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Novel approach of ethanol waste utilization: Biohydrogen production by mixed cultures of dark- and photo-fermentative bacteria using distillers grains
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 6 ) 1 e6
Available online at www.sciencedirect.com
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Novel approach of ethanol waste utilization: Biohydrogen production by mixed cultures of darkand photo-fermentative bacteria using distillers grains Harutyun Sargsyan, Karen Trchounian, Lilit Gabrielyan, Armen Trchounian* Department of Microbiology & Microbe and Plant Biotechnology, Biology Faculty, Yerevan State University, 1 A. Manoukian Str., 0025 Yerevan, Armenia
article info
abstract
Article history:
The combination of dark- and photo-fermentative bacteria is quite a new approach in
Received 4 September 2015
hydrogen (H2) production biotechnology, which offers potential efficient production of H2
Received in revised form
from renewable resources. This study is focused on the attempt to obtain high H2 yield by
3 November 2015
pure and mixed cultures of dark-fermentative (Escherichia coli) and photo-fermentative
Accepted 17 November 2015
(Rhodobacter sphaeroides) bacteria using distillers grains (DG), the ethanol fermentation
Available online xxx
waste. During the growth in diluted DG media H2 production by pure and mixed cultures started at 24 h growth. Moreover, the mixed culture produced significantly more H2 from
Keywords:
DG: 2-folds diluted media provided the ~1.5e3-folds higher H2 yield in comparison with
Distillers grains
pure culture. These effects were obtained for continuous H2 production at 72e96 h growth.
Biohydrogen production
DG dilution and neutralization to pH 7.0 were required. The results indicated that DG can
Escherichia coli
be used as effective and valuable source of organic substances in H2 production by bacteria.
Rhodobacter sphaeroides
This study can provide novel approach for an inexpensive energy generation, as well as to
Mixed cultures
resolve the problem of waste utilization. Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Dark- or photo-fermentation are the main pathways for biological hydrogen (H2) production. H2 generation through darkor photo-fermentation of organic compounds and wastes conversion is of great interest, thus acting as H2 production promising choice [1e3].
Constantly increasing demand for energy needs novel and cheaper substrates. Disposal of organic wastes, originating from food, sugar, wine and ethanol industry, makes a new promising approach of H2 production [1,4e6]. Distillers grains (DG) are industrial wastes generated during wine and ethanol fermentation. DG of wine fermentation has been used to produce H2 by mixed microflora [6]. However, undefined mixed cultures, heating (up to 95 C) treatment and acidic pH
* Corresponding author. Tel.: þ374 60 710520. E-mail address: [email protected] (A. Trchounian). http://dx.doi.org/10.1016/j.ijhydene.2015.11.082 0360-3199/Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Sargsyan H, et al., Novel approach of ethanol waste utilization: Biohydrogen production by mixed cultures of dark- and photo-fermentative bacteria using distillers grains, International Journal of Hydrogen Energy (2016), http:// dx.doi.org/10.1016/j.ijhydene.2015.11.082
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(pH 6.0) of DG used can limit application of this biotechnology developed. DG of ethanol fermentation, non-heating treatment and the other pH could lead to the other approach for H2 production using different identified dark- and photofermentative bacteria. The waste of ethanol fermentation can contain various sugars, proteins, amino acids, and trace elements such as calcium, iron, magnesium, and others, which are necessary for growth and H2 production by bacteria [7e9]. Using DG of ethanol fermentation as a cheaper (the cost in Armenia is 1.5 cent per liter) and valuable source of organic compounds for H2 production has the advantages of recycling wastes and protecting environment. H2 production by the most of dark-fermentative bacteria depends on the activity of different hydrogenases, which catalyze the oxidationereduction reaction of 2Hþ þ 2e / H2. Escherichia coli is applied to reveal mechanisms and regulation pathways of Н2 production, including hydrogenases and formate hydrogen lyases, as well as it can be used in Н2 production biotechnology [2,10,11]. Purple bacteria (e.g. Rhodobacter sp.) can produce H2 during photo-fermentation of organic acids using nitrogenase and hydrogenase [1e3,5]. Previously it was shown, that Rh. sphaeroides, isolated from mineral springs of Armenian mountains, were able to produce H2 in various conditions upon illumination [12e14]. Combination of dark- and photo-fermentation is quite a new approach in H2 production biotechnology, which is mainly achieved via a two- or one-stage process [15e19]. During the two-stage process, the dark-fermentation effluents provide sufficient amount of organic acids for the photo-fermentation. However, dark-fermentation effluents can contain various organic compounds such as ammonium, which significantly inhibits the nitrogenase activity of photo-fermentative bacteria [17]. Though, the system should be controlled to provide optimum media composition and environmental conditions for both bacteria [15,20,21]. In the one-stage (co-culture) process dark- and photo-fermentative bacteria are cultured in one system [19e21]. However, one-stage fermentation using mixed dark- and photo-fermentative bacteria has not yet been studied in details, in part owing to differences in growth conditions and requirements between the two types of microorganisms. This work was aimed to study H2 production by co-cultures of dark- (E. coli) and photo-fermentative (Rh. sphaeroides) bacteria using ethanol fermentation waste. Various parameters such as waste dilution and pH were optimized to reach a higher H2 yield. The changes of the external pH and redox potential (Eh) during the bacterial anaerobic growth were examined.
Materials and methods Bacterial strains, cultivation conditions and determination of growth E. coli BW 25113 wild type from Keio collection (Tsuruoka City, Yamagata, Japan) and Rh. sphaeroides strain MDC6521, isolated from Arzni mineral springs in Armenian mountains (Microbial Depository Center, National Academy of Sciences of Armenia,
Yerevan, Armenia, WDCM803) have been used. E. coli was grown in peptone medium with glucose (2 g L1) as carbon source, as described [11]. Rh. sphaeroides was cultivated in anaerobic conditions upon illumination in Ormerod medium with succinate (3.54 g L1) as carbon source, as detailed previously [12,13]. In the co-culture E. coli and Rh. sphaeroides were mixed, and mixed culture was also cultivated in anaerobic conditions upon illumination (~2000 lux), as applied before [12e14]. The growth of bacteria in batch cultures was measured by optical density (OD) changes OD at 600 nm for E. coli and at 660 nm for Rh. sphaeroides using Spectro UVeVis Auto spectrophotometer (Labomed, USA). Specific growth rate was calculated as described [13,14]. Wet DG from common (bread) wheat Triticum aestivum L. was obtained from “Alex Grig” Alcohol Plant Co. LTD (Yerevan, Armenia). DG is a yellow-brown color polydisperse system with yeast scent, in which compounds are dissolved and suspended (with visible milled caryopsis). DG was obtained during ethanol fermentation, which was performed by yeast Saccharomyces cerevisiae. In DG were found various sugars (glucose, xylose, arabinose and other), glycerol, fatty acids (linoleic, palmitic, oleic, and linolenic acids), different proteins (peptides) and 14 amino acids with a predominance of glutamate, including 8 essential amino acids: arginine, lysine, valine, histidine, threonine, phenylalanine, leucine, isoleucine, which can be used by bacteria as carbon and nitrogen source for H2 production [7e9]. The chemical composition of DG depends on the cereals fermentation conditions and the type of ethanol produced. The untreated DG was filtered through cotton wool, then paper filter, next sterilized by autoclaving at 120 C for 20 min. As pH of DG were ~3.5, before autoclaving the pH of the waste was adjusted to 7.0 by means of 0.1 M NaOH. DG were diluted 2-, 5-, and 10-folds using distilled water. In this study diluted DG was used without any medium supplements, because DG contained various carbon-containing compounds.
