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Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation
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Review Article
Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation Gopalakrishnan Kumar a,1, Si-Kyung Cho b,1, Periyasamy Sivagurunathan c, Parthiban Anburajan d, Durga Madhab Mahapatra e, Jeong-Hoon Park f, Arivalagan Pugazhendhi g,* a
Institute of Chemistry, Bioscience and Environmental Engineering, Faculty of Science and Technology, University of Stavanger, Box 8600 Forus, 4036, Stavanger, Norway b Department of Biological and Environmental Science, Dongguk University, 32 Dongguk-ro, Ilsandong-gu, Goyang, Gyeonggi-do, 10326, Republic of Korea c Faculty of Environment and Labour Safety, Ton Duc Thang University, Ho Chi Minh City, Viet Nam d School of Civil and Environmental Engineering, Yonsei University, Seoul, 03722, South Korea e Department of Biological and Ecological Engineering, Oregon State University, Corvallis, OR, 97331-3906, United States f Department of Civil, Environmental and Architectural Engineering, Korea University, Anam-Dong, Seongbuk-gu, Seoul, 02841, Republic of Korea g Innovative Green Product Synthesis and Renewable Environment Development Research Group, Faculty of Environment and Labour Safety, Ton Duc Thang University, Ho Chi Minh City, Viet Nam
article info
abstract
Article history:
Access to clean energy is vital to combat global warming and climate change, and nothing
Received 31 May 2018
but hydrogen could better deliver it with ease to secure future energy needs. Biohydrogen
Received in revised form
could be produced in different routes including photolysis, water-gas shift reaction, dark,
30 August 2018
photo-fermentation and combination of both. Dark fermentative hydrogen production
Accepted 6 September 2018
(DFHP) is efficient in comparison with photo-fermentation and utilizing organic waste
Available online xxx
ensures land usage and water for agriculture. Several microbes are involved in the process of biohydrogen production via dark fermentation and characterizing them at molecular
Keywords:
level unveils holistic approach and understanding. Limited resources were available in
Biohydrogen
terms of molecular tools for microbial characterization and this paper attempts to review
Organic waste
the evolution of advanced molecular techniques including their merits and demerits.
Microbiome
Understanding the composition of micro-flora is important in DFHP and could be classified
Molecular methodologies
as pure, co-cultures, enriched mixed cultures and mixed microbiota. These cultures act as seed sources for batch and continuous fermentations that help in understanding the
* Corresponding author. E-mail address: [email protected] (A. Pugazhendhi). 1 Authors have equal contribution to this work. https://doi.org/10.1016/j.ijhydene.2018.09.040 0360-3199/© 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Kumar G, et al., Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/ j.ijhydene.2018.09.040
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Dark fermentation
efficiency of these methods. The schematics and systematic assessment of the various
Continuous systems
molecular tools (cloning, PCR-DGGE, FISH, NGS, CE-SSCP) for quantification, identification, detection and characterization of the microbial cell activity have been elaborated. Lastly, a comparative tabulation recapitulates the merits and drawbacks of each technique discussed. This provides valued information for choosing the right kind of microbial and molecular assessment tool for future characterization. Such analysis aids in suitable identification and characterization of microflora as potential biocatalysts for biohydrogen production through dark fermentation. © 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Energy as backbone of present day economy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biohydrogen production methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrogen production via dark fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of microorganisms in hydrogen production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pure cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mixed cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Co-cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enriched mixed cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Granule producing microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular techniques in dark fermentative hydrogen production (DFHP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conventional techniques (cloning, T-RFLP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advanced molecular techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Denaturing Gradient Gel Electrophoresis (DGGE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quantitative Real-Time PCR (q-PCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fluorescence in situ Hybridization (FISH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Most advanced molecular techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Next Generation Sequencing (NGS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Capillary Electrophoresis Single-Strand Conformation Polymorphism (CE-SSCP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Illumina sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16S rRNA 454 pyrosequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction Energy as backbone of present day economy Petroleum derived fuels are the major sources of present day energy. Rampant use of fossil based energy sources besides causing concerns for the future energy security also pollutes the global resources with various contaminants and emissions [1,2]. It has been estimated that ~80% of the global warming is being caused due to greenhouse gas emissions related to the use of petroleum-derived fuels (PDF) for various purposes. This necessitates the exploration of sustainable and renewable alternative energy sources. Renewable energy sources including biohydrogen are gaining phenomenal interest as a substitute for the present day PDF. Hydrogen is one
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of the cleanest forms of energy (122 MJ/kg; 2.75 folds high heat value compared to hydrocarbons) compared to other sources which can be used for multiple applications including electricity, fuel and heat [3e6]. Compared to PDF, biohydrogen is C-neutral with no greenhouse gases produced during its combustion cycle [7]. Transformation of pure water to energy (biohydrogen) can be cyclically possible through harnessing hydrogen as the energy source and simultaneously using the part of the power derived from hydrogen to split water and regenerate hydrogen. Conventional hydrogen production includes a) coal gasification, b) electrolysis of water and c) steam reforming [8]. However, the above-mentioned methods exploit nonrenewable PDF energy for hydrogen production which is unsustainable. Thus, it becomes imperative to identify sustainable routes of hydrogen production from green and renewable
Please cite this article in press as: Kumar G, et al., Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/ j.ijhydene.2018.09.040
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sources. To accomplish this feat, a variety of hydrogen production technologies are under progress, especially using biomass as a substrate and biocatalyst. Local availability, reliability, decreased emissions of greenhouse gases and scope for jobs creation are some of the privileges of utilizing biomass for hydrogen production [9,10].
Biohydrogen production methods Thermochemical and biological routes are used predominantly for producing bio hydrogen from biomass. Thermochemical routes comprise of pyrolysis, liquefaction and gasification, while biological pathways include direct/indirect biophotolysis, photo/dark fermentation or a combination of these two [4,5,7,9,11]. Although several approaches to bio hydrogen production have been proposed, commercialization of these technologies needs further investigation. Using the 5R principles of sustainability, as recovering resources before it is land filled and using the wastes/substrates for energy generation including biohydrogen or methane. Such approaches not only curtail waste accumulation along with bioenergy production but also safeguard the environment with complete recycle and reuse of materials and thus paves way for sustainable resource management. In recent times, hydrogen production through various fermentative pathways, processes have been received greatly [12,13]. Fermentation processes use biomass as the feed for biological hydrogen production and are more sustainable owing to the use of water in photolysis process with regard to the increasing stress on water resources [14,15]. The primary benefit of the fermentative pathways is a high bio-hydrogen production rate [16]. Numerous pre-treatment techniques have evolved that make the biomass amenable to various reactions and compounds/enzymes for their bioconversions. Alternative to pretreatment methods, bio-augmentation by employing specific microbes, reduces the cost and energy requirements. Irrespective of the pre-treatment methods used, bio-augmentation helps in increasing the hydrogen yield through employing specific targeted microbes in fermentations [17,18]. Bio-augmentation had been a potential approach, as it a) b) c) d) e)
enhances the startup performance in a reactor [19], triggers the biosynthesis of essential metabolites [20], enhances reactor performance [21,22], avoids damaging existing microbial communities [20,23], recoups overloading [24].
Clostridium acetobutylicum showed higher hydrogen production via bio-augmentation [25]. To increase bio hydrogen yield, employing targeted microbes was a proven method at high organic loading [26]. Bio-augmentation of Lactobacillus delbrueckii sp. bulgaricus TISTR 895 yielded high biohydrogen production by Rhodobacter sphaeroides KKU-PS5 [18]. Numerous factors affect the bio-augmentation including a) the amount of time and existence of augmented biocatalyst b) specific targeting of the microbes for its desired function and c) enhancing the performance and yields [26]. Apparently, due to all these critical factors, bio-augmented species have failed to compete with the indigenous microflora [27] in few cases. Studies by
3
Goud et al [26] have documented the substantial standing of targeted microbes after generations, which is a notable success of bio-augmentation. Testing bio-augmentation by hydrogen producing bacteria on biogas production showed a positive response increasing the yield [28]. However, due to bio-augmentation of LX-B there was a prominent effect of the net hydrogen yield due to alterations in the indigenous microbial community that resulted in a shift towards the acidogenic pathway [29]. There are many theories for generation of biohydrogen based on a) metabolite consumption, b) safeguarding the reduced environment and c) maintaining the balance of the cellular internal environment. The most critical reasons of hydrogen biosynthesis are a) to dispose of the excess reducing equivalents and b) N fixation (hydrogen as a byproduct). There 6 different ways to improve hydrogen yield, which include isolating efficient microbes, optimization of processing and environmental stress, improvising utility of light and developing energy efficient and economical photobioreactor. More research and demonstrable pilot studies are necessary for the commercialization of bio hydrogen production. In this context, the present communication focuses on existing knowledge and frontiers of biological dark fermentation processes for bio hydrogen production and critically assesses the role of microbes involved in these processes through various strategies involved in growth, development and in vivo/vitro microbial characterization.
Hydrogen production via dark fermentation Dark fermentation is economically practical at bench scales due to the high hydrogen production rate albeit lower doubling time of microbes compared to photo-fermentation and bio-photolysis. In comparison to other non-fermentative pathways, hydrogen produced using dark fermentation is simple, robust and provide high yield [5,30,31]. Constant improvements and better understanding in microbial fuel cells will provide access and meet the demands of hydrogen production in the near future. Till date, microbial hydrogen production studies have primarily employed pure cultures [32e36], and have been conducted under mesophilic to thermophilic temperature ranges [36e38]. Facultative and obligate anaerobes are the promising candidates for hydrogen production using dark fermentation. In this process the organic matter are broken down by microbes for hydrogen production in the absence of light (without radiative energy transfer). Dark fermentation enables continuous hydrogen production in the absence of light, imparting a) higher production rates, b) ease of electron transfer and simple substrate transformations, c) lower net vitality data and d) exploitation of low-value waste raw materials [15]. Furthermore, dark fermentation encourages the use of mixed cultures, where strain selection is based on the feedstock/substrate complexity and prevailing bioreactor conditions [14]. Both liquid and solid substrates like wastewater [11,39,40] or solid sludge waste [30,41,42] are used as substrate for dark fermentative hydrogen production (DFHP). Table 1 shows the major pathways and biochemical reactions involved in DFHP.
