Keywords

3.1 Introduction

Global demand for energy sources, depletion of the fossil fuel resources and critical worry on the greenhouse gas release pusing scientific community looking for the alternative sources of green energy which can check the environmental issues (Sudheer et al. 2010; Sudheer Pamidimarri and Reddy 2014). The global decay of the earth’s environmental health and direct accumulation of the carbon footprints relesed by usage of fossil fuels; non-carbon energy source is said to be the way-out for the global crises of energy and to avoid the production of greenhouse gases (Hansel and Lindblad 1998). The non-carbon green fuels available in the present technology are hydraulic, wind, solar energies, and hydrogen fuel. Among these hydrogen fuel can readily answer the global environmental issues and have possibility to compensate the global energy demands (Dunn 2002).

Hydrogen (H2) produced from biological sources is considered as the cleanest energy. Biological hydrogen is generated from the biological source by the process where green energy is generated by environmentally friendly way and was credited with zero emissions of pollutants. Globally at present H2 is the most promising source in the succession of fuel evolution. Hydrogen fuel is encouraged throughout the globe because of several technical, socio-economic, and environmental benefits (Das and Veziroǧlu 2001). H2 gas is considered to be safer compared to the natural gas and better than domestic natural gas and is now universally accepted as environmentally safe. Moreover, hydrogen fuel could be generated from renewable source which can defy the greenhouse effect (Kumar and Kumar 2017). Presently, H2 is produced from various sources like natural gas, heavy oils, naptha, coal, and electrolysis which in turn contribute to greenhouse emissions. Microbial cell factories, unlike the chemical or electrochemical counterparts, generate no effluents and are environmentally safe. Biological production of hydrogen catalyzed by microorganisms in an aqueous environment at the ambient temperature and atmospheric pressure is a complete green process (Lynd et al. 2009; Chaubey et al. 2013).

Globally, the major share of energy utilization is for the transportation and it occupies the share of 65%. Petroleum based fuels are the sole source of transportation fuel presently used, which is causing the local and comprehensive climate change and air congestion in the urbanized areas (Kumar and Kumar 2017). This is causing the alarming disturbance in the air quality and making the metro cities unsuitable for the living. If the same continue further, the future position of the urban areas in prospective of living standards will be deteriorated and countries need to spend the major section of economy for the health care. Hence, replacing the traditional transportation fuel (petroleum and coal based) with hydrogen fueled transportation system will improve the situation and can make the metro and urban cities more human friendly (Das and Veziroǧlu 2001; Maeda et al. 2012; Kumar and Kumar 2017).

In the present era of biotechnology, the concept of microbial fuel cell is rising since the biomass requirement of the microbial cells is more flexible and the productivity is reached near to the theoretical values. This whole cell based catalysis for the production of fuel energy is supposed to be the most efficient system which can answer the present energy crisis. Hydrogen production from the microbial fuel cell is said to be a good concept of green fuel since the hydrogen fuel combustion results in no greenhouse gases. Moreover, the energy content per mass of the hydrogen energy is 142 MJ kg−1 which is better than biofuels like bio-ethanol and biodiesel (Maeda et al. 2012). This book chapter presents the microbial hydrogen fuel cells, their significance and production mechanism, will discuss further about the different microbial sources of hydrogen production, the biomass requirement, and prospective utilization of lignocellulosic biomass or other waste biomass. A separate section is dedicated for the biotechnological approaches for the improvement of hydrogen production in E. coli. The concluding part will include the future prospective of the microbial fuel cells and possible strategies for enhancing the hydrogen production and aspects of hydrogen economy for the implementation.

3.2 Hydrogen Production Sources

Currently, hydrogen production is by three major processes; these include electrochemical, thermochemical, and biological process. Superiority of these methods is always under debate since each method is having its own credits and demerits (Stojić et al. 2003; Turner 2004). Biological or microbial based hydrogen fuel production is encouraged globally for their independence of non-renewable substrates. In this section brief account of each method and their merits and demerits will be discussed and detailed discussion is made on microbial based hydrogen fuel production.

3.2.1 Electrochemical Process

Electrochemical process is the first process to be designed for the production of hydrogen from the source of water via electrolysis. It is the simple splitting of the water in to corresponding components by using the electrical energy (Stojić et al. 2003). There are majorly two types of the process involved in the electrolysis; these are by alkaline electrolyzer and the polymer electrolyte membrane (PEM) electrolyzer (Marcelo and Dell’Era 2008). The efficiencies of these processes are about 56–73%. Though, the H2 considered to be green energy source, however, the greenness of the process is mainly depending on the source of electricity utilized in the process. Hence the debate of the greenness of the process is still continuing. Utilizing solar energy for conducting the electrolysis is considered to be the best way for making whole process environmentally green. Considering the renewable source of electricity (via solar or wind power) the process can be most permissive in the view of carbon footprint. However, the investment is needed for shifting towards hydrogen renewable energies. In economic stand point for the production, cost per unit is very high and is not a method of choice for the commercial production. Moreover, the investment needed for this is very high and this will be added to the production cost.

3.2.2 Thermochemical Process

Unlike the electrochemical process thermochemical process is more suitable for the bulk production and will have possibility of scale-up to the commercial level due to its higher productivity and efficiency (Ohta 1979; Freni et al. 2000; Funk 2001). There are various thermochemical methodologies used to produce H2. These include thermal dissociation, thermal pretreatment (pyrolysis and gasification), and reforming. Among these three processes, only the thermal dissociation method uses direct splitting of water into corresponding elements and produces H2 as same as in case of electrochemical process (Utgikar and Thiesen 2006). Later two methods use either hydrocarbons or organic biomass as starting material for the production of H2 (Haryanto et al. 2005; Navarro et al. 2007). Thermal pretreatment method uses carbonaceous matter, and is first converted to smaller constituents which can be used for the production of H2 in the second phase. Pyrolysis is the popular method for converting the rice husk or similar biomass into hydrogen. Gasification is similar to reforming, where it uses steam or oxygen for the conversion of carbonaceous material or biomass into gaseous product (Vasudeva et al. 1996; Marquevich et al. 2000; Demirbas 2004; Czernik et al. 2007). However, these methods are under debate since all these discussed methods rely on energy input which may not be from the source of green process. Hence, there are many efforts were made to integrate renewable energy like solar energy for the production of heat energy which can be used in the process (Fujishima et al. 2000). Moreover, the process reforming and pyrolysis process use the hydrocarbons as raw material whose sources are non-renewable; hence, long-term production technologies using renewable biomass must be developed for the sustainable production of H2.