Determination of medium pH, Eh and H2 yield The pH of the growth medium was determined by a pH-meter (HI 122-02, HANNA Instruments, Portugal) with selective pH electrode, as described [13,14]. The medium Eh was measured during bacterial pure and mixed cultures anaerobic growth using a pair of redox electrodes: platinum (Pt) and titanium-silicate (TieSi) electrodes, as described [13,14]. In spite of TieSi electrode, Pt one was sensitive to H2. Therefore, the changes of Eh and the differences between these electrode readings provided information about not only redox processes, but also H2 yield, which was determined by the decrease of Eh to low negative values during bacterial growth [13,14,22]. This determination of H2 is close to the method with Clark-type electrode employed by the other authors [23]. The correlation between Eh and H2 production was shown; the supplementation of H2 didn't affect medium pH [13,14,23]. H2 production in gas phase was confirmed chemically by the method based on the bleaching of KMnO4 solution in H2SO4 with H2 as described [24].
Please cite this article in press as: Sargsyan H, et al., Novel approach of ethanol waste utilization: Biohydrogen production by mixed cultures of dark- and photo-fermentative bacteria using distillers grains, International Journal of Hydrogen Energy (2016), http:// dx.doi.org/10.1016/j.ijhydene.2015.11.082
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Reagents, data processing and others Various reagents of analytical grade were used. Each experiment was repeated three times to determine deviation, which is presented as error bars on Figures. Standard errors were calculated using Microsoft Excel 2013. Student criteria (p) was employed to validate the difference in average data between various series of experiments [13,14].
Results and discussion Effects of DG dilution on H2 yield in E. coli and Rh. sphaeroides pure and mixed cultures E. coli BW 25113 and Rh. sphaeroides MDC6521 were grown separately in the appropriate culture media with carbon sources e succinate and glucose. The H2 production ability of E. coli and Rh. sphaeroides pure cultures during DG dark- and photo-fermentation was determined. H2 production was detected during 96 h anaerobic growth. In diluted (2e10-folds) DG media E. coli produced H2 only during 24 h growth, and H2 production by Rh. sphaeroides was started at 24 h and continued till 96 h growth (Fig. 1a). It is interesting, that H2 production by Rh. sphaeroides control cells, grown in Ormerod medium, was detected at 48 h growth, whereas H2 production by E. coli cells, grown in peptone medium, was only observed in 24 h culture (Fig. 1a). The H2 yield in E. coli during growth in 2e5-folds diluted DG media was ~2-fold higher in comparison with culture, grown in peptone medium with glucose; the H2 yield was decreased at 10-fold dilution (Fig. 1a). H2 production was not observed, when undiluted DG were used (not shown). This is possible due to high organic compounds content in DG of ethanol fermentation [7e9]. It is known, that high content of sugars has inhibitory effect on bacteria growth and H2 production ability [25e27]. Thus, dilution of DG is necessary to optimize the organic compounds concentration for the growth and H2 production by bacteria. The highest H2 yields of ~5.2e6.3 mmol L1 were obtained in Rh. sphaeroides in the 2e5-folds diluted media after 48 h growth, whereas the highest H2 production by Rh. sphaeroides control cells was observed in 72 h culture (Fig. 1a). The H2 yields in 2- and 5-folds diluted media after 48 h anaerobic growth were ~4.0- and 4.8-folds higher in comparison with control cells, grown in Ormerod medium (Fig. 1a). DG contain various organic acids and amino acids, particularly glutamate, which can be used as the carbon and nitrogen sources for H2 production, and can affect the activity of the key H2-producing enzyme of purple bacteria e nitrogenase [12,28]. Thus, diluted DG have an evident effect on H2 yield in E. coli and Rh. sphaeroides pure cultures and can be efficiently used in H2 production. The H2 yield in E. coli and Rh. sphaeroides mixed culture using DG was also determined during 96 h growth. Mixed culture was cultivated in anaerobic conditions upon illumination. H2 production by mixed culture was also started at 24 h and continued during the growth up to 96 h (Fig. 1b). The H2 yield in co-culture during the first 24 h growth in 2e5-folds diluted DG media was lower ~1.1- and ~2.7-folds, in
Fig. 1 e The H2 yields of pure (a) and mixed (b) cultures of E. coli BW 25113 and Rh. sphaeroides MDC6521 during anaerobic growth in 2-, 5-, and 10-folds diluted DG.
comparison with E. coli and Rh. sphaeroides pure cultures, respectively. Then, H2 production by co-culture was increased. The H2 yield by mixed culture was higher than that of pure cultures: H2 yield in mixed culture during 72e96 h growth in 2-fold diluted DG media was increased ~1.5e3-folds in comparison with Rh. sphaeroides, grown in 2-fold diluted DG medium (Fig. 1b). The maximal rate of H2 production by mixed culture calculated was 5.16 mmol H2 per L and day. It should be noted that the highest H2 production rate was 7.9 mmol L1per day which has been reported with unidentified mixed culture and from DG of wine industry for heating treatment conditions and acidic pH [6]. The H2 yields in mixed culture of E. coli and Rh. sphaeroides in 5-fold diluted DG media were 1.1e1.7-folds higher than that of Rh. sphaeroides pure culture (Fig. 1b). E. coli can convert the DG to various organic acids and other products. Then, the DG fermentation endproducts can be utilized by Rh. sphaeroides for continued H2 generation. The increase of H2 yield can be also related with formation of reductive power and ATP synthesis [13,14]. Thus, the combination of dark- and photo-fermentative bacteria can enhance H2 yield.
