogenases 1 (hya) and 2 Appl Microbiol Biotechnol 2016;100:2917–26. (hyb). Int J Hydrogen Energy 2015;40:7459–64. [141] Niederman RA. Development and dynamics of the photosynthetic apparatus in [113] Murphy BJ, Sargent F, Armstrong FA. Transforming an oxygen-tolerant [NiFe] purple phototrophic bacteria. Biochim Biophys Acta (BBA) - Bioenerg uptake hydrogenase into a proficient, reversible hydrogen producer. Energy 2016;1857:232–46. Environ Sci 2014;7:1426–33. [142] Kars G, Gündüz U, Rakhely G, Yücel M, Ero ğlu İ, Kovacs KL. Improved hydrogen [114] Pinske C, Sargent F. Exploring the directionality of Escherichia coli formate production by uptake hydrogenase deficient mutant strain of Rhodobacter hydrogenlyase: a membrane‐bound enzyme capable of fixing carbon dioxide to sphaeroides OU 001. Int J Hydrogen Energy 2008;33:3056–60. organic acid. Microbiology Open 2016. http://dx.doi.org/10.1002/mbo3.365. [143] Sargsyan H, Gabrielyan L, Trchounian A. Concentration-dependent effects of [115] Trchounian K, Soboh B, Sawers RG, Trchounian A. Contribution of hydrogenase 2 metronidazole, inhibiting nitrogenase, on hydrogen photoproduction and proton-

to stationary phase H2 production by Escherichia coli during fermentation of translocating ATPase activity of Rhodobacter sphaeroides. Int J Hydrogen Energy glycerol. Cell Biochem Biophys 2013;66:103–8. 2014;39:100–6.

[116] Zbell AL, Maier RJ. Role of the Hya hydrogenase in recycling of anaerobically [144] Kars G, Gündüz U. Towards a super H2 producer: improvements in photofer- produced H2 in Salmonella enterica serovar typhimurium. Appl Environ mentative biohydrogen production by genetic manipulations. Int J Hydrogen

1215 K. Trchounian et al. Renewable and Sustainable Energy Reviews 80 (2017) 1201–1216

Energy 2010;35:6646–56. high frequency electromagnetic fields in the environment: what are their effects on [145] Oelze J, Klein G. Control of nitrogen fixation by oxygen in purple nonsulfur bacteria?. Appl Mcirobiol Biotechnol 2016;100:4761–71. bacteria. Arch Microbiol 1996;165:219–25. [171] Passos F, Solé M, García J, Ferrer I. Biogas production from microalgae grown in [146] Akköse S, Gündüz U, Yücel M, Eroglu I. Effects of ammonium ion, acetate and wastewater: effect of microwave pretreatment. Appl Energy 2013;108:168–75. aerobic conditions on hydrogen production and expression levels of nitrogenase [172] Pattanamanee W, Chisti Y, Choorit W. Photofermentive hydrogen production by genes in Rhodobacter sphaeroides OU 001. Int J Hydrogen Energy Rhodobacter sphaeroides S10 using mixed organic carbon: effects of the mixture 2009;34:8818–27. composition. Appl Energy 2015;157:245–54. [147] Koku H, Eroğlu I, Gündüz U, Yücel M, Türker L. Aspects of the metabolism of [173] Kuniyoshi TM, Balan A, Schenberg AC, Severino D, Hallenbeck PC. Heterologous

hydrogen production by Rhodobacter sphaeroides. Int J Hydrogen Energy expression of proteorhodopsin enhances H2 production in Escherichia coli when 2002;27:1315–29. endogenous Hyd-4 is overexpressed. J Biotechnol 2015;206:52–7.