Please cite this article in press as: Kumar G, et al., Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/ j.ijhydene.2018.09.040
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Table 1 e Major pathways and stoichiometric reactions involved in the DFHP. Reaction Complete oxidation of glucose Acetate production Butyrate production Ethanol production Acetate/ethanol production Lactate production Butanol production Propionate production Valerate production Homoacetogenesis Acetate production Methane production Anaerobic oxidation of acetate Anaerobic oxidation of butyrate Sulfate reduction
Stoichiometric reaction 12H2 O/6HCO 3
þ
C6 H12 O6 þ þ 6H þ 12H2 þ C6 H12 O6 þ 4H2 O/2CH3 COO þ 2HCO 3 þ 4H þ 4H2 þ C6 H12 O6 þ 2H2 O/2CH3 CH2 CH2 COO þ 2HCO 3 þ 3H þ 2H2 C6 H12 O6 þ 2H2 O/2CH3 CH2 OH þ 2HCO3 þ 2H2 þ C6 H12 O6 þ 3H2 O/CH3 COO þ CH3 CH2 OH þ 2HCO 3 þ 3H 2H2 þ C6 H12 O6 /2CH3 CHOHCOO þ 2H þ C6 H12 O6 þ H2 O/CH3 CH2 CH2 OH þ 2HCO 3 þ 2H þ C6 H12 O6 þ 2H2 /2CH3 CH2 COO þ 2H2 O þ 2H þ C6 H12 O6 þ H2 /CH3 CH2 CH2 CH2 COO þ HCO 3 þ 2H þ H2 O þ 2HCO3 þ H 4H2 /CH3 COO þ 4H2 O C6 H12 O6 /3CH3 COO þ 3Hþ 4H2 þ CO2 /CH4 þ 2H2 O þ CH3 COO þ H2 0/2HCO 3 þ H þ 4H2 þ CH3 CH2 CH2 COO þ 10H2 O/4HCO 3 þ 3H þ 10H2 þ þ H /HS þ 4H O 4H2 þ SO2 2 4
Role of microorganisms in hydrogen production Biological hydrogen production is accomplished by a wide variety of microorganisms that have different substrate, pH and temperature requirements [43e45]. Bacteria and archaea have been known to produce biohydrogen by dark fermentation over a broad temperature range [46,47]. Major groups of microbes that contribute to hydrogen production include Hydrolytic and fermentative (acidogenic) bacteria that hydrolyze and ferment organic substrates to volatile fatty acids (VFAs) with production or consumption of hydrogen, Acetogenic bacteria that transform VFAs into acetate and hydrogen, Methanogenic archaea (both acetoclastic and hydrogenotrophic) that convert acetate and H2/CO2 into CH4. Conventionally several microbial communities have been used for dark fermentation that include a) obligate anaerobes (Clostridia, methylotrophic methanogens, and rumenbacteria), b) facultative anaerobes (Escherichia coli, Enterobacter and Citrobacter sp.) and c) aerobes (under aerobic conditions e.g. Aeromonas) [48]. For hydrogen production, Clostridia (Gram positive, spore former) and Enteric bacteria (Gram negative, non-spore former) have been primarily investigated through dark fermentation [49,50]. Utilizing both facultative and obligate anaerobes is advantageous, as facultative anaerobes helps in converting oxygen to water which indeed maintain anaerobic conditions for obligate anaerobes thus reducing the need for supplements (reducing agents to the growth medium). Higher hydrogen yields have also been recorded from mixed microbial cultures from sludge.
Pure cultures Pure cultures through isolation and strain procurement have been extensively used for investigations on biohydrogen production. Nevertheless, pure cultures are highly susceptible to contaminants, for the reason which aseptic conditions are essential. Furthermore, developing pure cultures are time-
DGO kJ reaction1 þ3.2 206.3 254.8 235.0 215.7 198.1 280.5 359.0 330.9 104.6 310.6 131.0 þ104.6 þ257.3 151.0
consuming for growth and development to reach the optimal biomass or the desired products levels that decreases the profitability of the process [44]. Hydrogen production performances of various pure cultures and their major metabolic products/pathways involved in hydrogen production are tabulated (Table 2). Characteristics of pure cultures substrate selectivity, easier control of the metabolic pathway (flexibility in modifications to development conditions), reliable hydrogen yields due to ceasing undesired byproducts, reproducibility of the bioprocess and, true genetic material that can be modified and altered at a later stage. Dark fermentation using sugars such as sucrose or glucose employing Caldicellulosiruptor saccharolyticus and Thermotoga elfii as pure cultures at 70 C have been reported earlier [51]. Furthermore, van Niel et al. reported that un-dissociated acetate concentration did not actually repress hydrogen production at pH 6.5 and 7.2 at 70 C using Caldicellulosiruptor saccharolyticus [52]. This study also revealed that the accumulating acetate as the main inhibitor of hydrogen production. Interestingly, SCB hydrolysate was used for bio hydrogen generation with the SCUT27/Dldh [53]. The procedure and maintenance of pure cultures have been debated over various research domains even for biohydrogen production where the techno-economic analysis utilizing CG plays a crucial role in deciding the production feasibility [54]. Various alternative approaches and the combination of unit processes for the reduction of the production cost were elaborated [55]. Using mixed cultures reduced the processing cost over pure- or coculture microbes by reducing the use of growth media and special requirements for their incubation [56].
Mixed cultures Utilizing mixed cultures could be economically feasible and industrially viable for hydrogen production from organic wastes [5,31,57]. However, thermophilic dark fermentation of
Please cite this article in press as: Kumar G, et al., Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/ j.ijhydene.2018.09.040
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Table 2 e Hydrogen production performance of various pure cultures and their major the hydrogen producing metabolic products. Microorganisms
Facultative anaerobes Enterobacter aerogenes Enterobacter cloacae Escherichia coli Klebsiella pneumoniae Klebsiella oxytoca Citrobacter sp. Y19 Obligate anaerobes Clostridium acetobutylicum Clostridium beijerinckii Clostridium tyrobutyricum Clostridium butyricum Clostridium pasteurianum Ethanoligenens harbinense Thermophilic Clostridium thermocellum Thermoanaerobacterium thermosaccharolyticum Thermoanaerobacter mathranii Thermotoga neapolitana Caldicellulosiruptor saccharolyticus
Fermentation products
Substrate
Maximum References hydrogen (mol/mol substrate)
Hydrogen, acetate, butyrate, propionate Hydrogen, butyrate, ethanol, acetate, formate Hydrogen, formate, acetate, lactate Hydrogen, formate, acetate, lactate, ethanol, 2,3-butanediol Hydrogen, formate, acetate, lactate, ethanol, 2,3-butanediol, succinate Hydrogen, ethanol, acetate, propionate, butyrate, valerate
Xylose Glucose Glucose Glucose
1.92 3.1 1.44 2.07
[133] [134] [72] [135]
Glucose
1.01
[136]
Glucose
2.49
[137]
Hydrogen, acetate, butyrate, lactate, formate, propionate, ethanol, butanol Hydrogen, acetate, butyrate, lactate, formate, propionate, ethanol, butanol Hydrogen, acetate, butyrate, lactate, ethanol, formate Hydrogen, acetate, butyrate, lactate, formate Hydrogen, acetate, butyrate, lactate, formate Hydrogen, ethanol, acetate, butyrate
Glucose
1.8
[138]
Glucose
2.81
[138]
Glucose Glucose Glucose Glucose
1.47 2.29 1.19 2.2
[138] [138] [139] [140]
Hydrogen, lactate, acetate, ethanol Hydrogen, lactate, acetate, ethanol, butyrate
Cellulose Cellulose
0.8 1.8
[141] [141]
Hydrogen, acetate, butyrate, lactate
Glucose
2.64
[142]
Hydrogen, lactate, acetate, ethanol Hydrogen, lactate, acetate, ethanol
Carrot pulp Carrot pulp
2.4 1.3
[143] [143]
organic household waste (70 C) using mixed cultures has not been achieved thus far. But from the engineering point of view, mixed cultures are suitable for full scale industrial application. Mixed cultures maintenance and regulation need minimal upstream processing and features low operations and maintenance cost with involvement in simpler control systems for running the entire production pathway. Nonetheless, mixed cultures require critical platforms and a better decision support system for the usage [58]. Mixed cultures of Clostridium strains likely produce hydrogen in different processing conditions including mesophilic and thermophilic ranges of temperature with a pH range of 5.0 and 6.5. Darkfermentation with mixed cultures will encourage subsequent growth of unified bacterial assemblage in the fermenter that permits association of various species to effectively regulate the growth environment and transform organic waste materials into hydrogen, in an acidic environment created by natural acids [59]. Clostridium strains produce a combination of organic acids like butyrate and propionate [60]. These organic acids act as a precursor for hydrogen production by Rhodobacter [61]. Such mixed consortia can potentially be procured from an assortment of various natural organic waste sources comprising sewage sludge [49,62], anaerobically digested sludge [43,63], acclimated sludge [64,65], compost [66,67] and soil [16,68], or native microbes found in specific type of wastes [69e71]. Despite the benefits of mixed cultures, their utilization can mask the effects of essential non-hydrogen generating
species, which could lead to failure of the process. Pretreatment techniques could minimize such an incidence of undesired microorganisms, and aid in providing conditions, which support superior hydrogen production by the selected € la € et al. examined hydrogen producing species [44]. Seppa Clostridium butyricum and E. coli for steady-state hydrogen production both individually and as mixed cultures without any reducing agents [72]. Furthermore, Yokoi et al. studied oxygen resistance and hydrogen generation by mixed strains of C. butyricum and Enterobacter aerogenes in both batch and continuous modes of operation using starch as a feedstock (T37 C, pH 5.5e6.5) [61]. Puhakka et al. recorded a shift in microbial population observed from a mixed culture from hydrogen producing Clostridium sp. (37 Ce45 C) to Thermoanaerobacterium species at (60 C) [73]. Similarly, Sargsyan et al. explored the use of waste distiller grains in biohydrogen production by mixed cultures [74]. For the mixed-culture studies, cattle manure sludge, reactor wastes and various types of soil have often been utilized as seed inoculum [56]. The impurities in CG obtained from restaurant and meat processing wastes have increased inhibition effects on pure-/ co-cultures in contrast to the impurities in CG obtained from pure substrates [75]. Application of mixed-cultures nullifies the problem of contamination associated with the use of complex CG with synergistic impacts that are beneficial over pure and co-culture system framework amongst glycerol fermentation [76]. Studies have shown that mixed culture has the greatest hydrogen production and better inoculum
Please cite this article in press as: Kumar G, et al., Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/ j.ijhydene.2018.09.040
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compared with mono and co-culture methods. Heat pretreatment of mixed cultures is a simple, modest and mechanically appropriate approach for hydrogen production using pure or mixed cultures [56]. In the context of pretreatment, Farghaly et al. investigated the use of surfactantsupplemented with mixed bacterial cultures to produce hydrogen from paper board mill wastewater [77]. Similarly, Ghimire et al. investigated the scale-up and utilizing biomass for producing hydrogen under mixed cultures and dark fermentations [78].
Co-cultures From an economical perspective, co-cultures can be maintained under anaerobic conditions for optimal hydrogen production thus eliminating the need for expensive reducing agents. In principle, co-cultures (obligate and facultative anaerobes) can enhance the hydrolysis of complex sugars and are less energy intensive, when compared to other hydrogen production processes. Studies have shown that pure cultures of E. coli and C. butyricum yield 1.45 mol H2/molglucose and 2.09 mol H2/molglucose, while their co-cultivation resulted in 1.65 mol H2/molglucose. Similar findings have demonstrated the cost-effectiveness of using co-cultures as they eliminate the need for expensive reducing agents while yielding higher hydrogen production. Co-cultures of Bacillus and Enterobacter sp. were also shown to enhance the hydrogen production more significantly [79]. To date, limited studies have reported hydrogen production by co-culture utilizing organic sources such as starch/glucose as substrate [80,81]. Bio hydrogen production by co-fermenting crude glycerol and apple pomace hydrolysate using E. aerogenes and C. butyricum as has been shown successful with better hydrogen efficiency, although higher substrate inhibition was observed while using cocultures [82]. The production of biohydrogen from industrial wastes using defined microbial co-culture has also been found to provide significant results compared to mixed or pure cultures [83].
Enriched mixed cultures Enriched mixed culture (utilitarian consortium) are far more favourable compared to natural microflora (sludge/mixed culture) because of its highly selective population of hydrogen producing microorganisms [84]. Using enriched mixed cultures (EMC) and its recycling on glycerol provides a reliable consortium for hydrogen production [85]. Moreover, EMC development and transfer to a hydrogen fermenter enhance the hydrogen yield and production rate. A batch study with EMC was studied by Sivagurunathan et al. that revealed an integrative strategy with the varied microbial population for enhancement of hydrogen production [84]. Sivagurunathan et al. assessed the feasibility of EMC for continuous hydrogen fermentation of galactose and found that EMC obtained from batch process doesn't help in higher hydrogen production, with a drop in the HY in batch 1.05 mol H2/molgalactose added to 0.82 mol H2/molgalactose added in CSTR's [84]. These differences were owing to variations in microflora pattern with hydrogen producing Clostridium sp. dominating under batch mode, whereas lactate producing Sporolactobacillus sp. was
predominating in the CSTR's. Consequently, a necessary elimination of non-hydrogen producing bacteria is needed for efficient hydrogen production under continuous operation.