Please cite this article in press as: Sargsyan H, et al., Novel approach of ethanol waste utilization: Biohydrogen production by mixed cultures of dark- and photo-fermentative bacteria using distillers grains, International Journal of Hydrogen Energy (2016), http:// dx.doi.org/10.1016/j.ijhydene.2015.11.082
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Effect of DG dilution on medium Eh during pure and mixed cultures growth Eh of growth medium is considered as important environmental parameter, which can be defined as the biological system ability to reduce or oxidize various compounds [13,22]. Eh of E. coli control cells has decreased only up to 405 mV during growth till 24 h, whereas Eh of Rh. sphaeroides has gradually decreased up to 580 mV during the growth up to 72 h (Fig. 2). This negative value of Eh is coupled with H2 generation, because for the 2Hþ þ 2ee / H2 reaction Eh equals to 414 mV [13,22,29]. Eh of E. coli, grown in 2-fold diluted DG media, has decreased up to 505 mV during the growth up to 24 h, and then increased up to 60 mV (Fig. 2b). The same kinetics of Eh was obtained in 5- and 10-folds diluted DG media (Fig. 2a). The dilution of DG affected the Eh of pure cultures. Eh of Rh. sphaeroides cells, grown in 2e5-folds diluted DG media, have gradually decreased during the 48 h growth up to 610 to 680 mV, and then increased during the growth up to 96 h (Fig. 2b). Such changes of Eh can be connected with the reducing equivalents (NADH or FADH2) production. These compounds can have a pronounced effect on the bacterial metabolism, because greater availability of NADH considerably alters the nature of end-products. Eh of Rh. sphaeroides, grown in 10-fold diluted DG medium, has decreased during the 48 h growth up to 535 mV, and then increased up to 225 mV (Fig. 2b). The Eh in mixed culture using DG was also determined during anaerobic growth. Eh of mixed culture, grown on 2e5folds diluted DG medium, have gradually decreased during the growth till 72 h up to 570 to 610 mV (Fig. 2c). Such change of Eh also indicates H2 generation by mixed culture.
Effect of DG dilution on pure and mixed cultures growth properties and medium pH The growth properties and pH changes were monitored during bacteria cultivation on diluted DG media. E. coli and Rh. sphaeroides were unable to grow on undiluted medium (not shown). However, the growth yields of cells, grown in 2e10folds diluted DG media, were considerably lower than those of control cells (not shown). Specific growth rates of pure and mixed cultures have decreased during growth on DG media in dilution-dependent manner (not shown). Medium pH is another significant parameter, which affects the H2 production by bacteria, because it can change the activity of H2-producing enzymes, as well as the metabolic pathways [1e3,11]. In our previous studies we have shown the correlation between the decrease of Eh and the increase of pH in Rh. sphaeroides, which points out not only the fermentation end-products formation, but also the redox reactions on the bacterial membrane surface [13,22]. pH of E. coli, grown in peptone medium, up to 24 h has decreased from pH 7.0 to 6.0 (Fig. 3a). This decrease can be coupled with end products of glucose or glycerol fermentation, such as formic and other organic acids and H2 [10,11,22,25,27]. Value of pH after 24e96 h E. coli growth in DG media was higher in comparison with control (Fig. 3a). During the Rh. sphaeroides control cells growth till 72 h in Ormerod
Fig. 2 e Changes of medium Eh during E. coli BW 25113 pure culture (a), Rh. sphaeroides MDC6521 pure culture (b), and bacterial mixed culture (c) anaerobic growth in 2-, 5-, and 10-folds diluted DG.
medium the pH of medium increased from 7.0 (initial pH) to ~8.75 (Fig. 3b). In 2e10-folds diluted DG media pH of Rh. sphaeroides pure culture was increased to ~9.0e9.5 (Fig. 3b). Such changes of pH can be connected with the available substrates uptake and fermentation products formation, as
Please cite this article in press as: Sargsyan H, et al., Novel approach of ethanol waste utilization: Biohydrogen production by mixed cultures of dark- and photo-fermentative bacteria using distillers grains, International Journal of Hydrogen Energy (2016), http:// dx.doi.org/10.1016/j.ijhydene.2015.11.082
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and fermentation end products formation in E. coli and Rh. sphaeroides in mixed culture can change pH. This might change activity of hydrogenases and formate hydrogen lyase in E. coli [2,10,11,27,29] and possibly nitrogenase and hydrogenase in Rh. sphaeroides [1e3,5] and improve H2 production by mixed culture, as suggested [21].
Concluding remarks In the present study H2 production by pure and mixed cultures of dark- (E. coli) and photo-fermentative bacteria (Rh. sphaeroides) using DG of ethanol fermentation has been investigated during batch fermentation at pH 7.0. The H2 yields in pure cultures during growth up to 48 h in 2e5-folds diluted DG media were higher in comparison with cultures, grown in appropriate media. The mixed culture produced significantly more H2 from DG: 2-folds diluted media provided the ~1.5e3folds higher H2 yield in comparison with pure culture. Moreover, H2 production by mixed culture was continued during the growth up to 96 h. DG dilution and neutralization are necessary to adjust the organic acids concentration and the pH 7.0 for the optimal growth of dark- and photo-fermentative bacteria. The results have shown the possibility of using DG of ethanol industry (in optimal dilution), as an effective substrate for H2 production. This study can provide novel approach for an inexpensive energy generation using identified mixed cultures of dark- and photo-fermentative bacteria as well as it can resolve the problem of waste utilization. During this study potentially new experimental data have been obtained, which can develop the understanding of bacterial metabolism mechanisms through the optimization of the conditions for darkand photo-fermentative cooperation for efficient Н2 production.
Acknowledgments Thanks to Dr. V. Ohanyan for help in supplying distillers grains and to Dr. A. Poladyan for comments. This study was supported by Research Grants from the State Committee of Science, Ministry of Education and Science of Armenia, to AT (13-1F002) and from the Armenian National Science and Education Fund (ANSEF), based in New York, USA, to LG (NSBiotechnology-3777).