[148] Ghirardi ML. Implementation of photobiological H2 production: the O2 sensitivity [174] Lee HJ, Sekhon SS, Kim YS, Park JY, Kim YH, Min J. Enhanced hydrogen of hydrogenases. Photosynth Res 2015;125:383–93. production by co-cultures of hydrogenase and nitrogenase in Escherichia coli. [149] Mackenzie C, Choudhary M, Larimer FW, Predki PF, Stilwagen S, Armitage JP, Curr Microbiol 2016;72:242–7. Barber RD, Donohue TJ, Hosler JP, Newman JE, Shapleigh JP. The home stretch, [175] Zhou P, Wang Y, Gao R, Tong J, Yang Z. Transferring [NiFe] hydrogenase gene a first analysis of the nearly completed genome of Rhodobacter sphaeroides 2.4. 1. from Rhodopeseudomonas palustris into E. coli BL21 (DE3) for improving Photosynth Res 2001;70:19–41. hydrogen production. Int J Hydrogen Energy 2015;40:4329–36. [150] Kars G, Gündüz U, Yücel M, Türker L, Eroglu İ. Hydrogen production and [176] Zagrodnik R, Laniecki M. The role of pH control on biohydrogen production by transcriptional analysis of nifD, nifK and hupS genes in Rhodobacter sphaeroides single stage hybrid dark- and photo-fermentation. Bioresour Technol OU 001 grown in media with different concentrations of molybdenum and iron. 2015;194:187–95. Int J Hydrogen Energy 2006;31:1536–44. [177] Laurinavichene T, Tsygankov A. Different types of H 2 photoproduction by starch- [151] Kars G, Gündüz U, Yücel M, Rakhely G, Kovacs KL, Eroğlu İ. Evaluation of utilizing co-cultures of Clostridium butyricum and Rhodobacter sphaeroides. Int hydrogen production by Rhodobacter sphaeroides OU 001 and its hupSL deficient J Hydrogen Energy 2016;41:13419–25. mutant using acetate and malate as carbon sources. Int J Hydrogen Energy [178] Zagrodnik R, Laniecki M. The effect of pH on cooperation between dark-and 2009;34:2184–90. photo-fermentative bacteria in a co-culture process for hydrogen production from [152] Yang H, Zhang J, Wang X, Feng J, Yan W, Guo L. Coexpression of Mo-and Fe- starch. Int J Hydrogen Energy 2017. http://dx.doi.org/10.1016/j.ijhy- nitrogenase in Rhodobacter capsulatus enhanced its photosynthetic hydrogen dene.2016.12.150. production. Int J Hydrogen Energy 2015;40:927–34. [179] Chandra R, Nikhil GN, Mohan SV. Single-stage operation of hybrid dark-photo [153] Gabrielyan L, Sargsyan H, Hakobyan L, Trchounian A. Regulation of hydrogen fermentation to enhance biohydrogen production through regulation of system photoproduction in Rhodobacter sphaeroides batch culture by external oxidizers redox condition: evaluation with real-field wastewater. Int J Mol Sci and reducers. Appl Energy 2014;131:20–5. 2015;1:9540–56. [154] Gabrielyan L, Sargsyan H, Trchounian A. Novel properties of photofermentative [180] Guo XM, Trably E, Latrille E, Carrere H, Steyer JP. Predictive and explicative biohydrogen production by purple bacteria Rhodobacter sphaeroides:effects of models of fermentative hydrogen production from solid organic waste: role of protonophores and inhibitors of responsible enzymes. Microb Cell Fact butyrate and lactate pathways. Int J Hydrogen Energy 2014;39:7476–85. 2015;14:131–40. [181] Ghosh S, Chowdhury R, Bhattacharya P. Mixed consortia in bioprocesses: role of [155] Kelley BC, Nicholas DJD. Inhibition of nitrogenase activity by mitronidazole in microbial interactions. Appl Microbiol Biotechnol 2016;100:4283–95. Rhodopseudomonas capsulata. Arch Microbiol 1981;129:344–8. [182] Mudhoo A, Kumar S. Effects of heavy metals as stress factors on anaerobic [156] Hakobyan L, Gabrielyan L, Trchounian A. Proton motive force in Rhodobacter digestion processes and biogas production from biomass. Int J Environ Sci sphaeroides under anaerobic conditions in the dark. Curr Microbiol Technol 2013;10:1383–98. 2011;62:415–9. [183] Pinske C, Sawers G. The importance of iron in the biosynthesis and assembly of [157] Hakobyan L, Gabrielyan L, Trchounian A. Relationship of proton motive force and [NiFe]-hydrogenases. Biomol Concepts 2014;5:55–70.

the FOF1-ATPase with bio-hydrogen production activity of Rhodobacter sphaer- [184] Ogata H, Nishikawa K, Lubitz W. Hydrogens detected by subatomic resolution oides:effects of diphenylene iodonium, hydrogenase inhibitor, and its solvent protein crystallography in a [NiFe] hydrogenase. Nature 2015;520:571–4. dimethylsulphoxide. J Bioenerg Biomembr 2012;44:495–502. [185] Hasona A, Self WT, Ray RM, Shanmugam KT. Molybdate-dependent transcription [158] Tsygankov AA, Fedorov AS, Laurinavichene TV, Gogotov IN, Rao KK, Hall DO. of hyc and nar operons of Escherichia coli requires MoeA protein and ModE- Actual and potential rates of hydrogen photoproduction by continuous culture of molybdate. FEMS Microbiol Lett 1998;169:111–6. the purple non-sulphur bacterium Rhodobacter capsulatus. Appl Microbiol [186] Trchounian K, Poladyan A, Trchounian A. Optimizing strategy for Escherichia coli Biotechnol 1998;49:102–7. growth and hydrogen production during licerol fermentation in batch culture: [159] Sabourin-Provost G, Hallenbeck PC. High yield conversion of a crude glycerol effects of some heavy metal ions and their mixture. Appl Energy fraction from biodiesel production to hydrogen by photofermentation. Bioresour 2016;177:335–40. Technol 2009;100:3513–7. [187] Poladyan A, Trchounian K, Minasyants M, Trchounian A. Glycerol fermentation [160] Pott RW, Howe CJ, Dennis JS. Photofermentation of crude glycerol from biodiesel and molecular hydrogen production by Escherichia coli batch cultures affected by using Rhodopseudomonas palustris: comparison with organic acids and the some reducing reagents and heavy metal ions. In Black Sea energy resource identification of inhibitory compounds. Bioresour Technol 2013;130:725–30. development and hydrogen energy problems. Netherlands: Springer; 2013. p. [161] Buckel W, Thauer RK. Energy conservation via electron bifurcating ferredoxin 153–63. reduction and proton/Na+ translocating ferredoxin oxidation. Biochim Biophys [188] Trchounian K, Poladyan A, Trchounian A. Enhancement of Escherichia coli Acta (BBA) - Bioenerg 2013;1827:94–113. bacterial biomass and hydrogen production by some heavy metal ions and their