Granule producing microorganisms Enhancement of hydrogen production performance is plausible through the microbially mediated granulation process as it provides stable microbial populations/colonization and prevents microbial washout during long term operation. There are certain groups of granule forming facultative and obligate anaerobic bacteria that are often detected in the hydrogen producing granular systems (Table 3). For example, Xing et al. stated that the addition of Ethanoligenens harbinense promotes the self-flocculation process in a CSTR and is the dominant bacterial population under non-sterile conditions with stable hydrogen production over 21 days of operation [86]. Hung et al. further demonstrated interactions between Clostridium sp. and facultative anaerobes (Klebsiella and Streptococcus sp.) in an auto-formed granular sludge in an anaerobic granular sludge bed reactor with powdered activated carbon as a biological carrier for granules development [87]. Their results showed that low HRT enhanced the granule formation, where the Clostridium sp. formed net-like structures and Streptococcus sp. were self-aggregated to form a solid granular core inside the net. Sivagurunathan et al. observed that the formation of granular sludge occurred only at a low HRT of 2e3 h when Selenomonas sp. was present along with Clostridium populations [57]. Furthermore, reduction of the HRT to 1.5 h led to the wash-out of Selenomonas sp. populations and the disappearance of granular sludge, resulting in reduced total cell concentrations, thus explaining the role of Selenomonas sp. in maintaining the granular structures.
Molecular techniques in dark fermentative hydrogen production (DFHP) An increasingly enormous number of molecular biology methods have been applied to confirm the presence of hydrogen producing microbes, since the last decade. Each method has their pros and cons (Table 4). Since the organisms used for biohydrogen production are prokaryotes, molecular techniques are specifically used to target the 16S rRNA gene and functional specific genes such as DNA gyrase. Traditional hybridization techniques or modern PCR based techniques are widely used for altering gene to enhance the biohydrogen producing organisms.
Conventional techniques (cloning, T-RFLP) Terminal restriction fragment length polymorphism (T-RFLP) is a PCR based technique initially used as a semi-quantitative molecular fingerprint technique that is robust, cost effective and reproducible. T-RFLP has been used to identify the abundant population of bacteria detected in the bioreactors [88,89]. This technique relies on the position of a specific and unaltered site nearest to the labelled end of an amplified gene. T-RFLP targets intensification of the 16S rRNA gene utilizing
Please cite this article in press as: Kumar G, et al., Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/ j.ijhydene.2018.09.040
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Table 3 e Granules forming bacterial populations observed in hydrogen producing systems. Microorganism
Fermentation products
Streptococcus sp.
Hydrogen, formate, acetate, lactate
Selenomonas sp.
Hydrogen, lactate, acetate, propionate
Prevotellaceae sp.
H2S, acetic, butyric and isovaleric acids
Sporolactobacillaceae sp.
Hydrogen, lactate, acetate, butyrate
fluorescently labelled preliminary base pairs at 50 ends (Fig. 1). The technique is extremely sensitive with high reproducibility. The primary advantage of T-RFLP is the capacity to identify even the uncommon population of a specimen. However, the strategy is costly and tedious in comparison to DGGE. The exploratory section size may not be indistinguishable to the hypothetical length and can be considered a fault with the T-RFLP technique. Moreover, T-RFLP is also influenced by incomplete digestion and development of pseudo terminal-restriction fragments that lead to false
Roles of bacteria and mechanism
References
Facultative anaerobic hydrogen producing bacteria, often detected in hydrogen producing granular reactors, act as a seed for self-flocculated granule formation Self-flocculating and granule forming bacteria that enhance biomass productivity and help to maintain the structure of the granules Hydrogen sulphide producing bacteria with biofilm forming ability Lactate producing bacteria with granule forming ability
[98,135,144]
[57,145e147]
[148e150] [63,149,151]
positive results or over estimation of the biodiversity [90]. TRFLP investigation on microbial communities in bioreactors has shown Clostridium and Bacillus sp. as dominant populations. T-RFLP was reliable for use in fast analysis of variation in mixed cultures [91]. T-RFLP helps in finding mesophiles that produces hydrogen and their bacterial composition [28]. Castello et al. reported that sequences from fermentative microorganisms (Bifidobacterium, Lactobacillus, Clostridium, and Streptococcus) were correlated with the T-RFLP peaks using in silico analysis [89].
Table 4 e Comparison of different microbial characterization methods and their advantages and disadvantages. Microbial characterization technique
Advantages
DGGE T-RFLP
Taxonomic identification, large database Community profiling, taxonomic identification, high sensitivity, high reproducibility
qPCR
Quantify microbial community at lower concentration, fast simultaneous amplification and quantification of target DNA, microbial composition and dynamics can both be analyzed Fast and simple, direct observation of uncultured microbes, can be used to measure metabolic activity
FISH
NGS
Fast, simple, inexpensive, scalable, high yield
PCR-CESSP
The high sensitivity of multicolour (up to five different fluorochromes) laser-induced detection. Identification of unknown microorganisms from environmental samples by comparison of the mobility of the unknown DNA to that of known sequences. Suitable for population monitoring and identification of microorganisms. High reproducibility between runs; internal standard eliminates run-to-run variability. Automation allows easy and rapid processing of 96 samples at one time. Relative abundance of detected sequences (microorganisms) is determined by measuring the fluorescence of each peak relative to the sum of the fluorescence. Illumina MiSeq: Moderate cost of instrument and runs, low cost per Mb for a small platform, fastest Illumina run times and longest Illumina read lengths High throughput analysis: Completely identifies all microbial population
Illumina sequencing
454 Pyrosequencing
Disadvantages
Cumbersome and time-consuming Expensive, time-consuming, variations in experimental and theoretical size of fragments, incomplete digestion, the formation of pseudo TRFs. PCR biases, community profiles of several species laborious and expensive
In mixed culture background, autofluorescence effects FISH images, optimization of hybridization condition. Only useful on single stranded DNA High rate of sequencing errors Low yield of high-quality sequences CE-SSCP analysis suffers from the same drawbacks as all PCR-based community analysis techniques. Limited to DNA fragments of 200e400 bp, which limits phylogenetic characterization. Not quantitative, only semi-quantitative. Two different sequences may have the same electrophoretic mobility
Relatively few reads and higher cost per Mb compared to HiSeq More time e pyrophosphate bond formation and breakage
Please cite this article in press as: Kumar G, et al., Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/ j.ijhydene.2018.09.040
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Fig. 1 e A schematic representation of Terminal restriction fragment length polymorphism, T-RFLP.
Fig. 2 e A schematic representation of Denaturing Gradient Gel Electrophoresis (DGGE).
Advanced molecular techniques Denaturing Gradient Gel Electrophoresis (DGGE) Numerous microbes contribute to bio-hydrogen production in case of mixed cultures or bacterial consortia. DGGE acts as a quick screening, identification and enumeration tool for tracking these beneficial microorganisms (Fig. 2). This electrophoresis technique aids in the rapid identification of any significant transitions in the bacterial composition in response to changes and variations in growth and culture conditions during hydrogen production. DGGE has been applied in the analysis of variations in the microbial profile regarding distinct pre-treatment processes
and their impact on hydrogen creation [92]. In one such report, variations in the microbial profile during distinct pretreatment processes, their impact on hydrogen formation and the impact of substrate burden on microbial population were examined utilizing DGGE [93]. Hafez et al. utilized DGGE to demonstrate the variations in the microbial profile at varying organic loading rates (OLR) [94]. In another study, Carillo et al. utilized DGGE to investigate Buffalo fertilizer eubacteria responsible for hydrogen generation and their relationship with pH, temperature and various pretreatments [95]. Similarly, Xing et al. monitored microbial group structure and progression in a biohydrogen production reactor using DGGE [96].
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Polymerase chain reaction coupled with Denaturing gradient gel electrophoresis (PCR-DGGE) has been utilized as an exciting device for investigation of the hydrogen producing bacterial community structure [97]. However, a setback of this strategy is that it only allows identification of the major members of the population. PCR-DGGE demonstrated that Clostridium sp. was the prevalent microorganisms [98]. PCR reamplification and DGGE investigations were conducted to ensure the purity of reclaimed DNA in several studies. DGGE of the 16S rRNA genes for the identification of the microbes (bacteria) has revealed improvement in bacterial community structure in Peptone Yeast Extract Glucose Broth (PYG) compared to the basal medium. C. butyricum, C. saccharobutylicum, C. tertium and C. perfringens were found to be present in all the samples, regardless of the medium utilized, which shows the abundance of actual hydrogen producers in the enriched mixed cultures [62].
Quantitative Real-Time PCR (q-PCR) PCR based strategies are of significant interest because of their ability to distinguish DNAs of different organisms even at trace levels. Since, the normal PCR method is qualitative and time consuming, an improvised quantitative method has been introduced called the quantitative real-time PCR (qRTPCR; Fig. 3) or simply, real-time PCR that identifies and evaluates the microbial community in real time [99]. Numerous methods including metagenomics, phylogenetic microarrays, DNA fingerprinting strategies and qPCR have been used for characterization of microbiota that aids developing characteristic taxonomic and quantitative data describing the microbiota [100]. The qPCR has been utilized since the 1990s for its quantitative accuracy, high specificity, immaculate performance, wide element range, great reproducibility, and moderately low costs [101]. Overall, qPCR performs with in a low to medium throughput, since most qPCR stages have an ability organization of 96 or 384. Most qPCR investigations have been directed on investigations on gut microbiota normally with the help of universal bacterial
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primers [102]. Wang et al. evaluated the efficacy of dark hydrogen fermentation by indigenous C. butyricum based on the expression analysis of the hydrogenase gene using RTPCR [25]. The three strains C. butyricum, C. felsineum and C. pasteurianum were monitored throughout the fermentation process for which the recA and gyrA marker genes are used as internal controls [103].
Fluorescence in situ Hybridization (FISH) FISH is a nucleic acid based hybridization technique that employs group or species specific fluorescent labelled oligoprobes that aids in both identification and differentiation of microbial species. FISH offers an excellent way to distinguish microbes without the need for DNA extraction and PCR, consequently, it overcomes the problems associated with PCR based methods such as DGGE, RFLP, RISA, cloning and sequencing (Fig. 4). DeLong et al. first reported the use of fluorescent tagged rRNA for phylogenetic studies. The system is predominantly in view of the quickly expanding data on bacterial smaller subunit (16S rRNA) rRNA arrangements, which has been massively accumulated for the investigation of microbial phylogeny [104]. To a lesser degree, tests have been similarly built that encompasses larger subunit rRNA (23S rRNA) [105]. The labelled probes used in FISH are usually short DNA sequences with target sequence specificity, enabling identification of organisms at any taxonomic level. Hence, FISH can be potentially utilized to identify microbes during hydrogen production. FISH based on 16S rRNA for Thermoanaerobacterium, Caldicellulosiruptor and Thermoanaerobacterium thermosaccharolyticum were conducted to evaluate the spatial distribution of hydrogen producing microbes in the secretions and granules from anaerobic reactors [106]. PCR-RFLP and FISH were also utilized to distinguish Megasphaera elsdenii as a hydrogen producer [107]. Similarly, FISH and SEM (scanning electron microscopy) were used to identify predominant classes of HPB as E. aerogenes ATCC13048 and Firmicutes including Clostridium, Bacillus and Dialister sp. [108].