Fig. 3 e Changes of medium pH during E. coli BW 25113 pure culture (a), Rh. sphaeroides MDC6521 pure culture (b), and bacterial mixed culture (c) anaerobic growth in 2-, 5-, and 10-folds diluted DG.
polyhydroxybutyrate shown before [13,28]. The mixed culture pH (2e5-folds DG media) has decreased to 6.1e6.2 during the first 24 h, then gradually increased up to 8.3e8.75 (Fig. 3c). Besides, the disproportion between rates of substrates uptake
references
[1] Kapdan IK, Kargi F. Bio-hydrogen production from waste materials. Enzyme Microb Technol 2006;38:569e82. [2] Trchounian A. Mechanisms for hydrogen production by different bacteria during mixed-acid and photofermentation and perspectives of hydrogen production biotechnology. Crit Rev Biotechnol 2015:35,103e35113. [3] Gabrielyan L, Trchounian A. Purple bacteria and cyanobacteria as potential producers of molecular hydrogen: аn electrochemical and bioenergetic approach. In:
Please cite this article in press as: Sargsyan H, et al., Novel approach of ethanol waste utilization: Biohydrogen production by mixed cultures of dark- and photo-fermentative bacteria using distillers grains, International Journal of Hydrogen Energy (2016), http:// dx.doi.org/10.1016/j.ijhydene.2015.11.082
6
[4]
[5]
[6]
[7]
[8] [9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 6 ) 1 e6
Trchounian A, editor. Bacterial membranes, Kerala: research signpost; 2009. p. 233e73. Mekjinda N, Ritchie RJ. Breakdown of food waste by anaerobic fermentation and non-oxygen producing photosynthesis using a photosynthetic bacterium. Waste Manag 2015;35:199e206. Mirza SSh, Qazi JI, Zhao Q, Chen Sh. Photo-biohydrogen production potential of Rhodobacter capsulatus-PK from wheat straw. Biotechnol Biofuels 2013;6:144e56. Chuang Y-S, Huang Ch-Yu, Lay Ch-H, Chen Ch-Ch, Sen B, Lin Ch-Y. Fermentative bioenergy production from distillers grains using mixed microflora. Int J Hydrogen Energy 2012;37:15547e55. Kayshev AS, Kaysheva NS. Biologically active compounds of ethanol production waste. Pharm Pharmacol 2014;4:3e22 [in Russian)]. Liu K. Chemical composition of distillers grains, a review. J Agric Food Chem 2011;59:1508e26. Ch Wood, Rosentrater KA, Muthukumarappan K, Zh Gu. Quantification of physical and chemical properties, and identification of potentially valuable components from fuel ethanol process streams. Cereal Chem 2013;90:70e9. Trchounian K, Sargsyan H, Trchounian A. Hydrogen production by Escherichia coli depends on glucose concentration and its combination with glycerol at different pHs. Int J Hydrogen Energy 2014;39:6419e23. Trchounian K, Poladyan A, Vassilian A, Trchounian A. Multiple and reversible hydrogenases for hydrogen production by Escherichia coli: dependence on fermentation substrate, pH and F0F1-ATPase. Crit Rev Biochem Mol Biol 2012;47:236e49. Gabrielyan L, Torgomyan H, Trchounian A. Growth characteristics and hydrogen production by Rhodobacter sphaeroides using various amino acids as nitrogen sources and their combinations with carbon sources. Int J Hydrogen Energy 2010;35:12201e7. Gabrielyan L, Sargsyan H, Hakobyan L, Trchounian A. Regulation of hydrogen photoproduction in Rhodobacter sphaeroides batch culture by external oxidizers and reducers. Appl Energy 2014;131:20e5. Sargsyan H, Gabrielyan L, Hakobyan L, Trchounian A. Lightdark duration alternation effects on Rhodobacter sphaeroides growth, membrane properties and bio-hydrogen production in batch culture. Int J Hydrogen Energy 2015;40:4084e91. Argun H, Kargi F. Bio-hydrogen production by different operational modes of dark and photo-fermentation: an overview. Int J Hydrogen Energy 2011;36:7443e59. Chen Ch-Y, Yang M-H, Yeh K-L, Liu Ch-H, Chang J-Sh. Biohydrogen production using sequential two-stage dark and
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
photo fermentation processes. Int J Hydrogen Energy 2008;33:4755e62. Liu B-F, Xie G-J, Wang R-Q, Xing De-F, Ding J, Zhou Xu, et al. Simultaneous hydrogen and ethanol production from cascade utilization of mono-substrate in integrated dark and photo-fermentative reactor. Biotechnol Biofuels 2015;8:8e19. Lee Ch-M, Chen P-Ch, Wang Ch-Ch, Tung Y-Ch. Photohydrogen production using purple nonsulfur bacteria with hydrogen fermentation reactor effluent. Int J Hydrogen Energy 2002;27:1309e13. Su H, Cheng J, Zhou J, Song W, Cen K. Combination of darkand photo-fermentation to enhance hydrogen production and energy conversion efficiency. Int J Hydrogen Energy 2009;34:8846e53. Nath K, Das D. Effect of light intensity and initial pH during hydrogen production by an integrated dark and photofermentation process. Int J Hydrogen Energy 2009;34:7497e501. Zagrodnik R, Laniecki M. The role of pH control on biohydrogen production by single stage hybrid dark- and photo-fermentation. Bioresour Technol 2015;194:187e95. Poladyan A, Avagyan A, Vassilian A, Trchounian A. Oxidative and reductive routes of glycerol and glucose fermentation by Escherichia coli batch cultures and their regulation by oxidizing and reducing reagents at different pH. Curr Microbiol 2013;66:49e55. Fernandez VM. An electrochemical cell for reduction of biochemicals: its application to the study of the effect of pH and redox potential on the activity of hydrogenases. Anal Biochem 1983;130:54e9. Maeda T, Wood TK. Formate detection by potassium permanganate for enhanced hydrogen production in Escherichia coli. Int J Hydrogen Energy 2008;33:2409e12. Murarka A, Dharmadi Y, Yazdani SS, Gonzalez R. Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals. Appl Environ Microbiol 2008;74:1124e35. Rosales-Colunga LM, Rodriguez ADL. Escherichia coli and its application to biohydrogen production. Rev Environ Sci Biotechnol 2015;14:123e35. Trchounian K, Trchounian A. Hydrogen production from glycerol by Escherichia coli and other bacteria: an overview and perspectives. Appl Energy 2015;156:174e84. McDowall JS, Murphy BJ, Haumann M, Palmer T, Armsrtong FA, Sargent F. Bacterial formate hydrogenlyase complex. Proc Natl Acad Sci USA 2014;111:E3948e56. Khatipov E, Miyake M, Miyake J, Asada Y. Accumulation of poly-b-hydroxybutyrate by Rhodobacter sphaeroides on various carbon and nitrogen substrates. FEMS Microbiol Lett 1998;162:39e45.