3.2.3 Biological Process

Biological production of H2 is said to be the most prominent process since the technology involves complete green production and moreover the flexibility of starting material could be diverse based on the microbial source utilized for fermentation. The hydrogen producing microbes can be divided into two groups: photosynthetic and non-photosynthetic or fermentative hydrogen producers (Das and Veziroǧlu 2001). Both processes use renewable raw material for the biomass generation and hydrogen production. Superiority of any method is not relevant since both photosynthetic and fermentative process have own advantages and demerits. Hence the following section describes in details regarding biological H2 production.

3.3 Microbial Hydrogen Fuel Cells

In contrast with electrochemical or thermochemical hydrogen production microbial fuel cells for the H2 production is always given superiority because they are based on completely green process. Moreover, the process could be conducted in ambient condition without use of extreme temperatures and pressures. As mentioned earlier, among the photosynthetic and fermentative methods, much of the research is focused on the fermentative method because of the advantages like (1) this method does not depend on the presence of light for the H2 production, (2) its higher production rates, and (3) a variety of carbon energy sources like organic matter, low-cost carbohydrates, cellulosic, lignocellulosic, cellobiose, and other waste biomass could be used as carbon source to grow the microbial cell mass for the production of H2. In this section we will discuss both photosynthetic and fermentative methods of H2 production.

3.3.1 Photosynthetic H2 Production

Photosynthetic H2 production is carried out by various bacterial, algal, and cyanobacterial species. These microbes use diverse pathways and various machinery for the generation of cellular energy and H2 production, respectively. These photosynthetic H2 producing bacteria can be grouped majorly into two groups based on oxygen generation. Majority of algal and cyanobacterial species use photosystems for harvesting the energy, and electrons are donated by photolysis of water, importantly the O2 accept the electrons finally and these are called oxygenic photosynthetic H2 producers (Barbosa et al. 2001; Kovács et al. 2006). The other group depends on various organic acids for the electron donors and use nitrogenases for the production of H2 as a by-product during nitrogen fixation. In this section the mechanisms, advantages, technical limitations, and future prospective will be discussed in detail.

3.3.1.1 Oxygenic Photosynthetic H2 Production

Photosynthesis is the basic functional aspect of plants, algae, and cyanobacteria. In the process of oxygenic photosynthesis H2O is oxidized, generate O2 and the electrons will be used by photosystems for the reduction of NADP. The protons released during photolysis combined with the electrons passed to membrane, upon electron transport by reducing NADPH or ferredoxin will be used for the production of H2 by hydrogenases in many cyanobacteria and algae (Miyake et al. 1999) [Fig. 3.1]. In general, the photosynthetic system needs four electrons for a pair of electrons sequester from H2O and reduce NADP or to generate couple of H2 molecules. The major advantage of this process is, it utilizes the light energy for the splitting of H2O to O2 and H2 (Dutta et al. 2005; Lee et al. 2010a). This oxygen generating H2 production system is the only green energy produced from the renewable light energy without emission of CO2 and also it has a great importance of fixing the CO2 and also generates the algal biomass which could be used for many biotechnological and fermentative applications (Miyake et al. 1999; Dutta et al. 2005). Although oxygenic photosynthetic H2 production looks very promising, the major challenge in commercial implementation is especially in the context of engineering limitations for designing a suitable bioreactor for scale up to the level of industrial production. Since, the system needs the illumination of light, engineering a closed system with translucent glass reactor for the bulk production is necessary. Hence, there should be an innovative reactor model need to be designed for the bulk production and scale-up.

Fig. 3.1
figure 1

Photosynthetic oxygenic H2 production by microalgae and cyanobacteria. RI reactive intermediate, PS-I Photosystem 1, PSII Photosystem II, PQ Plastoquinone, Hydn Hydrogenase

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3.3.1.2 Non-oxygenic Photosynthetic H2 Production

Though, the oxygenic photosynthetic hydrogen production system is under major discussion; a separate group of bacterial species called non-oxygenic photosynthetic H2 producers comes under the group photosynthetic purple non-sulfur bacteria are also important group worth discussing in this section. The genera Rhodobacter, Rhodopseudomonas, and Rhodospirillum are the major representatives of photosynthetic purple bacteria that generate H2 without generating O2 (Lee et al. 2010a). These are the alternative photosynthetic H2 producers in place of oxygenic photosynthetic H2 producers. These utilize light as the energy source and organic acids most commonly carboxylic acids as electron donors. Since H2O does not act as the electron donor, hence no oxygen is released. The major benefit of this system is, in case of oxygenic photosynthetic H2 production, the sensitivity of hydrogenases towards the presence of O2 in high concentration will inhibit or lower in several folds the production efficiency. These non-oxygenic H2 producers do not generate O2 since this system uses nitrogenase in place of hydrogenases to generate H2 (Masepohl et al. 2002). This system can effectively bypass the issue of hydrogenase sensitivity to the O2 and can integrate with dark fermentation using organic acid containing effluents. This integrated system will be very valuable in harvesting energy from light; in addition it will help in effluent treatment and producing valuable green energy. The stoichiometry of moles of H2 released during the fixation of mole of N2 differs vastly. It ranges from 1 mol of H2 produced while fixing 1 mol of N2 by common Mo-containing nitrogenase to 9 mol of H2will be produced while fixing a mole of N2 by highly oxygen sensitive Fe-containing nitrogenase. Despite the unfavorable hydrogen production by nitrogen fixation, which may not be economically valuable; however, acceptable amount of H2 production is possible if an efficient reactor system is developed based on the utilization of waste organic effluent. This could harvest natural light can bring an economically feasible system for H2 production while treating effluent (Harwood 2008).