[162] Zhang Y, Yang H, Feng J, Guo L. Overexpressing F0/F1 operon of ATPase in mixtures during glycerol vs glucose fermentation at a relatively wide range of pH. Rhodobacter sphaeroides enhanced its photo-fermentative hydrogen production. Int J Hydrogen Energy 2017. http://dx.doi.org/10.1016/j.ijhydene.2017.02.003. Int J Hydrogen Energy 2016;41:6743–51. [189] Karadag D, Puhakka JA. Enhancement of anaerobic hydrogen production by iron [163] Sargsyan H, Gabrielyan L, Hakobyan L, Trchounian A. Light-dark duration and nickel. Int J Hydrogen Energy 2010;35:8554–60.

alternation effects on Rhodobacter sphaeroides growth, membrane properties and [190] Hakobyan L, Gabrielyan L, Trchounian A. Bio-hydrogen production and the FoF1- bio-hydrogen production in batch culture. Int J Hydrogen Energy ATPase activity of Rhodobacter sphaeroides:effects of various heavy metal ions. 2015;40:4084–91. Int J Hydrogen Energy 2012;37:17794–800. [164] Kim E-J, Kim J-S, Kim M-S, Lee JK. Effect of changes in the level of light [191] Hakobyan L, Gabrielyan L, Trchounian A. Ni (II) and Mg (II) ions as factors harvesting complexes of Rhodobacter sphaeroides on the photoheterotrophic enhancing biohydrogen production by Rhodobacter sphaeroides from mineral production of hydrogen. Int J Hydrogen Energy 2006;31:531–8. springs. Int J Hydrogen Energy 2012;37:7486–91. [165] Kim EJ, Kim MS, Lee JK. Hydrogen evolution under photoheterotrophic and dark [192] Hakobyan L, Gabrielyan L, Trchounian A. The effect of various metal ions on bio-

fermentative conditions by recombinant Rhodobacter sphaeroides containing the hydrogen production and FOF1-ATPase activity of Rhodobacter sphaeroides.In genes for fermentative pyruvate metabolism of Rhodospirillum rubrum. Int J Black Sea energy resource development and hydrogen energy problems. Hydrogen Energy 2008;33:5131–6. Netherlands: Springer; 2013. p. 165–77. [166] Li X, Wang Y, Zhang S, Chu J, Zhang M, Huang M, Zhuang Y. Eﬀects of light/dark [193] Gou C, Guo J, Lian J, Guo Y, Jiang Z, Yue L, Yang J. Characteristics and kinetics of

cycle, mixing pattern and partial pressure of H2 on biohydrogen production by biohydrogen production with Ni 2+ using hydrogen-producing bacteria. Int J Rhodobacter sphaeroides ZX-5. Bioresour Technol 2011;102:1142–8. Hydrogen Energy 2015;40:161–7. [167] Corona VM, Le Borgne S, Revah S, Morales M. Effect of light-dark cycles on [194] Kirakosyan G, Trchounian A. Redox sensing by Escherichia coli:effects of copper hydrogen and poly-β-hydroxybutyrate production by a photoheterotrophic culture ions as oxidizers on proton-coupled membrane transport. Bioelectrochemistry and Rhodobacter capsulatus using a dark fermentation effluent as substrate. 2007;70:58–63. Bioresour Technol 2017;226:238–46. [195] Sapra R, Bagramyan K, Adams MW. A simple energy-conserving system: proton [168] Kirakosyan G, Bagramyan K, Trchounian A. Redox sensing by Escherichia coli: reduction coupled to proton translocation. Proc Nat Acad Sci USA effects of dithiothreitol, a redox reagent reducing disulphides, on bacterial growth. 2003;100:7545–50. Biochem Biophys Res Commun 2004;325:803–6. [196] Hakobyan L, Sargsyan H, Gabrielyan L, Trchounian A. The effect of Cu (I) and Cu

[169] Gabrielyan L, Sargsyan H, Trchounian A. Biohydrogen production by purple non- (II) ions' low concentrations on growth, biohydrogen production and the FoF1- sulfur bacteria Rhodobacter sphaeroides:eﬀect of low-intensity electromagnetic ATPase activity of Rhodobacter sphaeroides. Int J Hydrogen Energy irradiation. J Photochem Photobiol B: Biol 2016;162:592–6. 2016;41:16807–12. [170] Soghomonyan D, Trchounian K, Trchounian A. Millimeter waves or extremely

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