Fig. 3 e A schematic representation of Quantitative Real-Time PCR (q-PCR). Please cite this article in press as: Kumar G, et al., Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/ j.ijhydene.2018.09.040
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Fig. 4 e A schematic representation of Fluorescence in Situ Hybridization (FISH).
Most advanced molecular techniques Next Generation Sequencing (NGS) In a typical hydrogen reactor, minor individuals from the population may also play a critical role in hydrogen production; accordingly, strategies that enable identification of the entire population are in need and one such strategy is to use the next-generation sequencing techniques. NGS is a higthroughout method that has wide sequence coverage without compromising the quality of the sequencing. NGS could be used to study the microbial diversity of the given environment either by focusing on their ribosomal RNA sequences alone [109e111] or by sequencing their whole genomes and comparing the genomes [112] for relatedness or evolutionary differences. Microbial communities unravelled by the high-throughput sequencing-based metagenomic evaluations showed a predominance of Clostridium sp. in the reactor environment, as the reactions of the variables explored were enhanced towards the highest hydrogen producing potential. High-throughput next generation metagenomic analyses are increasingly being used to better understand the key microbiological elements driving dark fermentative biohydrogen production. NGS based analyses are useful in identifying the minor components of the complex microbial communities, which are usually left out while analysing through standard or traditional microbiological methods that majorly concentrate on the dominant members alone. NGS is a general term that includes modern sequencing technologies like
Illumina (Solexa) sequencing Roche 454 sequencing Ion torrent: Proton/PGM sequencing SOLiD sequencing
Each of these techniques differ from each other in their working modalities yet delivering more or less the same outputs. Sequencing of a huge amount of DNA from an environmental sample is plausible with NGS enabling it to be completed in a day [113,114]. Since, NGS are high-throughput techniques, they generate humongous amount of sequence data that requires specialized bioinformatics tools and experts to decode the information it contained [113]. Nevertheless, the initiation and development of NGS have provided a great platform for a cost effective and a comprehensive characterization of complex microbial consortia compositions over conventional molecular techniques such as DGGE, RFLP and cloning followed by Sanger sequencing [115,116].
Capillary Electrophoresis Single-Strand Conformation Polymorphism (CE-SSCP) Capillary electrophoresis is a method that revolutionized many other techniques because of its uncomplicated workflow and high-throughput analysis. SSCP is a traditional method used to differentiate closely related organisms whose sequence details are unknown. CE-SSCP method is a valuable tool for (i) producing molecular fingerprints of 16S rRNA and/ or functional genes and (ii) comparing the shift in the bacterial population structure and diversity relative to the progression of ecological parameters [117,118]. The functional CE-SSCP strategy aided in effective discrimination between Clostridium strains and offered dependable and high throughput analysis of hydA genes in mixed-culture bioprocesses. me neur et al. showed the capability of utilizing the hydA Que based CE-SSCP to observe the structure and elements of hydrogen producing Clostridial populations, which play a key role in hydrogen production in bioreactors [119]. The study reported that components of functional gene based CE-SSCP profiles from pure strains are helpful for concentrating on
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the structure of functional populations [120]. In another study, me neur et al. evaluated the interference of fermentative Que hydrogen production by lignocellulose-derived compounds in mixed cultures, utilizing CE-SSCP strategy [121] (Fig. 5). Additionally, information on the hydrogen producers and their diversity and structure in various specimens of dark fermentation bioreactors have been demonstrated through hydA CESSCP strategy. Furthermore, it is increasingly being used to monitor environmental stress response i.e. impact of environmental parameters on microbial diversity and their structures [119,122]. It is interesting to note that every peak of the CE-SSCP profile corresponds to the gene fragment of a unique 16S rRNA gene sequence, and thus represents specific species or strain within the microbial community thus aiding their identification. The relative abundance of the corresponding microbial species in the community is gauged by the region under the peak [95].
Illumina sequencing Sequencing by synthesis (SBS) through Illumina sequencing technology, is by far the best and most often used nextgeneration sequencing (NGS) technology worldwide. This method has a wide variety of uses that deals with any related information on the genome, transcriptome, or epi-genome of diverse organisms. Zillions of genomes have been sequenced and are undergoing this process that aids in the rapid examination of DNA sequences to improve understanding of health, disease and diagnostics making it the least expensive than other accessible sequencing techniques. In addition to that, the state of the art microarray technologies (e.g., Illumina sequencing innovation) permit high-throughput performance and adequate precision [123]. The sequencing of the hyper variable regions of the 16S ribosomal rRNA gene through Illumina MiSeq sequencing was employed and proven handy to uncover the microbial diversity of the fermentation reactions [124e126]. Table 4 provides information about the pros and cons of various methods mentioned above.
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16S rRNA 454 pyrosequencing Next generation DNA sequencing investigations including 454-pyrosequencing have been utilized to break down the bacterial diversity of mixed cultures because they provide significantly more data than conventional techniques [127,128]. Pyrosequencing involves DNA synthesis monitoring in real time and is based on the principle of DNA polymerization tracked by measuring pyrophosphate production, which can be detected by light. In this process, the DNA polymerase synthesizes DNA by extending the 30 end of the initial strand using the information encoded in the template strand [128]. In pyrosequencing, the nucleotides are identified based on the pyrophosphates release upon addition of each base. Pyrophosphate moiety of each nucleotide is labelled with an unique fluorophore and the addition of the labelled nucleotides in the growing chain of nucleic acid governed by the polymerase, releases the labelled pyrophosphate. The released pyrophosphate will be detected by the laser detectors and the sequence of the nucleotides are deduced from that [127]. Improvements in technologies such as 454 pyrosequencing (Roche), helps in handling more sequences in a short time. This method is used to identify microbes in multi-disciplinary conditions. There are very few studies on microbial population in hydrogen production through pyrosequencing and thus have a lot of scope for future research in microbiomes for hydrogen production through high throughput pyrosequencing. It provides the complete picture of the bacterial microbiome with detailed dominant microbiota. Many microbial communities have been identified in various types of batch systems [129] that help in the process of fermentation and can be either dominant or mere present microflora. Although Thermoanaerobacterium and Thermohydrogenium bacterial populations are dominating under mesophilic and thermophilic conditions, there are many other bacterial members that are present, (belonging to both hydrogen producing and non-hydrogen producing communities). Abundance of various sub-dominant bacterial
Fig. 5 e A schematic representation of Capillary Electrophoresis Single-Strand Conformation Polymorphism (CE-SSCP). Please cite this article in press as: Kumar G, et al., Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/ j.ijhydene.2018.09.040
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Table 5 e Major HPBs and molecular techniques reported in the literature. Microbial source
Feedstock
Medium
AMC
Beverage wastewater
PYG
Cow dung compost, sewage sludge Activated sludge Activated sludge
Beverage wastewater
PYG
Mixed microflora (EMC)
Beverage industrial wastewater (BW) Synthetic wastewater
Basal endo medium
Anaerobic sludge
Maize silage LB De-oiled Jatropha waste Nutrient solution
nr
Anaerobic digested sludge Pure strain
Glucose
Endo medium
Glucose
MDt medium
Pure strain Pure strain Seed sludge
nr Pure glycerol Sugar beet molasses
Modified PYG Nutrient broth M9 medium
Granular sludge
Tequila vinasses
Hydrogen producing strain Anaerobic sludge, cassava (Cas) and cæcotrophs (Cæ) Anaerobic digested sludge
Sucrose
Mineral nutrient solution nr
Anaerobic sludge Anaerobic sludge Digested sludge
Glucose
Glucose-based medium
Fructose, glucose, sucrose, maltose, cellobiose, maltotriose Glucose Glucose Glucose
Culture medium
Major HPB reported
Clostridium sp., Enterobacter sp. C. buytricum, C. tertium, C. perfringens E. cloacae C. thermopalmarium, C. buytricum, B. ginsengihumi, B. coagulans Clostridium sp.
Molecular technique employed
References
PCR-DGGE
[84]
PCR-DGGE
[57]
T-RFLP, Real-Time PCR PCR-DGGE
[28] [93]
PCR-DGGE
[96]
Clostridium sp., DGGE Ethanologen bacterium sp. nr PCReDGGE
[97] [99]
C. butyricum, C. felsineum and C. pasteurianum Megasphaera sp. E. aerogenes ATCC 13048 Leuconostocaeae and Enterobacteriaceae Clostridium strains
FISH and qPCR
[103]
PCR-RFLP and FISH FISH PCReDGGE
[107] [108] [128]
DGGE
[127]
C. sporogenes
CE-SSCP
[119]
C. pasteurianum
CE-SSCP
[92]
CE-SSCP
[150]
Illumina MiSeq sequencing Illumina MiSeq sequencing Illumina MiSeq sequencing
[152] [153] [154]
C. acetobutylicum, C. cellulolyticum, C. kluyveri, C., C. sporogenes Synthetic wastewater Clostridium sp. nr Clostridium ljungdahlii Enriched Clostridium perfringens
communities in terms of OTUs (operational taxonomic units) that is considered as a surrogate of the bacterial abundance in the present genetic identification approaches [129]. In this context, significant progress has been made for the establishment of efficient and high throughput screening and characterization tools in molecular biology (Table 5). Apart from the different molecular biology or genetic engineering studies used to enhance biohydrogen production. Nowadays researchers are also improving biohydrogen productivity by involving nanotechnology using nanoparticles. Already nanomaterials have been used in producing electrical energy in some studies [6,8,130,131]. But as future perspective researchers are suggesting metallic or non-metallic nanoparticles like nitrogen doped graphitic carbon sheets and carbon encapsulated RuO2 nanorods for gas fuel production especially hydrogen [132]. Therefore, finding different techniques to amplify the production rate, reduce production cost and increase yield of production will be the future of biohydrogen.
hydrogen producing bacterial communities are highly essential. As the cleanest and powerful fuel, a biohydrogen economy ensures limitless energy for humanity and future energy security. Moreover, hydrogen as a fuel is apparently having zero environmental externalities thus paving the path for a sustainable bioenergy future. The present communication attempts to understand issues in discerning biohydrogen future through rapid dark fermentative pathways with a variety of microbial biocatalysts and numerous pre-treatment techniques applied. Additionally, the pros and cons of each molecular technique have been discussed for better understanding and improvisation. It is also envisioned that a combination of molecular techniques could be implemented to cross confirm the microbial population inside a reactor. A holistic approach is required for a clean and green hydrogen economy including developments in genetically designed microbes, advances in bioreactor design and economics, and finally, storage and distribution systems.
Conclusion
Acknowledgement
In the rapidly evolving microbial world, molecular tools for screening, enumeration and identification of potential
This work was supported by the Dongguk University Research fund of 2015.