3.3.2 Hydrogen Producing Machinery (Hydrogenases/Nitrogenases) in Photosynthetic hydrogen Production

The most common hydrogenases are Fe-Fe hydrogenases prominently present in most of the bacteria and eukaryote and followed by Ni-Fe hydrogenases present generally in Achaea and some species of bacteria. Among these Fe-Fe found to be more sensitive to oxygen compared to Ni-Fe hydrogenases. Fe-Fe hydrogenases are highly sensitive to oxygen and undergo denaturation even under trace concentrations of O2 in the cell. Ni-Fe hydrogenases found to be more stable in the presence of O2; in few cases up to minutes of exposure these remain stable and active (Stripp et al. 2009). Hence, Ni-Fe hydrogenase containing microbial source, in this case H2 production in micro-oxygenic conditions is more preferable than Fe-Fe hydrogenases. Moreover, unlike Fe-Fe hydrogenases, Ni-Fe hydrogenases upon long time exposure to O2 will get inactivate reversible rather than irreversible manner, hence, H2 production can be revived by removal of O2.However, the Fe-Fe hydrogenases have advantage of high rate of H2 production compared to the Ni-Fe hydrogenases (Ghirardi et al. 2007). In case of scale-up production in industrial scale, the hydrogenases with O2 stability will have better advantage, Ni-Fe hydrogenases are more preferred. These hydrogenases are taken as subject of studies in the aspects of molecular improvement and could be selected for the future protein engineering studies. The most promising virtue of enhancing the productivity is heterologous expression of more oxygen tolerant hydrogenases in efficient microbial system for the H2 production. Introducing gene cluster of tolerant hydrogenase gene cluster into target organism can be beneficial system for enhancing H2 production. However, expression of active hydrogenases is very difficult since the maturation of the hydrogenase apparatus to involve multiple steps to produce active protein. Hence along with hydrogenase gene cluster, the maturation proteins also need to express in the heterologous system. Few studies reported in this regard (Maeda et al. 2008; Vardar-Schara et al. 2008); however, the successful bench scale studies need to be scale up to the industrial level for the real economic success. The reactor engineering is the major part of research to be concentrated for making these lab scale studies to get commercial success.

In evolution, purple bacteria generally produce H2via nitrogen fixation; hence, the hydrogenases are replaced with nitrogenases and H2 produced as by-product during nitrogen fixation. Nitrogenases catalyze high energy implicated, electron intensive N2–fixation and there is no oxygen involvement in this process. Like in case of hydrogenases, nitrogenases are also oxygen sensitive and need to be protected from oxygen for their normal functions. Majorly two types of nitrogenases understood and they are Mo-containing nitrogenases and Fe-containing nitrogenases. In virtue of productivity Fe-containing nitrogenases produce high stoichiometric (9 mol) H2production of per 1 mol of N2 fixation. In this regard, Mo-containing nitrogen fixation found to be more energy intensive (use 16ATP) for the production of 1 mol of H2 (Harwood 2008). Unlike in case of photo-chemical H2 production, where the electron donor is by photolysis of water; purple bacteria needs organic acid for the electron to be provided to the microorganisms. Hence, the economic feasibility is under debate unless the carbon source is derived from the waste biomass or from organic effluent. So, key challenge here is to integrate the waste biomass and/or effluent carbon source with light harvesting bioreactor for efficient and economically viable hydrogen production by purple bacteria.

3.4 Fermentative Hydrogen Production

H2 production via fermentation which does not need any light energy, more specifically it is also called as dark fermentation. The hydrogen is produced in the dark fermentation by taking H2 as electron sink and is possible via anaerobic fermentation. These microbes are divided into two major groups; (1) Obligate anaerobe H2 producers and (2) facultative anaerobe H2 producers. The obligate anaerobes are strict anaerobes that will harvest the electron from pyruvate oxidation, then use these electrons for the oxidation of ferredoxin (Fd), further these electrons travel to the hydrogenases where H2 will be produced. The best examples of this category are Clostridium, Ethanoligenens, and Desulfovibrio. The second group is facultative anaerobes which produce H2 via formate oxidation. In this process formate is electron donor and produces hydrogen through formate hydrogen lyase. The major group of microbes fall under this system are Enterobacter, Citrobacter, Klebsiella, Escherichia coli, and Bacillus species (Brosseau and Zajic 1982; Kapdan and Kargi 2006). The dark fermentation takes up a pair of electrons and the ultimate sink of the electron is not always H2. Only a part of electrons will be parted to produce H2. In many cases only 17% of electrons are ended up in producing H2 and other will be accepted by other organic side products. The best example is, up on glucose fermentation by E. coli only the theoretical yields of H2 are 2 mol per 1 mol of glucose and many other organic products act as electron sinks and will be accumulated in the culture medium. Ethanol and lactic acid are popular among those. To push maximum metabolic flux towards the H2 production, many researchers made efforts in metabolic engineering and successfully made recombinant E. coli strain to make the H2 production near to theoretical yields. Moreover, many organisms have hydrogenases which also conduct reversible reaction which utilize H2 for the electron generation and utilize the protons for the reduction of co-factors (Hallenbeck 2012). Hence, the gene product needs to be removed in the cell via gene knockout for stabilizing the produced H2. There are prominent studies conducted in this aspect and will be discussed in the preceding section in detail.

3.4.1 H2 Production by Microbes and Productivity

Hydrogen energy by dark fermentation was studied from past couple of decades. However, the research was more confined to the laboratory. There are very limited studies promoted up to pilot scale level. Though the technologies demonstrated in the laboratory, the major success in scale-up will depend on the efficient bioreactor engineering. Many times though successful hydrogen is generated through the fermentation, instability to maintain the produced hydrogen is also a major issue since the microbial hydrogenases are equipped with reversible reaction to take up the H2 back and release protons for reducing the co-factor. In nature dark fermentation occurs in a larger quantity utilizing the organic matter releasing H2 in the environment by various processes. This process is called anaerobic digestion (Antonopoulou et al. 2008; Ren et al. 2011). During this process hydrogen is produced as a by-product; however, the produced product will be immediately utilized by other microbes producing methane and CO2 as an end product. In this process many microbial communities are involved, namely hydrolyzers, acetogens, facultative anaerobic H2 producers combined with methanogens and Archaea bacterial communities (Tapia-Venegas et al. 2015). Though the synthetic anaerobic digestion systems are reported for H2 production by many researchers, these processes will be discussed in the later part of this section.