Please cite this article in press as: Sargsyan H, et al., Novel approach of ethanol waste utilization: Biohydrogen production by mixed cultures of dark- and photo-fermentative bacteria using distillers grains, International Journal of Hydrogen Energy (2016), http:// dx.doi.org/10.1016/j.ijhydene.2015.11.082
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/he
Novel approach of ethanol waste utilization: Biohydrogen production by mixed cultures of darkand photo-fermentative bacteria using distillers grains Harutyun Sargsyan, Karen Trchounian, Lilit Gabrielyan, Armen Trchounian* Department of Microbiology & Microbe and Plant Biotechnology, Biology Faculty, Yerevan State University, 1 A. Manoukian Str., 0025 Yerevan, Armenia
article info
abstract
Article history:
The combination of dark- and photo-fermentative bacteria is quite a new approach in
Received 4 September 2015
hydrogen (H2) production biotechnology, which offers potential efficient production of H2
Received in revised form
from renewable resources. This study is focused on the attempt to obtain high H2 yield by
3 November 2015
pure and mixed cultures of dark-fermentative (Escherichia coli) and photo-fermentative
Accepted 17 November 2015
(Rhodobacter sphaeroides) bacteria using distillers grains (DG), the ethanol fermentation
Available online xxx
waste. During the growth in diluted DG media H2 production by pure and mixed cultures started at 24 h growth. Moreover, the mixed culture produced significantly more H2 from
Keywords:
DG: 2-folds diluted media provided the ~1.5e3-folds higher H2 yield in comparison with
Distillers grains
pure culture. These effects were obtained for continuous H2 production at 72e96 h growth.
Biohydrogen production
DG dilution and neutralization to pH 7.0 were required. The results indicated that DG can
Escherichia coli
be used as effective and valuable source of organic substances in H2 production by bacteria.
Rhodobacter sphaeroides
This study can provide novel approach for an inexpensive energy generation, as well as to
Mixed cultures
resolve the problem of waste utilization. Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Dark- or photo-fermentation are the main pathways for biological hydrogen (H2) production. H2 generation through darkor photo-fermentation of organic compounds and wastes conversion is of great interest, thus acting as H2 production promising choice [1e3].
Constantly increasing demand for energy needs novel and cheaper substrates. Disposal of organic wastes, originating from food, sugar, wine and ethanol industry, makes a new promising approach of H2 production [1,4e6]. Distillers grains (DG) are industrial wastes generated during wine and ethanol fermentation. DG of wine fermentation has been used to produce H2 by mixed microflora [6]. However, undefined mixed cultures, heating (up to 95 C) treatment and acidic pH
* Corresponding author. Tel.: þ374 60 710520. E-mail address: [email protected] (A. Trchounian). http://dx.doi.org/10.1016/j.ijhydene.2015.11.082 0360-3199/Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Sargsyan H, et al., Novel approach of ethanol waste utilization: Biohydrogen production by mixed cultures of dark- and photo-fermentative bacteria using distillers grains, International Journal of Hydrogen Energy (2016), http:// dx.doi.org/10.1016/j.ijhydene.2015.11.082
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(pH 6.0) of DG used can limit application of this biotechnology developed. DG of ethanol fermentation, non-heating treatment and the other pH could lead to the other approach for H2 production using different identified dark- and photofermentative bacteria. The waste of ethanol fermentation can contain various sugars, proteins, amino acids, and trace elements such as calcium, iron, magnesium, and others, which are necessary for growth and H2 production by bacteria [7e9]. Using DG of ethanol fermentation as a cheaper (the cost in Armenia is 1.5 cent per liter) and valuable source of organic compounds for H2 production has the advantages of recycling wastes and protecting environment. H2 production by the most of dark-fermentative bacteria depends on the activity of different hydrogenases, which catalyze the oxidationereduction reaction of 2Hþ þ 2e / H2. Escherichia coli is applied to reveal mechanisms and regulation pathways of Н2 production, including hydrogenases and formate hydrogen lyases, as well as it can be used in Н2 production biotechnology [2,10,11]. Purple bacteria (e.g. Rhodobacter sp.) can produce H2 during photo-fermentation of organic acids using nitrogenase and hydrogenase [1e3,5]. Previously it was shown, that Rh. sphaeroides, isolated from mineral springs of Armenian mountains, were able to produce H2 in various conditions upon illumination [12e14]. Combination of dark- and photo-fermentation is quite a new approach in H2 production biotechnology, which is mainly achieved via a two- or one-stage process [15e19]. During the two-stage process, the dark-fermentation effluents provide sufficient amount of organic acids for the photo-fermentation. However, dark-fermentation effluents can contain various organic compounds such as ammonium, which significantly inhibits the nitrogenase activity of photo-fermentative bacteria [17]. Though, the system should be controlled to provide optimum media composition and environmental conditions for both bacteria [15,20,21]. In the one-stage (co-culture) process dark- and photo-fermentative bacteria are cultured in one system [19e21]. However, one-stage fermentation using mixed dark- and photo-fermentative bacteria has not yet been studied in details, in part owing to differences in growth conditions and requirements between the two types of microorganisms. This work was aimed to study H2 production by co-cultures of dark- (E. coli) and photo-fermentative (Rh. sphaeroides) bacteria using ethanol fermentation waste. Various parameters such as waste dilution and pH were optimized to reach a higher H2 yield. The changes of the external pH and redox potential (Eh) during the bacterial anaerobic growth were examined.
Materials and methods Bacterial strains, cultivation conditions and determination of growth E. coli BW 25113 wild type from Keio collection (Tsuruoka City, Yamagata, Japan) and Rh. sphaeroides strain MDC6521, isolated from Arzni mineral springs in Armenian mountains (Microbial Depository Center, National Academy of Sciences of Armenia,
Yerevan, Armenia, WDCM803) have been used. E. coli was grown in peptone medium with glucose (2 g L1) as carbon source, as described [11]. Rh. sphaeroides was cultivated in anaerobic conditions upon illumination in Ormerod medium with succinate (3.54 g L1) as carbon source, as detailed previously [12,13]. In the co-culture E. coli and Rh. sphaeroides were mixed, and mixed culture was also cultivated in anaerobic conditions upon illumination (~2000 lux), as applied before [12e14]. The growth of bacteria in batch cultures was measured by optical density (OD) changes OD at 600 nm for E. coli and at 660 nm for Rh. sphaeroides using Spectro UVeVis Auto spectrophotometer (Labomed, USA). Specific growth rate was calculated as described [13,14]. Wet DG from common (bread) wheat Triticum aestivum L. was obtained from “Alex Grig” Alcohol Plant Co. LTD (Yerevan, Armenia). DG is a yellow-brown color polydisperse system with yeast scent, in which compounds are dissolved and suspended (with visible milled caryopsis). DG was obtained during ethanol fermentation, which was performed by yeast Saccharomyces cerevisiae. In DG were found various sugars (glucose, xylose, arabinose and other), glycerol, fatty acids (linoleic, palmitic, oleic, and linolenic acids), different proteins (peptides) and 14 amino acids with a predominance of glutamate, including 8 essential amino acids: arginine, lysine, valine, histidine, threonine, phenylalanine, leucine, isoleucine, which can be used by bacteria as carbon and nitrogen source for H2 production [7e9]. The chemical composition of DG depends on the cereals fermentation conditions and the type of ethanol produced. The untreated DG was filtered through cotton wool, then paper filter, next sterilized by autoclaving at 120 C for 20 min. As pH of DG were ~3.5, before autoclaving the pH of the waste was adjusted to 7.0 by means of 0.1 M NaOH. DG were diluted 2-, 5-, and 10-folds using distilled water. In this study diluted DG was used without any medium supplements, because DG contained various carbon-containing compounds.