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[146] Dighe AS, Shouche YS, Ranade DR. Selenomonas lipolytica sp. nov., an obligately anaerobic bacterium possessing lipolytic activity. Int J Syst Evol Microbiol 1998;48:783e91. [147] Xing D, Ren N, Rittmann BE. Genetic diversity of hydrogenproducing bacteria in an acidophilic ethanol-H2coproducing system, analyzed using the [Fe]-hydrogenase gene. Appl Environ Microbiol 2008;74:1232e9. [148] Harding G, Sutter V, Finegold S, Bricknell K. Characterization of bacteroides melaninogenicus. J Clin Microbiol 1976;4:354e9. [149] Kumar G, Bakonyi P, Kobayashi T, Xu K-Q, Sivagurunathan P, Kim S-H, et al. Enhancement of biofuel production via microbial augmentation: the case of dark fermentative hydrogen. Renew Sustain Energy Rev 2016;57:879e91. [150] Washio J, Sato T, Koseki T, Takahashi N. Hydrogen sulfideproducing bacteria in tongue biofilm and their relationship with oral malodour. J Med Microbiol 2005;54:889e95. [151] Fang HH, Liu H, Zhang T. Characterization of a hydrogenproducing granular sludge. Biotechnol Bioeng 2002;78:44e52. [152] Si B, Liu Z, Zhang Y, Li J, Xing X-H, Li B, et al. Effect of reaction mode on biohydrogen production and its microbial diversity. Int J Hydrogen Energy 2015;40:3191e200. [153] Si B, Li J, Li B, Zhu Z, Shen R, Zhang Y, et al. The role of hydraulic retention time on controlling methanogenesis and homoacetogenesis in biohydrogen production using upflow anaerobic sludge blanket (UASB) reactor and packed bed reactor (PBR). Int J Hydrogen Energy 2015;40:11414e21. [154] Yin Y, Wang J. Changes in microbial community during biohydrogen production using gamma irradiated sludge as inoculum. Bioresour Technol 2016;200:217e22.
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Review Article
Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation Gopalakrishnan Kumar a,1, Si-Kyung Cho b,1, Periyasamy Sivagurunathan c, Parthiban Anburajan d, Durga Madhab Mahapatra e, Jeong-Hoon Park f, Arivalagan Pugazhendhi g,* a
Institute of Chemistry, Bioscience and Environmental Engineering, Faculty of Science and Technology, University of Stavanger, Box 8600 Forus, 4036, Stavanger, Norway b Department of Biological and Environmental Science, Dongguk University, 32 Dongguk-ro, Ilsandong-gu, Goyang, Gyeonggi-do, 10326, Republic of Korea c Faculty of Environment and Labour Safety, Ton Duc Thang University, Ho Chi Minh City, Viet Nam d School of Civil and Environmental Engineering, Yonsei University, Seoul, 03722, South Korea e Department of Biological and Ecological Engineering, Oregon State University, Corvallis, OR, 97331-3906, United States f Department of Civil, Environmental and Architectural Engineering, Korea University, Anam-Dong, Seongbuk-gu, Seoul, 02841, Republic of Korea g Innovative Green Product Synthesis and Renewable Environment Development Research Group, Faculty of Environment and Labour Safety, Ton Duc Thang University, Ho Chi Minh City, Viet Nam
article info
abstract
Article history:
Access to clean energy is vital to combat global warming and climate change, and nothing
Received 31 May 2018
but hydrogen could better deliver it with ease to secure future energy needs. Biohydrogen
Received in revised form
could be produced in different routes including photolysis, water-gas shift reaction, dark,
30 August 2018
photo-fermentation and combination of both. Dark fermentative hydrogen production
Accepted 6 September 2018
(DFHP) is efficient in comparison with photo-fermentation and utilizing organic waste
Available online xxx
ensures land usage and water for agriculture. Several microbes are involved in the process of biohydrogen production via dark fermentation and characterizing them at molecular
Keywords:
level unveils holistic approach and understanding. Limited resources were available in
Biohydrogen
terms of molecular tools for microbial characterization and this paper attempts to review
Organic waste
the evolution of advanced molecular techniques including their merits and demerits.
Microbiome
Understanding the composition of micro-flora is important in DFHP and could be classified
Molecular methodologies
as pure, co-cultures, enriched mixed cultures and mixed microbiota. These cultures act as seed sources for batch and continuous fermentations that help in understanding the
* Corresponding author. E-mail address: [email protected] (A. Pugazhendhi). 1 Authors have equal contribution to this work. https://doi.org/10.1016/j.ijhydene.2018.09.040 0360-3199/© 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Kumar G, et al., Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/ j.ijhydene.2018.09.040
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Dark fermentation
efficiency of these methods. The schematics and systematic assessment of the various
Continuous systems
molecular tools (cloning, PCR-DGGE, FISH, NGS, CE-SSCP) for quantification, identification, detection and characterization of the microbial cell activity have been elaborated. Lastly, a comparative tabulation recapitulates the merits and drawbacks of each technique discussed. This provides valued information for choosing the right kind of microbial and molecular assessment tool for future characterization. Such analysis aids in suitable identification and characterization of microflora as potential biocatalysts for biohydrogen production through dark fermentation. © 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Energy as backbone of present day economy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biohydrogen production methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hydrogen production via dark fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Role of microorganisms in hydrogen production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pure cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mixed cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Co-cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enriched mixed cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Granule producing microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Molecular techniques in dark fermentative hydrogen production (DFHP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conventional techniques (cloning, T-RFLP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advanced molecular techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Denaturing Gradient Gel Electrophoresis (DGGE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Quantitative Real-Time PCR (q-PCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fluorescence in situ Hybridization (FISH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Most advanced molecular techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Next Generation Sequencing (NGS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Capillary Electrophoresis Single-Strand Conformation Polymorphism (CE-SSCP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Illumina sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16S rRNA 454 pyrosequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction Energy as backbone of present day economy Petroleum derived fuels are the major sources of present day energy. Rampant use of fossil based energy sources besides causing concerns for the future energy security also pollutes the global resources with various contaminants and emissions [1,2]. It has been estimated that ~80% of the global warming is being caused due to greenhouse gas emissions related to the use of petroleum-derived fuels (PDF) for various purposes. This necessitates the exploration of sustainable and renewable alternative energy sources. Renewable energy sources including biohydrogen are gaining phenomenal interest as a substitute for the present day PDF. Hydrogen is one
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of the cleanest forms of energy (122 MJ/kg; 2.75 folds high heat value compared to hydrocarbons) compared to other sources which can be used for multiple applications including electricity, fuel and heat [3e6]. Compared to PDF, biohydrogen is C-neutral with no greenhouse gases produced during its combustion cycle [7]. Transformation of pure water to energy (biohydrogen) can be cyclically possible through harnessing hydrogen as the energy source and simultaneously using the part of the power derived from hydrogen to split water and regenerate hydrogen. Conventional hydrogen production includes a) coal gasification, b) electrolysis of water and c) steam reforming [8]. However, the above-mentioned methods exploit nonrenewable PDF energy for hydrogen production which is unsustainable. Thus, it becomes imperative to identify sustainable routes of hydrogen production from green and renewable
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sources. To accomplish this feat, a variety of hydrogen production technologies are under progress, especially using biomass as a substrate and biocatalyst. Local availability, reliability, decreased emissions of greenhouse gases and scope for jobs creation are some of the privileges of utilizing biomass for hydrogen production [9,10].
Biohydrogen production methods Thermochemical and biological routes are used predominantly for producing bio hydrogen from biomass. Thermochemical routes comprise of pyrolysis, liquefaction and gasification, while biological pathways include direct/indirect biophotolysis, photo/dark fermentation or a combination of these two [4,5,7,9,11]. Although several approaches to bio hydrogen production have been proposed, commercialization of these technologies needs further investigation. Using the 5R principles of sustainability, as recovering resources before it is land filled and using the wastes/substrates for energy generation including biohydrogen or methane. Such approaches not only curtail waste accumulation along with bioenergy production but also safeguard the environment with complete recycle and reuse of materials and thus paves way for sustainable resource management. In recent times, hydrogen production through various fermentative pathways, processes have been received greatly [12,13]. Fermentation processes use biomass as the feed for biological hydrogen production and are more sustainable owing to the use of water in photolysis process with regard to the increasing stress on water resources [14,15]. The primary benefit of the fermentative pathways is a high bio-hydrogen production rate [16]. Numerous pre-treatment techniques have evolved that make the biomass amenable to various reactions and compounds/enzymes for their bioconversions. Alternative to pretreatment methods, bio-augmentation by employing specific microbes, reduces the cost and energy requirements. Irrespective of the pre-treatment methods used, bio-augmentation helps in increasing the hydrogen yield through employing specific targeted microbes in fermentations [17,18]. Bio-augmentation had been a potential approach, as it a) b) c) d) e)
enhances the startup performance in a reactor [19], triggers the biosynthesis of essential metabolites [20], enhances reactor performance [21,22], avoids damaging existing microbial communities [20,23], recoups overloading [24].
Clostridium acetobutylicum showed higher hydrogen production via bio-augmentation [25]. To increase bio hydrogen yield, employing targeted microbes was a proven method at high organic loading [26]. Bio-augmentation of Lactobacillus delbrueckii sp. bulgaricus TISTR 895 yielded high biohydrogen production by Rhodobacter sphaeroides KKU-PS5 [18]. Numerous factors affect the bio-augmentation including a) the amount of time and existence of augmented biocatalyst b) specific targeting of the microbes for its desired function and c) enhancing the performance and yields [26]. Apparently, due to all these critical factors, bio-augmented species have failed to compete with the indigenous microflora [27] in few cases. Studies by
3
Goud et al [26] have documented the substantial standing of targeted microbes after generations, which is a notable success of bio-augmentation. Testing bio-augmentation by hydrogen producing bacteria on biogas production showed a positive response increasing the yield [28]. However, due to bio-augmentation of LX-B there was a prominent effect of the net hydrogen yield due to alterations in the indigenous microbial community that resulted in a shift towards the acidogenic pathway [29]. There are many theories for generation of biohydrogen based on a) metabolite consumption, b) safeguarding the reduced environment and c) maintaining the balance of the cellular internal environment. The most critical reasons of hydrogen biosynthesis are a) to dispose of the excess reducing equivalents and b) N fixation (hydrogen as a byproduct). There 6 different ways to improve hydrogen yield, which include isolating efficient microbes, optimization of processing and environmental stress, improvising utility of light and developing energy efficient and economical photobioreactor. More research and demonstrable pilot studies are necessary for the commercialization of bio hydrogen production. In this context, the present communication focuses on existing knowledge and frontiers of biological dark fermentation processes for bio hydrogen production and critically assesses the role of microbes involved in these processes through various strategies involved in growth, development and in vivo/vitro microbial characterization.
Hydrogen production via dark fermentation Dark fermentation is economically practical at bench scales due to the high hydrogen production rate albeit lower doubling time of microbes compared to photo-fermentation and bio-photolysis. In comparison to other non-fermentative pathways, hydrogen produced using dark fermentation is simple, robust and provide high yield [5,30,31]. Constant improvements and better understanding in microbial fuel cells will provide access and meet the demands of hydrogen production in the near future. Till date, microbial hydrogen production studies have primarily employed pure cultures [32e36], and have been conducted under mesophilic to thermophilic temperature ranges [36e38]. Facultative and obligate anaerobes are the promising candidates for hydrogen production using dark fermentation. In this process the organic matter are broken down by microbes for hydrogen production in the absence of light (without radiative energy transfer). Dark fermentation enables continuous hydrogen production in the absence of light, imparting a) higher production rates, b) ease of electron transfer and simple substrate transformations, c) lower net vitality data and d) exploitation of low-value waste raw materials [15]. Furthermore, dark fermentation encourages the use of mixed cultures, where strain selection is based on the feedstock/substrate complexity and prevailing bioreactor conditions [14]. Both liquid and solid substrates like wastewater [11,39,40] or solid sludge waste [30,41,42] are used as substrate for dark fermentative hydrogen production (DFHP). Table 1 shows the major pathways and biochemical reactions involved in DFHP.