Pure cultures are always advantageous for study and implication in any microbial based fermentation system because of their consistent results, and easy for the storage and reproduction of the process. Pure cultures are significant in the aspect of metabolic control, easy for the establishing optimized conditions, also suitable for the molecular manipulations for enhancing the H2 production by diversion of metabolic flux towards H2 production either by addition of heterologous genes or knockout of the unwanted genes in the genome. In a dark fermentation process by a pure culture, the possible complete oxidation of glucose can result up to 12 molecules of hydrogen. However, this is true when only complete energy is released as H2 gas. In dark fermentation the H2 production in any microorganism is only a by-product during production of fermentation products like ethanol, acetate, formate, or butanol, etc. In this dark fermentation the maximum yields of H2 production can reach to 4 mole of H2 from any hexose sugar. Moreover, sugar as a carbon source will be utilized for the biomass generation. Hence, even if theoretical stoichiometry is followed, still the H2 productivity using glucose will not be economically feasible compared to other commercial system through which H2 is generated presently. There should be a cheap and/or waste biomass should be implied to make the technology economic then it can compete with present technologies (Kim et al. 2006a; Ghimire et al. 2015) (Table 3.1).

Table 3.1 Yields of H2 production using dark fermentation process with different carbon sources and different type of cultures (adopted from Łukajtis et al. 2018)
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3.4.2 Metabolic Pathway of H2 Production in Microbial Cell

The simple hexose sugar glucose is a basic sugar used as carbon source by microbes. The microbes follow majorly two routes for the production of H2. As mentioned earlier, H2 is the by-product of dark fermentation and the final fermentation product is organic acids like acetic acid, butyric acid, lactic acid or alcohol like ethanol or butanol [Fig. 3.2]. In majority of microbes the glucose degradation leads to the pyruvate production via basic pathway of glycolysis. It results in the production of cellular energy, i.e., ATP and reduction of NAD to form NADH. This pyruvate now either converted in to acetyl-CoA and CO2 or acetyl-CoA and formate. In the first case, the reduce ferredoxin molecule will be oxidized to produce H2 by pyruvate ferredoxin oxidoreductase (PFOR). In the later situation, the formate was converted to H2 and CO2 by formate hydrogen lyase (FHL) system and whole pathway is called pyruvate formate lyase (PFL) pathway (Cai et al. 2011; Hallenbeck et al. 2012). The most popular organisms follow these pathways are Clostridium sp., being an obligate anaerobe follow the former one and E. coli as a facultative anaerobe will follow the later pathway produce H2 from formate using FHL system. The productivity of these two pathways differs significantly. The production of H2 by facultative anaerobes using FHL system depends on formate dependent [Fe-Fe] hydrogenases in most cases will not use NADH produced during glycolysis; hence, various products (ethanol or lactate) will be formed upon oxidizing the NAD. Hence, the final product of this pathway is only 2 moles of H2 for 1 mole of glucose utilized. Unlike FHL system which follows PFL pathway, in case of PFOR pathway hydrogen production results by oxidation of reduced ferredoxin (Fdred) with the help of ferredoxin dependent [FeFe] hydrogenase. Moreover, two more H2 can also be generated by oxidation of NAPH with the help of NADH dependent [Fe-Fe] hydrogenase or NADH-Fdred dependent [Fe-Fe] hydrogenase. Hence, here the productivity can be 2–4 mole of hydrogen from 1 mol of glucose. This shows the potentiality of the POFL pathway in efficient production of hydrogen (Tapia-Venegas et al. 2015).

Fig. 3.2
figure 2

Various pathways used by different microbial species for the production of hydrogen from basic hexose sugar (Glucose)

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3.4.3 Dark Fermentation: An Economic Prospective of H2 Production

Using simple sugars like glucose, sucrose, and lactose are generally studied in the lab scale for understanding the efficiency and stoichiometry of H2 production. The majority reports demonstrated in the lab scale utilized these model sugars. These sugars are readily acceptable for many microbes and their utilization in metabolic pathway is well known, and manipulating the condition for the better productivity is convenient. However, in economic point of view, the production cost of H2 using these sugars as carbon source cannot compete the present commercial H2 production cost. Hence, the technology needs to be developed to replace these costly model sugars with low-cost renewable carbon sources. Utilization of lignocellulosic biomass, crude glycerol generated during biodiesel production, industrial waste water containing different organic acids which can directly enter into the metabolic pathway for the production of H2, waste biomass having high content of biodegradable sugars looks very promising and many researchers conducted valuable studies utilizing these low cost or waste carbon source for the production of H2. Dark fermentation found to be very promising concept of H2 production by utilizing the waste biomass for the H2 production from industrial waste biomass [Table 3.2] (Łukajtis et al. 2018; Toledo-Alarcón et al. 2018).

Table 3.2 Hydrogen production achieved from wastewater by different industries (Adopted from Tapia-Venegas et al. 2015)
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Theoretically, dark fermentation is capable of producing the biological hydrogen from any waste biomass. In the literature, the reports are seen where the hydrogen was derived via biological means by dark fermentation utilizing renewable waste carbon source derived from agriculture, food industry, dairy whey, distillery industry, breviary, pulp and paper industry. Those waste biomasses are rich in starch, cellulose, and lignocelluloses which can be utilized as carbon source in anaerobic fermentation or by dark fermentation via mixed culture anaerobic digestion. The theoretical yield of the H2 production in dark fermentation depends on the ultimate electron acceptor during the anaerobic fermentation. As shown in the Eqs. (3.1) and (3.2) theoretical yields of the H2 production depends on the type of fermentation carried by the microorganism producing H2.

$$ {\mathrm{C}}_6{\mathrm{H}}_{12}{\mathrm{O}}_6+{6\mathrm{H}}_2\mathrm{O}\to {2\mathrm{CO}}_2+{2\mathrm{CH}}_3\mathrm{COOH}+{4\mathrm{H}}_2 $$
(3.1)
$$ {\mathrm{C}}_6{\mathrm{H}}_{12}{\mathrm{O}}_6+{6\mathrm{H}}_2\mathrm{O}\to {2\mathrm{CO}}_2+{\mathrm{C}\mathrm{H}}_3{\mathrm{C}\mathrm{H}}_2{\mathrm{C}\mathrm{H}}_2\mathrm{CO}\mathrm{OH}+{2\mathrm{H}}_2 $$
(3.2)

Though the theoretical yields are either 4 or 2 moles of H2 from the 1 mol of glucose, the final fermentation yields is always lower than the theoretical yields since the accumulation of different organic acids accumulated as electron acceptors. Moreover, the carbon sources are also used to build up the microbial cell biomass generation. With respective to the results obtained during the fermentation the experimental yields of the H2 in anaerobic fermentation vary from 1 to 1.5 mole. In economic prospective the conversion of 60 to 80% biomass energy to H2 said to be a cost-effective process. Possible use of organic acids accumulated during the fermentation for other process could decrease the cost of the production. A number of factors influence in the yields of the H2 production and in this section few of the important factors were discussed (Levin et al. 2006; Hawkes et al. 2007).