Determination of medium pH, Eh and H2 yield The pH of the growth medium was determined by a pH-meter (HI 122-02, HANNA Instruments, Portugal) with selective pH electrode, as described [13,14]. The medium Eh was measured during bacterial pure and mixed cultures anaerobic growth using a pair of redox electrodes: platinum (Pt) and titanium-silicate (TieSi) electrodes, as described [13,14]. In spite of TieSi electrode, Pt one was sensitive to H2. Therefore, the changes of Eh and the differences between these electrode readings provided information about not only redox processes, but also H2 yield, which was determined by the decrease of Eh to low negative values during bacterial growth [13,14,22]. This determination of H2 is close to the method with Clark-type electrode employed by the other authors [23]. The correlation between Eh and H2 production was shown; the supplementation of H2 didn't affect medium pH [13,14,23]. H2 production in gas phase was confirmed chemically by the method based on the bleaching of KMnO4 solution in H2SO4 with H2 as described [24].
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Reagents, data processing and others Various reagents of analytical grade were used. Each experiment was repeated three times to determine deviation, which is presented as error bars on Figures. Standard errors were calculated using Microsoft Excel 2013. Student criteria (p) was employed to validate the difference in average data between various series of experiments [13,14].
Results and discussion Effects of DG dilution on H2 yield in E. coli and Rh. sphaeroides pure and mixed cultures E. coli BW 25113 and Rh. sphaeroides MDC6521 were grown separately in the appropriate culture media with carbon sources e succinate and glucose. The H2 production ability of E. coli and Rh. sphaeroides pure cultures during DG dark- and photo-fermentation was determined. H2 production was detected during 96 h anaerobic growth. In diluted (2e10-folds) DG media E. coli produced H2 only during 24 h growth, and H2 production by Rh. sphaeroides was started at 24 h and continued till 96 h growth (Fig. 1a). It is interesting, that H2 production by Rh. sphaeroides control cells, grown in Ormerod medium, was detected at 48 h growth, whereas H2 production by E. coli cells, grown in peptone medium, was only observed in 24 h culture (Fig. 1a). The H2 yield in E. coli during growth in 2e5-folds diluted DG media was ~2-fold higher in comparison with culture, grown in peptone medium with glucose; the H2 yield was decreased at 10-fold dilution (Fig. 1a). H2 production was not observed, when undiluted DG were used (not shown). This is possible due to high organic compounds content in DG of ethanol fermentation [7e9]. It is known, that high content of sugars has inhibitory effect on bacteria growth and H2 production ability [25e27]. Thus, dilution of DG is necessary to optimize the organic compounds concentration for the growth and H2 production by bacteria. The highest H2 yields of ~5.2e6.3 mmol L1 were obtained in Rh. sphaeroides in the 2e5-folds diluted media after 48 h growth, whereas the highest H2 production by Rh. sphaeroides control cells was observed in 72 h culture (Fig. 1a). The H2 yields in 2- and 5-folds diluted media after 48 h anaerobic growth were ~4.0- and 4.8-folds higher in comparison with control cells, grown in Ormerod medium (Fig. 1a). DG contain various organic acids and amino acids, particularly glutamate, which can be used as the carbon and nitrogen sources for H2 production, and can affect the activity of the key H2-producing enzyme of purple bacteria e nitrogenase [12,28]. Thus, diluted DG have an evident effect on H2 yield in E. coli and Rh. sphaeroides pure cultures and can be efficiently used in H2 production. The H2 yield in E. coli and Rh. sphaeroides mixed culture using DG was also determined during 96 h growth. Mixed culture was cultivated in anaerobic conditions upon illumination. H2 production by mixed culture was also started at 24 h and continued during the growth up to 96 h (Fig. 1b). The H2 yield in co-culture during the first 24 h growth in 2e5-folds diluted DG media was lower ~1.1- and ~2.7-folds, in
Fig. 1 e The H2 yields of pure (a) and mixed (b) cultures of E. coli BW 25113 and Rh. sphaeroides MDC6521 during anaerobic growth in 2-, 5-, and 10-folds diluted DG.
comparison with E. coli and Rh. sphaeroides pure cultures, respectively. Then, H2 production by co-culture was increased. The H2 yield by mixed culture was higher than that of pure cultures: H2 yield in mixed culture during 72e96 h growth in 2-fold diluted DG media was increased ~1.5e3-folds in comparison with Rh. sphaeroides, grown in 2-fold diluted DG medium (Fig. 1b). The maximal rate of H2 production by mixed culture calculated was 5.16 mmol H2 per L and day. It should be noted that the highest H2 production rate was 7.9 mmol L1per day which has been reported with unidentified mixed culture and from DG of wine industry for heating treatment conditions and acidic pH [6]. The H2 yields in mixed culture of E. coli and Rh. sphaeroides in 5-fold diluted DG media were 1.1e1.7-folds higher than that of Rh. sphaeroides pure culture (Fig. 1b). E. coli can convert the DG to various organic acids and other products. Then, the DG fermentation endproducts can be utilized by Rh. sphaeroides for continued H2 generation. The increase of H2 yield can be also related with formation of reductive power and ATP synthesis [13,14]. Thus, the combination of dark- and photo-fermentative bacteria can enhance H2 yield.