Please cite this article in press as: Kumar G, et al., Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/ j.ijhydene.2018.09.040
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Table 1 e Major pathways and stoichiometric reactions involved in the DFHP. Reaction Complete oxidation of glucose Acetate production Butyrate production Ethanol production Acetate/ethanol production Lactate production Butanol production Propionate production Valerate production Homoacetogenesis Acetate production Methane production Anaerobic oxidation of acetate Anaerobic oxidation of butyrate Sulfate reduction
Stoichiometric reaction 12H2 O/6HCO 3
þ
C6 H12 O6 þ þ 6H þ 12H2 þ C6 H12 O6 þ 4H2 O/2CH3 COO þ 2HCO 3 þ 4H þ 4H2 þ C6 H12 O6 þ 2H2 O/2CH3 CH2 CH2 COO þ 2HCO 3 þ 3H þ 2H2 C6 H12 O6 þ 2H2 O/2CH3 CH2 OH þ 2HCO3 þ 2H2 þ C6 H12 O6 þ 3H2 O/CH3 COO þ CH3 CH2 OH þ 2HCO 3 þ 3H 2H2 þ C6 H12 O6 /2CH3 CHOHCOO þ 2H þ C6 H12 O6 þ H2 O/CH3 CH2 CH2 OH þ 2HCO 3 þ 2H þ C6 H12 O6 þ 2H2 /2CH3 CH2 COO þ 2H2 O þ 2H þ C6 H12 O6 þ H2 /CH3 CH2 CH2 CH2 COO þ HCO 3 þ 2H þ H2 O þ 2HCO3 þ H 4H2 /CH3 COO þ 4H2 O C6 H12 O6 /3CH3 COO þ 3Hþ 4H2 þ CO2 /CH4 þ 2H2 O þ CH3 COO þ H2 0/2HCO 3 þ H þ 4H2 þ CH3 CH2 CH2 COO þ 10H2 O/4HCO 3 þ 3H þ 10H2 þ þ H /HS þ 4H O 4H2 þ SO2 2 4
Role of microorganisms in hydrogen production Biological hydrogen production is accomplished by a wide variety of microorganisms that have different substrate, pH and temperature requirements [43e45]. Bacteria and archaea have been known to produce biohydrogen by dark fermentation over a broad temperature range [46,47]. Major groups of microbes that contribute to hydrogen production include Hydrolytic and fermentative (acidogenic) bacteria that hydrolyze and ferment organic substrates to volatile fatty acids (VFAs) with production or consumption of hydrogen, Acetogenic bacteria that transform VFAs into acetate and hydrogen, Methanogenic archaea (both acetoclastic and hydrogenotrophic) that convert acetate and H2/CO2 into CH4. Conventionally several microbial communities have been used for dark fermentation that include a) obligate anaerobes (Clostridia, methylotrophic methanogens, and rumenbacteria), b) facultative anaerobes (Escherichia coli, Enterobacter and Citrobacter sp.) and c) aerobes (under aerobic conditions e.g. Aeromonas) [48]. For hydrogen production, Clostridia (Gram positive, spore former) and Enteric bacteria (Gram negative, non-spore former) have been primarily investigated through dark fermentation [49,50]. Utilizing both facultative and obligate anaerobes is advantageous, as facultative anaerobes helps in converting oxygen to water which indeed maintain anaerobic conditions for obligate anaerobes thus reducing the need for supplements (reducing agents to the growth medium). Higher hydrogen yields have also been recorded from mixed microbial cultures from sludge.
Pure cultures Pure cultures through isolation and strain procurement have been extensively used for investigations on biohydrogen production. Nevertheless, pure cultures are highly susceptible to contaminants, for the reason which aseptic conditions are essential. Furthermore, developing pure cultures are time-
DGO kJ reaction1 þ3.2 206.3 254.8 235.0 215.7 198.1 280.5 359.0 330.9 104.6 310.6 131.0 þ104.6 þ257.3 151.0
consuming for growth and development to reach the optimal biomass or the desired products levels that decreases the profitability of the process [44]. Hydrogen production performances of various pure cultures and their major metabolic products/pathways involved in hydrogen production are tabulated (Table 2). Characteristics of pure cultures substrate selectivity, easier control of the metabolic pathway (flexibility in modifications to development conditions), reliable hydrogen yields due to ceasing undesired byproducts, reproducibility of the bioprocess and, true genetic material that can be modified and altered at a later stage. Dark fermentation using sugars such as sucrose or glucose employing Caldicellulosiruptor saccharolyticus and Thermotoga elfii as pure cultures at 70 C have been reported earlier [51]. Furthermore, van Niel et al. reported that un-dissociated acetate concentration did not actually repress hydrogen production at pH 6.5 and 7.2 at 70 C using Caldicellulosiruptor saccharolyticus [52]. This study also revealed that the accumulating acetate as the main inhibitor of hydrogen production. Interestingly, SCB hydrolysate was used for bio hydrogen generation with the SCUT27/Dldh [53]. The procedure and maintenance of pure cultures have been debated over various research domains even for biohydrogen production where the techno-economic analysis utilizing CG plays a crucial role in deciding the production feasibility [54]. Various alternative approaches and the combination of unit processes for the reduction of the production cost were elaborated [55]. Using mixed cultures reduced the processing cost over pure- or coculture microbes by reducing the use of growth media and special requirements for their incubation [56].
Mixed cultures Utilizing mixed cultures could be economically feasible and industrially viable for hydrogen production from organic wastes [5,31,57]. However, thermophilic dark fermentation of
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Table 2 e Hydrogen production performance of various pure cultures and their major the hydrogen producing metabolic products. Microorganisms
Facultative anaerobes Enterobacter aerogenes Enterobacter cloacae Escherichia coli Klebsiella pneumoniae Klebsiella oxytoca Citrobacter sp. Y19 Obligate anaerobes Clostridium acetobutylicum Clostridium beijerinckii Clostridium tyrobutyricum Clostridium butyricum Clostridium pasteurianum Ethanoligenens harbinense Thermophilic Clostridium thermocellum Thermoanaerobacterium thermosaccharolyticum Thermoanaerobacter mathranii Thermotoga neapolitana Caldicellulosiruptor saccharolyticus
Fermentation products
Substrate
Maximum References hydrogen (mol/mol substrate)
Hydrogen, acetate, butyrate, propionate Hydrogen, butyrate, ethanol, acetate, formate Hydrogen, formate, acetate, lactate Hydrogen, formate, acetate, lactate, ethanol, 2,3-butanediol Hydrogen, formate, acetate, lactate, ethanol, 2,3-butanediol, succinate Hydrogen, ethanol, acetate, propionate, butyrate, valerate
Xylose Glucose Glucose Glucose
1.92 3.1 1.44 2.07
[133] [134] [72] [135]
Glucose
1.01
[136]
Glucose
2.49
[137]
Hydrogen, acetate, butyrate, lactate, formate, propionate, ethanol, butanol Hydrogen, acetate, butyrate, lactate, formate, propionate, ethanol, butanol Hydrogen, acetate, butyrate, lactate, ethanol, formate Hydrogen, acetate, butyrate, lactate, formate Hydrogen, acetate, butyrate, lactate, formate Hydrogen, ethanol, acetate, butyrate
Glucose
1.8
[138]
Glucose
2.81
[138]
Glucose Glucose Glucose Glucose
1.47 2.29 1.19 2.2
[138] [138] [139] [140]
Hydrogen, lactate, acetate, ethanol Hydrogen, lactate, acetate, ethanol, butyrate
Cellulose Cellulose
0.8 1.8
[141] [141]
Hydrogen, acetate, butyrate, lactate
Glucose
2.64
[142]
Hydrogen, lactate, acetate, ethanol Hydrogen, lactate, acetate, ethanol
Carrot pulp Carrot pulp
2.4 1.3
[143] [143]
organic household waste (70 C) using mixed cultures has not been achieved thus far. But from the engineering point of view, mixed cultures are suitable for full scale industrial application. Mixed cultures maintenance and regulation need minimal upstream processing and features low operations and maintenance cost with involvement in simpler control systems for running the entire production pathway. Nonetheless, mixed cultures require critical platforms and a better decision support system for the usage [58]. Mixed cultures of Clostridium strains likely produce hydrogen in different processing conditions including mesophilic and thermophilic ranges of temperature with a pH range of 5.0 and 6.5. Darkfermentation with mixed cultures will encourage subsequent growth of unified bacterial assemblage in the fermenter that permits association of various species to effectively regulate the growth environment and transform organic waste materials into hydrogen, in an acidic environment created by natural acids [59]. Clostridium strains produce a combination of organic acids like butyrate and propionate [60]. These organic acids act as a precursor for hydrogen production by Rhodobacter [61]. Such mixed consortia can potentially be procured from an assortment of various natural organic waste sources comprising sewage sludge [49,62], anaerobically digested sludge [43,63], acclimated sludge [64,65], compost [66,67] and soil [16,68], or native microbes found in specific type of wastes [69e71]. Despite the benefits of mixed cultures, their utilization can mask the effects of essential non-hydrogen generating
species, which could lead to failure of the process. Pretreatment techniques could minimize such an incidence of undesired microorganisms, and aid in providing conditions, which support superior hydrogen production by the selected € la € et al. examined hydrogen producing species [44]. Seppa Clostridium butyricum and E. coli for steady-state hydrogen production both individually and as mixed cultures without any reducing agents [72]. Furthermore, Yokoi et al. studied oxygen resistance and hydrogen generation by mixed strains of C. butyricum and Enterobacter aerogenes in both batch and continuous modes of operation using starch as a feedstock (T37 C, pH 5.5e6.5) [61]. Puhakka et al. recorded a shift in microbial population observed from a mixed culture from hydrogen producing Clostridium sp. (37 Ce45 C) to Thermoanaerobacterium species at (60 C) [73]. Similarly, Sargsyan et al. explored the use of waste distiller grains in biohydrogen production by mixed cultures [74]. For the mixed-culture studies, cattle manure sludge, reactor wastes and various types of soil have often been utilized as seed inoculum [56]. The impurities in CG obtained from restaurant and meat processing wastes have increased inhibition effects on pure-/ co-cultures in contrast to the impurities in CG obtained from pure substrates [75]. Application of mixed-cultures nullifies the problem of contamination associated with the use of complex CG with synergistic impacts that are beneficial over pure and co-culture system framework amongst glycerol fermentation [76]. Studies have shown that mixed culture has the greatest hydrogen production and better inoculum
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compared with mono and co-culture methods. Heat pretreatment of mixed cultures is a simple, modest and mechanically appropriate approach for hydrogen production using pure or mixed cultures [56]. In the context of pretreatment, Farghaly et al. investigated the use of surfactantsupplemented with mixed bacterial cultures to produce hydrogen from paper board mill wastewater [77]. Similarly, Ghimire et al. investigated the scale-up and utilizing biomass for producing hydrogen under mixed cultures and dark fermentations [78].
Co-cultures From an economical perspective, co-cultures can be maintained under anaerobic conditions for optimal hydrogen production thus eliminating the need for expensive reducing agents. In principle, co-cultures (obligate and facultative anaerobes) can enhance the hydrolysis of complex sugars and are less energy intensive, when compared to other hydrogen production processes. Studies have shown that pure cultures of E. coli and C. butyricum yield 1.45 mol H2/molglucose and 2.09 mol H2/molglucose, while their co-cultivation resulted in 1.65 mol H2/molglucose. Similar findings have demonstrated the cost-effectiveness of using co-cultures as they eliminate the need for expensive reducing agents while yielding higher hydrogen production. Co-cultures of Bacillus and Enterobacter sp. were also shown to enhance the hydrogen production more significantly [79]. To date, limited studies have reported hydrogen production by co-culture utilizing organic sources such as starch/glucose as substrate [80,81]. Bio hydrogen production by co-fermenting crude glycerol and apple pomace hydrolysate using E. aerogenes and C. butyricum as has been shown successful with better hydrogen efficiency, although higher substrate inhibition was observed while using cocultures [82]. The production of biohydrogen from industrial wastes using defined microbial co-culture has also been found to provide significant results compared to mixed or pure cultures [83].