3.4.3.1 Substrates for the Dark Fermentation

The carbohydrates are the major source for the microbes to use for their metabolism and produce H2 in dark fermentation. Simple monosaccharides such as glucose, xylose, ribose, and disaccharides such as sucrose and lactose are the sugars readily utilized by most of the microbes and produce H2. In the reports shows that the highest yield of 6 mole H2 was obtained by utilizing mole of sucrose [83], in case of lactose up to 3 moles/mole of lactose. However, simple carbohydrates are not suitable carbon source in economic point of view because of their cost. Hence, use of these simple sugars makes unprofitable in industrial scale. Continuous and profitable production of the H2 needs the use of renewable and non-edible sugars. Lignocelluloses or starch polymer derived from the various agriculture and food waste are the good source of alternative to simple sugars and also they act as renewable carbon source for the industry [Table 3.3] (Logan et al. 2002; Hawkes et al. 2007). The major hindrance in utilizing lignocelluloses is, in many instances these carbon sources are not suitable to use directly for dark fermentation due to their polymer nature and slow microbial degradation process. The yields are also very slow which adds the operational cost for the production of H2 (Hallenbeck et al. 2012). The researchers come with the idea of pretreatment using chemically or biological means. In case of biological pretreatment, the biomass is subjected to pretreatment with many fungal species that will release the simple sugars like xylose and ribose which could be easily integrated in to metabolism by microbial species for the H2 production. The use of other waste carbon sources like organic waste derivatives, cheese whey, milk waste, crude glycerol obtained as by-product during biodiesel production could be best alternatives for the direct use in dark fermentation for the H2 production. The glycerol after a simple purification by neutralizing with the mild acid followed by heating and filtration will result in the purified form which is devoid of contaminants derived during biodiesel preparation (Sudheer et al. 2018). In this study authors successfully demonstrated the utilization of crude glycerol generated during synthesis of biodiesel from Jatropha seed oil. This work proving the potential of utilization of waste crude glycerol as biomass for the many fermentation process and could be also implemented for the dark fermentation to generate H2 gas (Hawkes et al. 2007; Ren et al. 2011).

Table 3.3 Hydrogen production by dark fermentation with different renewable waste carbon resources (Adopted from Łukajtis et al. 2018)
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Organic waste generated from domestic kitchen, food industry, bravery industry, and restaurants is also rich in carbon source in the form of simple sugars, cellulose, hemicelluloses, proteins, and lipid (Jayalakshmi et al. 2009). This waste biomass not only fulfills the carbon source but also some part as nitrogen supplement. These organic wastes are also very much suitable for the microbial fermentation. Moreover, the dark fermentation utilization of mixed culture fermentation results in the green manure rich in the form of simple nutrients. By using these waste biomasses to produce the H2 gas will have two-way advantages. One is, released to environment these will be taken up by methanogens and result in release of methane which in turn increase the carbon footprint. Utilizing it for the H2 production will result in green fuel (H2) with nil carbon footprint upon combustion (Guo et al. 2008). Many researchers also consider the municipal waste also organic waste, since it is rich in carbohydrates, disaccharides, proteins, and peptides. In addition, sewage sludge is of rich in the microbial community and no need to add externally any microbial inoculum. However, the sludge should be pretreated to remove the hydrogen utilizers like methano-bacteria. Various methods are suggested to remove these methano-bacteria. The simple methods are treating the sludge by microwave or ultrasound, acid or alkaline treatment. Guo et al. have studied in details and found that sludge treated by microwave and ultrasound treatment provided highest yields of H2 production (15 cm3 H2/g COD) (Valdez-Vazquez et al. 2005; Karlsson et al. 2008).

3.4.3.2 Microbial Type and Source

As introduced about the microbial types for the H2 production in the earlier section, in this section the details of the microbial system for H2 synthesis will be discussed in detail. The hydrogen gas production is purely of anaerobic fermentation and the cultures to be used should perform the anaerobic fermentation. This can be done by both obligate (strictly sensitive to oxygen) and facultative (grow in both in presence and absence of oxygen) anaerobic bacteria. Dark fermentation can be carried in either pure cultures or mixed cultures. Both systems have their superiorities and disadvantages [Table 3.1]. The pure cultures fermentation is a single bacterial strain that will involve in the fermentation utilizing a metabolizable carbon sugars. The best example of bacterial genus is Clostridium sp. Clostridium sp. is an obligatory anaerobic bacterium that utilizes many simple sugars and produces H2 via dark fermentation. The major characteristic feature of this species is, it performs the fermentation in variable carbon sources and also it has the ability to survive in difficult conditions such as high temperatures, pH, and presence of toxic substances. The major disadvantage with this species is; it produce the H2 during the log phase and once reach to stationary phase the metabolic flux will be shifted towards accumulation of organic compounds. Depending on the substrate used for the fermentation, Clostridium produces H2 along with accumulation of organic acids like acetic acid and butyric acid. Though wide variety of species like Methylotrophs, enteric bacteria like E. coli, Enterobacter, Citrobacter, Alcaligenes, Bacillus are capable of performing the dark fermentation as a pure culture; mixed culture fermentation has its superiority in H2 production from a complex organic or carbon source derived from waste biomass (Kapdan and Kargi 2006; Hallenbeck et al. 2012; Łukajtis et al. 2018).