Please cite this article in press as: Sargsyan H, et al., Novel approach of ethanol waste utilization: Biohydrogen production by mixed cultures of dark- and photo-fermentative bacteria using distillers grains, International Journal of Hydrogen Energy (2016), http:// dx.doi.org/10.1016/j.ijhydene.2015.11.082
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Effect of DG dilution on medium Eh during pure and mixed cultures growth Eh of growth medium is considered as important environmental parameter, which can be defined as the biological system ability to reduce or oxidize various compounds [13,22]. Eh of E. coli control cells has decreased only up to 405 mV during growth till 24 h, whereas Eh of Rh. sphaeroides has gradually decreased up to 580 mV during the growth up to 72 h (Fig. 2). This negative value of Eh is coupled with H2 generation, because for the 2Hþ þ 2ee / H2 reaction Eh equals to 414 mV [13,22,29]. Eh of E. coli, grown in 2-fold diluted DG media, has decreased up to 505 mV during the growth up to 24 h, and then increased up to 60 mV (Fig. 2b). The same kinetics of Eh was obtained in 5- and 10-folds diluted DG media (Fig. 2a). The dilution of DG affected the Eh of pure cultures. Eh of Rh. sphaeroides cells, grown in 2e5-folds diluted DG media, have gradually decreased during the 48 h growth up to 610 to 680 mV, and then increased during the growth up to 96 h (Fig. 2b). Such changes of Eh can be connected with the reducing equivalents (NADH or FADH2) production. These compounds can have a pronounced effect on the bacterial metabolism, because greater availability of NADH considerably alters the nature of end-products. Eh of Rh. sphaeroides, grown in 10-fold diluted DG medium, has decreased during the 48 h growth up to 535 mV, and then increased up to 225 mV (Fig. 2b). The Eh in mixed culture using DG was also determined during anaerobic growth. Eh of mixed culture, grown on 2e5folds diluted DG medium, have gradually decreased during the growth till 72 h up to 570 to 610 mV (Fig. 2c). Such change of Eh also indicates H2 generation by mixed culture.
Effect of DG dilution on pure and mixed cultures growth properties and medium pH The growth properties and pH changes were monitored during bacteria cultivation on diluted DG media. E. coli and Rh. sphaeroides were unable to grow on undiluted medium (not shown). However, the growth yields of cells, grown in 2e10folds diluted DG media, were considerably lower than those of control cells (not shown). Specific growth rates of pure and mixed cultures have decreased during growth on DG media in dilution-dependent manner (not shown). Medium pH is another significant parameter, which affects the H2 production by bacteria, because it can change the activity of H2-producing enzymes, as well as the metabolic pathways [1e3,11]. In our previous studies we have shown the correlation between the decrease of Eh and the increase of pH in Rh. sphaeroides, which points out not only the fermentation end-products formation, but also the redox reactions on the bacterial membrane surface [13,22]. pH of E. coli, grown in peptone medium, up to 24 h has decreased from pH 7.0 to 6.0 (Fig. 3a). This decrease can be coupled with end products of glucose or glycerol fermentation, such as formic and other organic acids and H2 [10,11,22,25,27]. Value of pH after 24e96 h E. coli growth in DG media was higher in comparison with control (Fig. 3a). During the Rh. sphaeroides control cells growth till 72 h in Ormerod
Fig. 2 e Changes of medium Eh during E. coli BW 25113 pure culture (a), Rh. sphaeroides MDC6521 pure culture (b), and bacterial mixed culture (c) anaerobic growth in 2-, 5-, and 10-folds diluted DG.
medium the pH of medium increased from 7.0 (initial pH) to ~8.75 (Fig. 3b). In 2e10-folds diluted DG media pH of Rh. sphaeroides pure culture was increased to ~9.0e9.5 (Fig. 3b). Such changes of pH can be connected with the available substrates uptake and fermentation products formation, as
Please cite this article in press as: Sargsyan H, et al., Novel approach of ethanol waste utilization: Biohydrogen production by mixed cultures of dark- and photo-fermentative bacteria using distillers grains, International Journal of Hydrogen Energy (2016), http:// dx.doi.org/10.1016/j.ijhydene.2015.11.082
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and fermentation end products formation in E. coli and Rh. sphaeroides in mixed culture can change pH. This might change activity of hydrogenases and formate hydrogen lyase in E. coli [2,10,11,27,29] and possibly nitrogenase and hydrogenase in Rh. sphaeroides [1e3,5] and improve H2 production by mixed culture, as suggested [21].
Concluding remarks In the present study H2 production by pure and mixed cultures of dark- (E. coli) and photo-fermentative bacteria (Rh. sphaeroides) using DG of ethanol fermentation has been investigated during batch fermentation at pH 7.0. The H2 yields in pure cultures during growth up to 48 h in 2e5-folds diluted DG media were higher in comparison with cultures, grown in appropriate media. The mixed culture produced significantly more H2 from DG: 2-folds diluted media provided the ~1.5e3folds higher H2 yield in comparison with pure culture. Moreover, H2 production by mixed culture was continued during the growth up to 96 h. DG dilution and neutralization are necessary to adjust the organic acids concentration and the pH 7.0 for the optimal growth of dark- and photo-fermentative bacteria. The results have shown the possibility of using DG of ethanol industry (in optimal dilution), as an effective substrate for H2 production. This study can provide novel approach for an inexpensive energy generation using identified mixed cultures of dark- and photo-fermentative bacteria as well as it can resolve the problem of waste utilization. During this study potentially new experimental data have been obtained, which can develop the understanding of bacterial metabolism mechanisms through the optimization of the conditions for darkand photo-fermentative cooperation for efficient Н2 production.
Acknowledgments Thanks to Dr. V. Ohanyan for help in supplying distillers grains and to Dr. A. Poladyan for comments. This study was supported by Research Grants from the State Committee of Science, Ministry of Education and Science of Armenia, to AT (13-1F002) and from the Armenian National Science and Education Fund (ANSEF), based in New York, USA, to LG (NSBiotechnology-3777).