Enriched mixed cultures Enriched mixed culture (utilitarian consortium) are far more favourable compared to natural microflora (sludge/mixed culture) because of its highly selective population of hydrogen producing microorganisms [84]. Using enriched mixed cultures (EMC) and its recycling on glycerol provides a reliable consortium for hydrogen production [85]. Moreover, EMC development and transfer to a hydrogen fermenter enhance the hydrogen yield and production rate. A batch study with EMC was studied by Sivagurunathan et al. that revealed an integrative strategy with the varied microbial population for enhancement of hydrogen production [84]. Sivagurunathan et al. assessed the feasibility of EMC for continuous hydrogen fermentation of galactose and found that EMC obtained from batch process doesn't help in higher hydrogen production, with a drop in the HY in batch 1.05 mol H2/molgalactose added to 0.82 mol H2/molgalactose added in CSTR's [84]. These differences were owing to variations in microflora pattern with hydrogen producing Clostridium sp. dominating under batch mode, whereas lactate producing Sporolactobacillus sp. was
predominating in the CSTR's. Consequently, a necessary elimination of non-hydrogen producing bacteria is needed for efficient hydrogen production under continuous operation.
Granule producing microorganisms Enhancement of hydrogen production performance is plausible through the microbially mediated granulation process as it provides stable microbial populations/colonization and prevents microbial washout during long term operation. There are certain groups of granule forming facultative and obligate anaerobic bacteria that are often detected in the hydrogen producing granular systems (Table 3). For example, Xing et al. stated that the addition of Ethanoligenens harbinense promotes the self-flocculation process in a CSTR and is the dominant bacterial population under non-sterile conditions with stable hydrogen production over 21 days of operation [86]. Hung et al. further demonstrated interactions between Clostridium sp. and facultative anaerobes (Klebsiella and Streptococcus sp.) in an auto-formed granular sludge in an anaerobic granular sludge bed reactor with powdered activated carbon as a biological carrier for granules development [87]. Their results showed that low HRT enhanced the granule formation, where the Clostridium sp. formed net-like structures and Streptococcus sp. were self-aggregated to form a solid granular core inside the net. Sivagurunathan et al. observed that the formation of granular sludge occurred only at a low HRT of 2e3 h when Selenomonas sp. was present along with Clostridium populations [57]. Furthermore, reduction of the HRT to 1.5 h led to the wash-out of Selenomonas sp. populations and the disappearance of granular sludge, resulting in reduced total cell concentrations, thus explaining the role of Selenomonas sp. in maintaining the granular structures.
Molecular techniques in dark fermentative hydrogen production (DFHP) An increasingly enormous number of molecular biology methods have been applied to confirm the presence of hydrogen producing microbes, since the last decade. Each method has their pros and cons (Table 4). Since the organisms used for biohydrogen production are prokaryotes, molecular techniques are specifically used to target the 16S rRNA gene and functional specific genes such as DNA gyrase. Traditional hybridization techniques or modern PCR based techniques are widely used for altering gene to enhance the biohydrogen producing organisms.
Conventional techniques (cloning, T-RFLP) Terminal restriction fragment length polymorphism (T-RFLP) is a PCR based technique initially used as a semi-quantitative molecular fingerprint technique that is robust, cost effective and reproducible. T-RFLP has been used to identify the abundant population of bacteria detected in the bioreactors [88,89]. This technique relies on the position of a specific and unaltered site nearest to the labelled end of an amplified gene. T-RFLP targets intensification of the 16S rRNA gene utilizing
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Table 3 e Granules forming bacterial populations observed in hydrogen producing systems. Microorganism
Fermentation products
Streptococcus sp.
Hydrogen, formate, acetate, lactate
Selenomonas sp.
Hydrogen, lactate, acetate, propionate
Prevotellaceae sp.
H2S, acetic, butyric and isovaleric acids
Sporolactobacillaceae sp.
Hydrogen, lactate, acetate, butyrate
fluorescently labelled preliminary base pairs at 50 ends (Fig. 1). The technique is extremely sensitive with high reproducibility. The primary advantage of T-RFLP is the capacity to identify even the uncommon population of a specimen. However, the strategy is costly and tedious in comparison to DGGE. The exploratory section size may not be indistinguishable to the hypothetical length and can be considered a fault with the T-RFLP technique. Moreover, T-RFLP is also influenced by incomplete digestion and development of pseudo terminal-restriction fragments that lead to false
Roles of bacteria and mechanism
References
Facultative anaerobic hydrogen producing bacteria, often detected in hydrogen producing granular reactors, act as a seed for self-flocculated granule formation Self-flocculating and granule forming bacteria that enhance biomass productivity and help to maintain the structure of the granules Hydrogen sulphide producing bacteria with biofilm forming ability Lactate producing bacteria with granule forming ability
[98,135,144]
[57,145e147]
[148e150] [63,149,151]
positive results or over estimation of the biodiversity [90]. TRFLP investigation on microbial communities in bioreactors has shown Clostridium and Bacillus sp. as dominant populations. T-RFLP was reliable for use in fast analysis of variation in mixed cultures [91]. T-RFLP helps in finding mesophiles that produces hydrogen and their bacterial composition [28]. Castello et al. reported that sequences from fermentative microorganisms (Bifidobacterium, Lactobacillus, Clostridium, and Streptococcus) were correlated with the T-RFLP peaks using in silico analysis [89].
Table 4 e Comparison of different microbial characterization methods and their advantages and disadvantages. Microbial characterization technique
Advantages
DGGE T-RFLP
Taxonomic identification, large database Community profiling, taxonomic identification, high sensitivity, high reproducibility
qPCR
Quantify microbial community at lower concentration, fast simultaneous amplification and quantification of target DNA, microbial composition and dynamics can both be analyzed Fast and simple, direct observation of uncultured microbes, can be used to measure metabolic activity
FISH
NGS
Fast, simple, inexpensive, scalable, high yield
PCR-CESSP
The high sensitivity of multicolour (up to five different fluorochromes) laser-induced detection. Identification of unknown microorganisms from environmental samples by comparison of the mobility of the unknown DNA to that of known sequences. Suitable for population monitoring and identification of microorganisms. High reproducibility between runs; internal standard eliminates run-to-run variability. Automation allows easy and rapid processing of 96 samples at one time. Relative abundance of detected sequences (microorganisms) is determined by measuring the fluorescence of each peak relative to the sum of the fluorescence. Illumina MiSeq: Moderate cost of instrument and runs, low cost per Mb for a small platform, fastest Illumina run times and longest Illumina read lengths High throughput analysis: Completely identifies all microbial population
Illumina sequencing
454 Pyrosequencing
Disadvantages
Cumbersome and time-consuming Expensive, time-consuming, variations in experimental and theoretical size of fragments, incomplete digestion, the formation of pseudo TRFs. PCR biases, community profiles of several species laborious and expensive
In mixed culture background, autofluorescence effects FISH images, optimization of hybridization condition. Only useful on single stranded DNA High rate of sequencing errors Low yield of high-quality sequences CE-SSCP analysis suffers from the same drawbacks as all PCR-based community analysis techniques. Limited to DNA fragments of 200e400 bp, which limits phylogenetic characterization. Not quantitative, only semi-quantitative. Two different sequences may have the same electrophoretic mobility
Relatively few reads and higher cost per Mb compared to HiSeq More time e pyrophosphate bond formation and breakage
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Fig. 1 e A schematic representation of Terminal restriction fragment length polymorphism, T-RFLP.
Fig. 2 e A schematic representation of Denaturing Gradient Gel Electrophoresis (DGGE).
Advanced molecular techniques Denaturing Gradient Gel Electrophoresis (DGGE) Numerous microbes contribute to bio-hydrogen production in case of mixed cultures or bacterial consortia. DGGE acts as a quick screening, identification and enumeration tool for tracking these beneficial microorganisms (Fig. 2). This electrophoresis technique aids in the rapid identification of any significant transitions in the bacterial composition in response to changes and variations in growth and culture conditions during hydrogen production. DGGE has been applied in the analysis of variations in the microbial profile regarding distinct pre-treatment processes
and their impact on hydrogen creation [92]. In one such report, variations in the microbial profile during distinct pretreatment processes, their impact on hydrogen formation and the impact of substrate burden on microbial population were examined utilizing DGGE [93]. Hafez et al. utilized DGGE to demonstrate the variations in the microbial profile at varying organic loading rates (OLR) [94]. In another study, Carillo et al. utilized DGGE to investigate Buffalo fertilizer eubacteria responsible for hydrogen generation and their relationship with pH, temperature and various pretreatments [95]. Similarly, Xing et al. monitored microbial group structure and progression in a biohydrogen production reactor using DGGE [96].
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Polymerase chain reaction coupled with Denaturing gradient gel electrophoresis (PCR-DGGE) has been utilized as an exciting device for investigation of the hydrogen producing bacterial community structure [97]. However, a setback of this strategy is that it only allows identification of the major members of the population. PCR-DGGE demonstrated that Clostridium sp. was the prevalent microorganisms [98]. PCR reamplification and DGGE investigations were conducted to ensure the purity of reclaimed DNA in several studies. DGGE of the 16S rRNA genes for the identification of the microbes (bacteria) has revealed improvement in bacterial community structure in Peptone Yeast Extract Glucose Broth (PYG) compared to the basal medium. C. butyricum, C. saccharobutylicum, C. tertium and C. perfringens were found to be present in all the samples, regardless of the medium utilized, which shows the abundance of actual hydrogen producers in the enriched mixed cultures [62].
Quantitative Real-Time PCR (q-PCR) PCR based strategies are of significant interest because of their ability to distinguish DNAs of different organisms even at trace levels. Since, the normal PCR method is qualitative and time consuming, an improvised quantitative method has been introduced called the quantitative real-time PCR (qRTPCR; Fig. 3) or simply, real-time PCR that identifies and evaluates the microbial community in real time [99]. Numerous methods including metagenomics, phylogenetic microarrays, DNA fingerprinting strategies and qPCR have been used for characterization of microbiota that aids developing characteristic taxonomic and quantitative data describing the microbiota [100]. The qPCR has been utilized since the 1990s for its quantitative accuracy, high specificity, immaculate performance, wide element range, great reproducibility, and moderately low costs [101]. Overall, qPCR performs with in a low to medium throughput, since most qPCR stages have an ability organization of 96 or 384. Most qPCR investigations have been directed on investigations on gut microbiota normally with the help of universal bacterial
9
primers [102]. Wang et al. evaluated the efficacy of dark hydrogen fermentation by indigenous C. butyricum based on the expression analysis of the hydrogenase gene using RTPCR [25]. The three strains C. butyricum, C. felsineum and C. pasteurianum were monitored throughout the fermentation process for which the recA and gyrA marker genes are used as internal controls [103].