The mixed consortia under a strict controlled condition can perform dark fermentation on complex organic carbon source and produce H2. These enriched consortia perform the dark fermentation utilizing broad spectrum of carbon source like industrial waste, animal waste manure, agricultural waste, sewage sludge, compost, and domestic kitchen waste. Upon the dark fermentation via mixed consortia will generate acetic acid, formic acid, butyric acid, and CO2 along with H2. The mixed culture fermentation has the advantage of utilizing the waste biomass like cellulose and lignocellulosic biomass directly without the pretreatment since metabolic cooperation one species with other will help in utilization of complex carbon sources. Hence, the mixed consortia based dark fermentation is the best way of utilization of waste biomass for the production of biohydrogen (Miyake et al. 1999; Logan et al. 2002; Ren et al. 2011; Łukajtis et al. 2018).

The other group of bacteria, i.e., the facultative anaerobes utilize oxygen for the generation of ATP and switch to anaerobic conditions in the absence of oxygen. The best example of hydrogen producing facultative anaerobes is Enterobacteriaceae group. The major system of hydrogen production in this group is via formate hydrogen lyase (FHL) system; where the hydrogen and CO2 are released by utilizing formic acid as the substrate. The base pathway of formate generation studied via glucose metabolism; where maximum theoretical hydrogen yields are 2 moles of H2 per mole of glucose. The final electron acceptor in the metabolism is most of the times organic acids or ethanol. Hence, at the end of the fermentation these organic acids are generated as end products along with hydrogen. To enhance the productivity and diverting the metabolic flux towards useful organic acids many researchers utilized molecular approaches, and details of this genetically modified strains for enhancing the hydrogen are described in the coming section (section details).

3.4.3.3 Fermentation Conditions Which Influence H2 Production

Dark fermentation utilizing the mixed culture or pure cultures, the comprehensive reactions flow involved in the microbes for the production of hydrogen are thermodynamically favorable; however, they are controlled via biological regulators by various mechanisms in microbial cells and need to have favorable conditions to attain maximum productivity. The optimal growth and production conditions should be maintained to get maximum productivity during fermentation process. Three major factors which influence the fermentation conditions are (a) temperature, (b) pH, and (c) gas partial pressures. In this section we will give details of these conditions and how they influence the end productivity of H2 in the fermentation.

3.4.3.3.1 Temperature

The crucial factor in any fermentation system is the temperature in which the fermentation system is operating. The productivity affected to the level of 100% or up to nil if favorable temperatures are not provided. There are no generalized temperatures defined for the H2 production. It ranges from ambient (20 °C) to as high as 80 °C. The optimum temperatures depend on the type of organism and/or crucial bacterial species whose hydrogenase system responsible for the H2production in context of mixed fermentation. Basically, bacterial species fall under three temperature groups and reports show that in each group of bacteria, ability of H2 production is reported. The suitable growth conditions like low temperature (5–20 °C) in case of psychrophiles, ambient temperatures to moderately high temperature (25–45 °C) for mesophiles, and high temperatures (65–80 °C) for thermophiles (Levin et al. 2004).

Selection of optimum temperatures for biohydrogen production depends on species in the culture or mixed culture used for the fermentation. And also, the production of H2 varies with the substrate used as carbon source. In many cases the cell growth and H2 production temperatures differ since the optimum growth of the cell need not be the favorable temperature for the hydrogenase enzyme which produces H2. Hence, crucial optimizations are very much necessary for the cell mass generation and H2 production. Pakarinen et al. (Levin et al. 2004) found that 70 °C is the optimum temperatures for the maximum productivity of H2 production; however, the cell mass generation is at the highest temperature of 50 °C. The multiple studies confirm that, thermophilic conditions are favorable for the substrates need to undergo hydrolysis during fermentation, and ambient conditions are sufficient for the simple sugars. This is because the high temperatures favor the hydrolyzing enzymes responsible for hydrolysis of complex substrates. One more reason for the enhanced productivity of H2 in high temperatures is because of low solubility of gases at low temperatures; hence the growth inhibition of microbes will be minimum in low dissolved aqueous medium (Wong et al. 2014). Though in context of H2 productivity, the high temperatures are favorable; however, in context of energy investment the profitability of process will be low (Azbar et al. 2009).

3.4.3.3.2 pH

In any fermentation system pH plays a significant role in cell growth and productivity, since all the metabolic processes are based on the enzyme activity of particular reaction at specific pH. The majority of the enzymes have a specific pH range; when the productivity of a target depends on multiple metabolic reactions, an optimum temperature needs to be studied to get a maximum productivity. The same concept is applicable to produce H2. Moreover, pH affects the growth of microbes whether it is pure culture or mixed cultures. In mixed culture fermentation, lower pH value favors for the production of H2 and limits the methanogens to utilize the produced H2. However, maintaining at specific pH during fermentation is very important. The production of hydrogen is accompanied by the accumulation of organic acids (acetic, lactic, butyric, and propionic) which will lower the pH of the medium makes it unfavorable for hydrogenase complex to produce H2 gas. Hence, the pH lower than 5 is not advised for the H2 production (Bowles and Ellefson 1985). It is also noted that both initial pH and the operational pH are important; in case of batch fermentation, initial pH at neutral is favorable. In case of continuous mode, maintaining the nutral pH will favor the maximum productivity (Wang and Wan 2009; Jung et al. 2011). The initial and optimal operational pH to be maintained vary with the kind of microbial strains selected for the fermentation or source of microbial consortia (in case of mixed culture), kind of substrate selected, mode of fermentation (batch/continues) system will determine the pH to be applied for the best productivity.

In general the pH range for the H2 production is reported to be in the range between 5.0 and 7.0 corresponding to the growth of the bacterial growth (Li and Fang 2007). The optimum pH differs with the substrate used for the fermentation; the neutral pH is suitable for the livestock waste, pH 6.5–7.0 is favorable for the crop/agriculture waste, pH 5–6 is good for the food waste (Liu and Shen 2004; Li and Fang 2007: Guo et al. 2010). However, some studies reported that 7–8 pH conditions also favorable for some mixed bacterial cultures, e.g. the studies of Liu and Shen explained that, the mixed culture fermentation of corn starch substrate gave best hydrogen production at pH 7 and 8 and the production was 103 and 120 mL H2/g substrate, respectively.