Fig. 3 e Changes of medium pH during E. coli BW 25113 pure culture (a), Rh. sphaeroides MDC6521 pure culture (b), and bacterial mixed culture (c) anaerobic growth in 2-, 5-, and 10-folds diluted DG.
polyhydroxybutyrate shown before [13,28]. The mixed culture pH (2e5-folds DG media) has decreased to 6.1e6.2 during the first 24 h, then gradually increased up to 8.3e8.75 (Fig. 3c). Besides, the disproportion between rates of substrates uptake
references
[1] Kapdan IK, Kargi F. Bio-hydrogen production from waste materials. Enzyme Microb Technol 2006;38:569e82. [2] Trchounian A. Mechanisms for hydrogen production by different bacteria during mixed-acid and photofermentation and perspectives of hydrogen production biotechnology. Crit Rev Biotechnol 2015:35,103e35113. [3] Gabrielyan L, Trchounian A. Purple bacteria and cyanobacteria as potential producers of molecular hydrogen: аn electrochemical and bioenergetic approach. In:
Please cite this article in press as: Sargsyan H, et al., Novel approach of ethanol waste utilization: Biohydrogen production by mixed cultures of dark- and photo-fermentative bacteria using distillers grains, International Journal of Hydrogen Energy (2016), http:// dx.doi.org/10.1016/j.ijhydene.2015.11.082
6
[4]
[5]
[6]
[7]
[8] [9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 6 ) 1 e6
Trchounian A, editor. Bacterial membranes, Kerala: research signpost; 2009. p. 233e73. Mekjinda N, Ritchie RJ. Breakdown of food waste by anaerobic fermentation and non-oxygen producing photosynthesis using a photosynthetic bacterium. Waste Manag 2015;35:199e206. Mirza SSh, Qazi JI, Zhao Q, Chen Sh. Photo-biohydrogen production potential of Rhodobacter capsulatus-PK from wheat straw. Biotechnol Biofuels 2013;6:144e56. Chuang Y-S, Huang Ch-Yu, Lay Ch-H, Chen Ch-Ch, Sen B, Lin Ch-Y. Fermentative bioenergy production from distillers grains using mixed microflora. Int J Hydrogen Energy 2012;37:15547e55. Kayshev AS, Kaysheva NS. Biologically active compounds of ethanol production waste. Pharm Pharmacol 2014;4:3e22 [in Russian)]. Liu K. Chemical composition of distillers grains, a review. J Agric Food Chem 2011;59:1508e26. Ch Wood, Rosentrater KA, Muthukumarappan K, Zh Gu. Quantification of physical and chemical properties, and identification of potentially valuable components from fuel ethanol process streams. Cereal Chem 2013;90:70e9. Trchounian K, Sargsyan H, Trchounian A. Hydrogen production by Escherichia coli depends on glucose concentration and its combination with glycerol at different pHs. Int J Hydrogen Energy 2014;39:6419e23. Trchounian K, Poladyan A, Vassilian A, Trchounian A. Multiple and reversible hydrogenases for hydrogen production by Escherichia coli: dependence on fermentation substrate, pH and F0F1-ATPase. Crit Rev Biochem Mol Biol 2012;47:236e49. Gabrielyan L, Torgomyan H, Trchounian A. Growth characteristics and hydrogen production by Rhodobacter sphaeroides using various amino acids as nitrogen sources and their combinations with carbon sources. Int J Hydrogen Energy 2010;35:12201e7. Gabrielyan L, Sargsyan H, Hakobyan L, Trchounian A. Regulation of hydrogen photoproduction in Rhodobacter sphaeroides batch culture by external oxidizers and reducers. Appl Energy 2014;131:20e5. Sargsyan H, Gabrielyan L, Hakobyan L, Trchounian A. Lightdark duration alternation effects on Rhodobacter sphaeroides growth, membrane properties and bio-hydrogen production in batch culture. Int J Hydrogen Energy 2015;40:4084e91. Argun H, Kargi F. Bio-hydrogen production by different operational modes of dark and photo-fermentation: an overview. Int J Hydrogen Energy 2011;36:7443e59. Chen Ch-Y, Yang M-H, Yeh K-L, Liu Ch-H, Chang J-Sh. Biohydrogen production using sequential two-stage dark and
[17]
[18]
[19]
[20]
[21]
[22]
[23]
[24]
[25]
[26]
[27]
[28]
[29]
photo fermentation processes. Int J Hydrogen Energy 2008;33:4755e62. Liu B-F, Xie G-J, Wang R-Q, Xing De-F, Ding J, Zhou Xu, et al. Simultaneous hydrogen and ethanol production from cascade utilization of mono-substrate in integrated dark and photo-fermentative reactor. Biotechnol Biofuels 2015;8:8e19. Lee Ch-M, Chen P-Ch, Wang Ch-Ch, Tung Y-Ch. Photohydrogen production using purple nonsulfur bacteria with hydrogen fermentation reactor effluent. Int J Hydrogen Energy 2002;27:1309e13. Su H, Cheng J, Zhou J, Song W, Cen K. Combination of darkand photo-fermentation to enhance hydrogen production and energy conversion efficiency. Int J Hydrogen Energy 2009;34:8846e53. Nath K, Das D. Effect of light intensity and initial pH during hydrogen production by an integrated dark and photofermentation process. Int J Hydrogen Energy 2009;34:7497e501. Zagrodnik R, Laniecki M. The role of pH control on biohydrogen production by single stage hybrid dark- and photo-fermentation. Bioresour Technol 2015;194:187e95. Poladyan A, Avagyan A, Vassilian A, Trchounian A. Oxidative and reductive routes of glycerol and glucose fermentation by Escherichia coli batch cultures and their regulation by oxidizing and reducing reagents at different pH. Curr Microbiol 2013;66:49e55. Fernandez VM. An electrochemical cell for reduction of biochemicals: its application to the study of the effect of pH and redox potential on the activity of hydrogenases. Anal Biochem 1983;130:54e9. Maeda T, Wood TK. Formate detection by potassium permanganate for enhanced hydrogen production in Escherichia coli. Int J Hydrogen Energy 2008;33:2409e12. Murarka A, Dharmadi Y, Yazdani SS, Gonzalez R. Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals. Appl Environ Microbiol 2008;74:1124e35. Rosales-Colunga LM, Rodriguez ADL. Escherichia coli and its application to biohydrogen production. Rev Environ Sci Biotechnol 2015;14:123e35. Trchounian K, Trchounian A. Hydrogen production from glycerol by Escherichia coli and other bacteria: an overview and perspectives. Appl Energy 2015;156:174e84. McDowall JS, Murphy BJ, Haumann M, Palmer T, Armsrtong FA, Sargent F. Bacterial formate hydrogenlyase complex. Proc Natl Acad Sci USA 2014;111:E3948e56. Khatipov E, Miyake M, Miyake J, Asada Y. Accumulation of poly-b-hydroxybutyrate by Rhodobacter sphaeroides on various carbon and nitrogen substrates. FEMS Microbiol Lett 1998;162:39e45.
Please cite this article in press as: Sargsyan H, et al., Novel approach of ethanol waste utilization: Biohydrogen production by mixed cultures of dark- and photo-fermentative bacteria using distillers grains, International Journal of Hydrogen Energy (2016), http:// dx.doi.org/10.1016/j.ijhydene.2015.11.082