Fluorescence in situ Hybridization (FISH) FISH is a nucleic acid based hybridization technique that employs group or species specific fluorescent labelled oligoprobes that aids in both identification and differentiation of microbial species. FISH offers an excellent way to distinguish microbes without the need for DNA extraction and PCR, consequently, it overcomes the problems associated with PCR based methods such as DGGE, RFLP, RISA, cloning and sequencing (Fig. 4). DeLong et al. first reported the use of fluorescent tagged rRNA for phylogenetic studies. The system is predominantly in view of the quickly expanding data on bacterial smaller subunit (16S rRNA) rRNA arrangements, which has been massively accumulated for the investigation of microbial phylogeny [104]. To a lesser degree, tests have been similarly built that encompasses larger subunit rRNA (23S rRNA) [105]. The labelled probes used in FISH are usually short DNA sequences with target sequence specificity, enabling identification of organisms at any taxonomic level. Hence, FISH can be potentially utilized to identify microbes during hydrogen production. FISH based on 16S rRNA for Thermoanaerobacterium, Caldicellulosiruptor and Thermoanaerobacterium thermosaccharolyticum were conducted to evaluate the spatial distribution of hydrogen producing microbes in the secretions and granules from anaerobic reactors [106]. PCR-RFLP and FISH were also utilized to distinguish Megasphaera elsdenii as a hydrogen producer [107]. Similarly, FISH and SEM (scanning electron microscopy) were used to identify predominant classes of HPB as E. aerogenes ATCC13048 and Firmicutes including Clostridium, Bacillus and Dialister sp. [108].
Fig. 3 e A schematic representation of Quantitative Real-Time PCR (q-PCR). Please cite this article in press as: Kumar G, et al., Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/ j.ijhydene.2018.09.040
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Fig. 4 e A schematic representation of Fluorescence in Situ Hybridization (FISH).
Most advanced molecular techniques Next Generation Sequencing (NGS) In a typical hydrogen reactor, minor individuals from the population may also play a critical role in hydrogen production; accordingly, strategies that enable identification of the entire population are in need and one such strategy is to use the next-generation sequencing techniques. NGS is a higthroughout method that has wide sequence coverage without compromising the quality of the sequencing. NGS could be used to study the microbial diversity of the given environment either by focusing on their ribosomal RNA sequences alone [109e111] or by sequencing their whole genomes and comparing the genomes [112] for relatedness or evolutionary differences. Microbial communities unravelled by the high-throughput sequencing-based metagenomic evaluations showed a predominance of Clostridium sp. in the reactor environment, as the reactions of the variables explored were enhanced towards the highest hydrogen producing potential. High-throughput next generation metagenomic analyses are increasingly being used to better understand the key microbiological elements driving dark fermentative biohydrogen production. NGS based analyses are useful in identifying the minor components of the complex microbial communities, which are usually left out while analysing through standard or traditional microbiological methods that majorly concentrate on the dominant members alone. NGS is a general term that includes modern sequencing technologies like
Illumina (Solexa) sequencing Roche 454 sequencing Ion torrent: Proton/PGM sequencing SOLiD sequencing
Each of these techniques differ from each other in their working modalities yet delivering more or less the same outputs. Sequencing of a huge amount of DNA from an environmental sample is plausible with NGS enabling it to be completed in a day [113,114]. Since, NGS are high-throughput techniques, they generate humongous amount of sequence data that requires specialized bioinformatics tools and experts to decode the information it contained [113]. Nevertheless, the initiation and development of NGS have provided a great platform for a cost effective and a comprehensive characterization of complex microbial consortia compositions over conventional molecular techniques such as DGGE, RFLP and cloning followed by Sanger sequencing [115,116].
Capillary Electrophoresis Single-Strand Conformation Polymorphism (CE-SSCP) Capillary electrophoresis is a method that revolutionized many other techniques because of its uncomplicated workflow and high-throughput analysis. SSCP is a traditional method used to differentiate closely related organisms whose sequence details are unknown. CE-SSCP method is a valuable tool for (i) producing molecular fingerprints of 16S rRNA and/ or functional genes and (ii) comparing the shift in the bacterial population structure and diversity relative to the progression of ecological parameters [117,118]. The functional CE-SSCP strategy aided in effective discrimination between Clostridium strains and offered dependable and high throughput analysis of hydA genes in mixed-culture bioprocesses. me neur et al. showed the capability of utilizing the hydA Que based CE-SSCP to observe the structure and elements of hydrogen producing Clostridial populations, which play a key role in hydrogen production in bioreactors [119]. The study reported that components of functional gene based CE-SSCP profiles from pure strains are helpful for concentrating on
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the structure of functional populations [120]. In another study, me neur et al. evaluated the interference of fermentative Que hydrogen production by lignocellulose-derived compounds in mixed cultures, utilizing CE-SSCP strategy [121] (Fig. 5). Additionally, information on the hydrogen producers and their diversity and structure in various specimens of dark fermentation bioreactors have been demonstrated through hydA CESSCP strategy. Furthermore, it is increasingly being used to monitor environmental stress response i.e. impact of environmental parameters on microbial diversity and their structures [119,122]. It is interesting to note that every peak of the CE-SSCP profile corresponds to the gene fragment of a unique 16S rRNA gene sequence, and thus represents specific species or strain within the microbial community thus aiding their identification. The relative abundance of the corresponding microbial species in the community is gauged by the region under the peak [95].
Illumina sequencing Sequencing by synthesis (SBS) through Illumina sequencing technology, is by far the best and most often used nextgeneration sequencing (NGS) technology worldwide. This method has a wide variety of uses that deals with any related information on the genome, transcriptome, or epi-genome of diverse organisms. Zillions of genomes have been sequenced and are undergoing this process that aids in the rapid examination of DNA sequences to improve understanding of health, disease and diagnostics making it the least expensive than other accessible sequencing techniques. In addition to that, the state of the art microarray technologies (e.g., Illumina sequencing innovation) permit high-throughput performance and adequate precision [123]. The sequencing of the hyper variable regions of the 16S ribosomal rRNA gene through Illumina MiSeq sequencing was employed and proven handy to uncover the microbial diversity of the fermentation reactions [124e126]. Table 4 provides information about the pros and cons of various methods mentioned above.
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16S rRNA 454 pyrosequencing Next generation DNA sequencing investigations including 454-pyrosequencing have been utilized to break down the bacterial diversity of mixed cultures because they provide significantly more data than conventional techniques [127,128]. Pyrosequencing involves DNA synthesis monitoring in real time and is based on the principle of DNA polymerization tracked by measuring pyrophosphate production, which can be detected by light. In this process, the DNA polymerase synthesizes DNA by extending the 30 end of the initial strand using the information encoded in the template strand [128]. In pyrosequencing, the nucleotides are identified based on the pyrophosphates release upon addition of each base. Pyrophosphate moiety of each nucleotide is labelled with an unique fluorophore and the addition of the labelled nucleotides in the growing chain of nucleic acid governed by the polymerase, releases the labelled pyrophosphate. The released pyrophosphate will be detected by the laser detectors and the sequence of the nucleotides are deduced from that [127]. Improvements in technologies such as 454 pyrosequencing (Roche), helps in handling more sequences in a short time. This method is used to identify microbes in multi-disciplinary conditions. There are very few studies on microbial population in hydrogen production through pyrosequencing and thus have a lot of scope for future research in microbiomes for hydrogen production through high throughput pyrosequencing. It provides the complete picture of the bacterial microbiome with detailed dominant microbiota. Many microbial communities have been identified in various types of batch systems [129] that help in the process of fermentation and can be either dominant or mere present microflora. Although Thermoanaerobacterium and Thermohydrogenium bacterial populations are dominating under mesophilic and thermophilic conditions, there are many other bacterial members that are present, (belonging to both hydrogen producing and non-hydrogen producing communities). Abundance of various sub-dominant bacterial
Fig. 5 e A schematic representation of Capillary Electrophoresis Single-Strand Conformation Polymorphism (CE-SSCP). Please cite this article in press as: Kumar G, et al., Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/ j.ijhydene.2018.09.040
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Table 5 e Major HPBs and molecular techniques reported in the literature. Microbial source
Feedstock
Medium
AMC
Beverage wastewater
PYG
Cow dung compost, sewage sludge Activated sludge Activated sludge
Beverage wastewater
PYG
Mixed microflora (EMC)
Beverage industrial wastewater (BW) Synthetic wastewater
Basal endo medium
Anaerobic sludge
Maize silage LB De-oiled Jatropha waste Nutrient solution
nr
Anaerobic digested sludge Pure strain
Glucose
Endo medium
Glucose
MDt medium
Pure strain Pure strain Seed sludge
nr Pure glycerol Sugar beet molasses
Modified PYG Nutrient broth M9 medium
Granular sludge
Tequila vinasses
Hydrogen producing strain Anaerobic sludge, cassava (Cas) and cæcotrophs (Cæ) Anaerobic digested sludge
Sucrose
Mineral nutrient solution nr
Anaerobic sludge Anaerobic sludge Digested sludge
Glucose
Glucose-based medium
Fructose, glucose, sucrose, maltose, cellobiose, maltotriose Glucose Glucose Glucose
Culture medium
Major HPB reported
Clostridium sp., Enterobacter sp. C. buytricum, C. tertium, C. perfringens E. cloacae C. thermopalmarium, C. buytricum, B. ginsengihumi, B. coagulans Clostridium sp.
Molecular technique employed
References
PCR-DGGE
[84]
PCR-DGGE
[57]
T-RFLP, Real-Time PCR PCR-DGGE
[28] [93]
PCR-DGGE
[96]
Clostridium sp., DGGE Ethanologen bacterium sp. nr PCReDGGE
[97] [99]
C. butyricum, C. felsineum and C. pasteurianum Megasphaera sp. E. aerogenes ATCC 13048 Leuconostocaeae and Enterobacteriaceae Clostridium strains
FISH and qPCR
[103]
PCR-RFLP and FISH FISH PCReDGGE
[107] [108] [128]
DGGE
[127]
C. sporogenes
CE-SSCP
[119]
C. pasteurianum
CE-SSCP
[92]
CE-SSCP
[150]
Illumina MiSeq sequencing Illumina MiSeq sequencing Illumina MiSeq sequencing
[152] [153] [154]
C. acetobutylicum, C. cellulolyticum, C. kluyveri, C., C. sporogenes Synthetic wastewater Clostridium sp. nr Clostridium ljungdahlii Enriched Clostridium perfringens
communities in terms of OTUs (operational taxonomic units) that is considered as a surrogate of the bacterial abundance in the present genetic identification approaches [129]. In this context, significant progress has been made for the establishment of efficient and high throughput screening and characterization tools in molecular biology (Table 5). Apart from the different molecular biology or genetic engineering studies used to enhance biohydrogen production. Nowadays researchers are also improving biohydrogen productivity by involving nanotechnology using nanoparticles. Already nanomaterials have been used in producing electrical energy in some studies [6,8,130,131]. But as future perspective researchers are suggesting metallic or non-metallic nanoparticles like nitrogen doped graphitic carbon sheets and carbon encapsulated RuO2 nanorods for gas fuel production especially hydrogen [132]. Therefore, finding different techniques to amplify the production rate, reduce production cost and increase yield of production will be the future of biohydrogen.
hydrogen producing bacterial communities are highly essential. As the cleanest and powerful fuel, a biohydrogen economy ensures limitless energy for humanity and future energy security. Moreover, hydrogen as a fuel is apparently having zero environmental externalities thus paving the path for a sustainable bioenergy future. The present communication attempts to understand issues in discerning biohydrogen future through rapid dark fermentative pathways with a variety of microbial biocatalysts and numerous pre-treatment techniques applied. Additionally, the pros and cons of each molecular technique have been discussed for better understanding and improvisation. It is also envisioned that a combination of molecular techniques could be implemented to cross confirm the microbial population inside a reactor. A holistic approach is required for a clean and green hydrogen economy including developments in genetically designed microbes, advances in bioreactor design and economics, and finally, storage and distribution systems.
Conclusion
Acknowledgement
In the rapidly evolving microbial world, molecular tools for screening, enumeration and identification of potential
This work was supported by the Dongguk University Research fund of 2015.
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