3.4.3.3.3 Partial Pressure of H2

The partial pressure of hydrogen (PPH) in the reactor is very crucial factor that affect the productivity. The hydrogen produced in the microbes is the result of the ferredoxin reduction up on oxidation enzyme hydrogenase. The hydrogenase also participates in reversible reaction up on higher availability of hydrogen gas, hence at high partial pressure of H2 in the reactor the production rate will reduce and metabolic flux will move towards other products such as organic acids, ethanol, and butanol (Abo-Hashesh and Hallenbeck 2012; Hallenbeck 2012; Ghimire et al. 2015). There are two ways to deal with high PPH in the reactor and make system continue with high productivity. One is reducing the partial pressures of hydrogen produced in the reactor by sparging with inert gas most frequently nitrogen or removing of gas released in the system by application of vacuum. The earlier method where reducing the PPH by sparging is effective, the results vary with the type of gas applied for sparging. Kim et al. (2006b) applied CO2 as sparging gas and observed a better productivity compared with the nitrogen sparging. The yields were up to1.68 moles H2/mole of hexoseconsumed compared to nitrogen sparging which yielded 0.95 moles H2/mole of hexoseconsumed. However, the major disadvantage of sparging system is the product will be diluted with the sparging gas and hydrogen separation will become tedious, time consuming, and require cost input. This all make the sparging system non-economic system which make final cost not competitive in commercial prospective. The alternative method as discussed is the removal of the generated gas in the reactor by applying vacuum. Theoretically this looks more beneficial than sparging; however, very limited studies were made in this aspect (Lee et al. 2012).

An alternative to above two methods is proposed by Teplyakov et al. (2002) and Nielsen et al. (2001) using activated selective membrane to hydrogen. The reactor equipped with the membrane system will remove the hydrogen which in turn will reduce the PPH. However, the membranes are effected with biofilms formed by microbes will have to be replaced often. Though many techniques are evolved to reduce the PPH, still much of the research is needed for handling high PPH in the reactor for the better productivity with inexpensive method which is economically competitive.

3.5 Engineered Bacterial System for Improving Hydrogen Productivity

In advance in the molecular biology, availability of genome sequencing system and evolution of various techniques for genome facilitated various researchers to engineer the available microbial sources rather than isolate new microbes with better productivity. The first choice of any researcher for microbial engineering is E. coli since much of the molecular information is explored and many tools were developed for the genome manipulation. Moreover, metabolic pathways were well characterized and information is available for easy manipulation for metabolic engineering. The majority of the work in strain engineering for understanding the microbial hydrogen production and/or improving the hydrogen productivity is made in E. coli. In this section much of the discussion will be made with the view of E. coli.

E. coli is a facultative anaerobe belongs to Enterobacteriaceae family have the intrinsic ability to produce hydrogen. The hydrogen producing apparatus of E. coli includes FHL (Formate Hydrogen Lyase) system. FHL system consists of hydrogenase 3 (hycABCDEFGHI) (Bagramyan and Trchounian 2003) and formate dehydrogenase-H (fdhF) (Axley et al. 1990). HycA protein acts as repressor of the FHL system. The FhlA will up regulate the FHL system and in turn will help in accumulation of H2. However, E. coli consume hydrogen produced by the FHL system by hydrogenase 1 (hyaABCDEF) and 2 (hybOABCDEFG). The efficient production of hydrogen by E. coli is controlled by the availability of formate to FHL system. There are two formate dehydrogenases, such as, formate dehydrogenase-N and formate dehydrogenase-O and formate transporter (FocA and FocB) (Rossmann et al. 1991; Suppmann and Sawers 1994; Andrews et al. 1997). Moreover, the cells having sufficient amount of formate can divert metabolic flux to produce and enhance the H2 productivity.The majority of the strain engineering aspects were designed based on the deletion of hydrogen utilizing genes, over expression of FhlA to upregulate the FHL system in turn to enhance the hydrogen production, and making formate available to the FHL system for increasing the productivity. In addition, the hydrogenases which utilize the produced hydrogen via the FHL system need to delete to avoid the reutilization of produced H2.

3.5.1 Metabolic Engineering of E. coli for Better Productivity

Theoretically, the productivity of hydrogen is formed from basic energy molecules such as 2 mole of glucose and 1 mole of formate. Reaching to the theoretical yields in the system is practically not possible, since the microbial cell utilizes much of the carbon source for the growth and cell biomass generation. Hence, many studies are made in the view of hydrogen production always towards getting near to theoretical yields. Maeda et al. (2007a, b, 2008, 2012, 2018) contributed major input on the metabolic engineering of E. coli for the hydrogen production. Their studies first time reported to reach the theoretical values when formate was used as the substrate for the hydrogen production. In this study, Maeda et al. (2008, 2018) explained to the theoretical values (Maeda et al. 2012) of over-expressed fhlA and deleted the HycA repressor for enriching the FHL complex cell. The hydrogen uptake activity was eliminated by gene deletion of larger subunits (hyaB and hybC) of hydrogenase 1and 2, respectively. In addition the metabolic flux from formate to H2 production was enhanced by deleting fdoG gene; this will inactivate the FDH which is responsible to convert formate into CO2 without H2 production (Maeda et al. 2008; Maeda et al. 2018).

Glucose is the being the starting carbon moiety and less expensive than formate, many researchers taken interest on metabolic engineering of E. coli utilizing glucose as the substrate to produce H2. The basic principle most of the strategies were designed to increase the metabolic flux towards enhancing the formate availability to FHL system for the hydrogen production. As mentioned earlier, the base strain selected always with inactive hydrogenase 1 and 2 to eliminate reutilization of H2 produced by FHL and FHL repressor (hycA) (E. coli - hyaB−, hybC− and hycA−). These mutations also showed that there is enhancement in H2 production using glucose as the substrate (Penfold et al. 2003; Yoshida et al. 2006; Maeda et al. 2007; Turcot et al. 2008; Fan et al. 2009; Kim et al. 2009; Mathews et al. 2010). In addition, the H2 production was further improved by over expressing FhlA with N-terminal truncation (Self et al. 2001; Turcot et al. 2008).

In E. coli, the glucose metabolism leads the formation of the phosphoenolpyruvate and pyruvate. The pyruvate is converted in to formate; subsequently, formate is transformed in to succinate and lactate as by-products. Hence, it is necessary to divert the metabolic flux towards formate and eliminate the succinate and lactate accumulation during fermentation for enhancing H2 production. The studies based on these were made by various researchers and enabled the recombinant E. coli to produce H2 from glucose (Yoshida et al. 2006; Maeda et al. 2007; Manish et al. 2007; Fan et al. 2009; Kim et al. 2009). These studies targeted genes deletion of ppc encodes phosphoenolpyruvate, frd ABCD fumarate reductase, idhA lactate dehydrogenase (Maeda et al. 2007). Table 3.4 comprehended the information of various studied done in E. coli and productivity achieved by various engineered E. coli strains. In addition to these, expressing Fnr a global DNA-binding transcriptional global regulator also found to enhance the H2 productivity (Fan et al. 2009). Comprehending all, the best H2 productivity was obtained with the E. coli holding knockout of seven genes (hyaB, hybC, hycA, fdoG, ldhA, frdC, and aceE) five gene inactivation by (hyaAB, hybABC, hycA, ldhA, and frdBC/hycA, hya, hyb, ldhA, and frdAB) (Kim et al. 2009; Mathews et al. 2010) and three gene inactivation (hya, hyb, and ldhA) (Turcot et al. 2008).

Table 3.4 Comparison of In-vivo Hydrogen production by engineered E. coli (Reproduced from Maeda et al. 2012)
Full size table

As discussed in earlier section about the application of various carbon substrates like crude glycerol or lignocellulosic biomass, application of these components as carbon source will be economically beneficial. In this regard, glycerol fermentation was initially ruled out since the glycerol fermentation do not favor H2 production. However, these studies made by Dharmadi et al. (2006) and Gonzalez et al. (2008) showed that at alkaline pH was favored the hydrogen production in the presence of potassium and phosphate. Despite the theoretical understanding of anaerobic fermentation by utilizing glycerol have the benefit of extra NADPH+ generation; however, there are many genes whose expression will be shutdown which are based on glucose metabolism. Despite of handful studies on glycerol fermentation by E. coli are available; the information existing for hydrogen production is far from the understanding when compared to glucose and other monosaccharaides. This is because of contradictory studies by various researchers and also experimental yields are considerably limited.

Metabolic engineering is a good way to make E. coli to produce good amount of H2 from glycerol. A powerful approach was made by Tran et al. (2014, 2015). In this study the knockout mutant of E. coli with seven genes which are mostly participating in enhancing the formate accumulation and blocking the metabolic flux in synthesis of by-products like methylglyoxal. The selected genes deleted are fumarate reductase (encoded by frdC), lactate dehydrogenase (ldhA), formate dehydrogenase (fdnG), phosphoenolpyruvate (ppc), nitrate reductase (narG), methylglyoxal synthase (mgsA), and the regulator of the transcriptional regulator FhlA (hycA). The resulted strain is able to produce the hydrogen near to the theoretical value (1 mole of H2 for 1 mole of glycerol). Instead of targeted gene deletions, Tran et al. applied random mutagenesis for looking genes responsible for hydrogen production in glycerol fermentation (Tran et al. 2014, 2015). In this study four genes were identified which involved in hydrogen production. The individual mutant of the following four genes, namely aroM, gatZ, ycgR, and yfgI enhanced the hydrogen production up to 1.6fold. Moreover, the mutants not only enhanced the hydrogen production but also increased the growth rate of the mutant strains compared to wild type under glycerol fermentation in anaerobic conditions. In addition to adoptive mutagenesis, Hu and Wood isolated a mutant strain named as HW2 which is holding the ability to produce 20 times more productivity and fivefold higher cell growth than original strain BW25113 ΔfrdC (Hu and Wood 2010). Further transcriptome analysis of this strain showed that the isolated mutant defective in fructose-1,6-bisphosphatase (encoded by fbp), formate transportation (focA), and tagatose-1,6-bisphosphate aldolase (gatYZ). These studies gave a better picture on glycerol metabolism in hydrogen production; however, more comprehensive data is needed to link all these studies for elaborated understanding glycerol metabolism and hydrogen production for better productivity with less energy investment which can lead to a technology which can be as competitive as commercial production presently followed (Akhtar and Jones 2008a).

In addition, with the strategies based on the deletion of targeted genes, adoptive mutagenesis and random mutagenesis; few studies are also made for enhancing the hydrogen production by heterologous expression of various clusters of genes. Among these studies, expression of hydrogenases genes isolated from various strains in E. coli is important and results in enhanced H2 production. In this regard the expression of hydrogenases derived from the microbial species like Enterobacter cloacae (Mishra et al. 2004; Chittibabu et al. 2006), Ethanoligenens harbinense (Zhao et al. 2010), Rhodobacter sphaeroides (Lee et al. 2010b), Clostridium acetobutylicum, and C. pasteurianum (Akhtar and Jones 2008b, 2009) were heterologous expressed in E. coli BL21 with no ability to produce hydrogen. The heterologous expression of hydrogenases resulted in H2 production by BL21 strain. Along with hydrogenases heterologous expression, few researchers also tried co-expression of other genes involved in the transportation of substrates and substrate utilizing enzymes which will divert in the core cellular metabolism tried for enhancing the hydrogen production. Few among these a significant study is expression of scrB (encode β-D-fructofuranosidefructohydrolase catalyzes the hydrolysis of sucrose 6-phosphate to β-D-fructose and α-D-glucose 6-phosphate) and scrR (encodes the negative repressor of the scr regulon) which enhanced the hydrogen productivity up to twofold from sucrose (Penfold et al. 2003).

3.6 Future Prospects

Hydrogen being the only green fuel which does not release any carbon footprint up on combustion is the next generation fuel for the future environmental outlook. To make this fuel as alternative fuel for the transportation and other industrial applications, the production cost must come down as competitive as commercial available hydrocarbon based fuels. The key points to be looked in the aspects of biohydrogen production is (1) innovative methodologies to be developed to utilize waste biomass and industrial waste water effluents, (2) isolating and developing efficient strains which could be used for hydrogen production utilizing more diverse carbon substrates, (3) engineering the microbial system for enhancing the productivity, resistance to growth retarding fermentative by-products, increasing growth rate, imparting ability to utilizing complex substrates, and accumulating useful by-products, and (4) innovative reactor designs. Looking at these aspects future research goals need be put forward to generate a sustainable biological hydrogen producing system prospective to forecast energy needs and for environmental safety.