1. Introduction
The growing gap between the energy demand of the world and an insufficient energy supply has caused a steep increase in fossil fuel use. As a result, we encounter the severe constraints imposed by an alarming increase in pollution levels around the world along with the depletion of fossil fuels. Additionally, the continuous increase in the levels of greenhouse gases (GHGs) released from the combustion of fossil fuels aggravates the problems of global warming. Currently, the CO
2 concentration exceeds 350 parts per million (ppm) by volume, and the increase in concentration potentially increases the greenhouse effect, which results in increasing global temperatures [
1,
2]. In recent decades, the organic carbon released by human activities is equivalent to that which was accumulated over millions of years. The limited availability of global fossil fuel reserves and concerns about global climate change from GHG emissions prompted notable interest in the investigation and development of eco-friendly, renewable energy alternatives to fulfill the growing energy demands [
3]. Therefore, in the current global energy scenario, the diversification of energy and fuel options is an essential requirement [
4]. To diversify, bio-based energy is a sustainable and promising alternative to fossil fuel-based energy; this alternative energy can defend against a crisis in the energy supply and can protect the world from the approaching environmental calamity. Recently, global attention focused on hydrogen (H
2) gas as one of the most promising, eco-friendly, and renewable energy sources. H
2 is a potentially versatile energy currency that could alter the use of liquid fossil fuels because the fuel has a high-energy yield per unit mass of 122 kJ/g, which is 2.75-fold higher than that of hydrocarbon fuels [
5,
6]. Additionally, the combustion of H
2 with O
2 produces water (H
2O) as the only by-product, an obviously favorable outcome for a reduction in GHG emissions. In particular, H
2 is the pre-eminent choice for an energy carrier because it is more similar to electricity than fossil fuels in the framework of energy systems [
7].
Currently, molecular H
2 is primarily produced from the use of fossil fuels through steam reforming of natural gas or methane (CH
4). The worldwide production of H
2 currently exceeds 1 billion m
3/day of which 48% is produced from natural gas, 30% from oil, 18% from coal, and the remaining 4% is produced from H
2O-splitting electrolysis [
2,
8]. In combination with steam reforming, the production of pure H
2 is also achieved with an H
2O-gas shift reaction, which is one of the important industrial reactions used specifically for ammonia production. The other thermochemical methods available for the production of H
2 include thermal decomposition, autothermal reforming, catalytic oxidation, pyrolysis, and steam gasification [
2,
9,
10]. However, the production of H
2 based on fossil fuel resources increases the emissions of GHGs. Alternatively, the production of H
2 from biomass through biological pathways is an emerging technology because it is sustainable and eco-friendly. Indeed, a scientometric analysis that used the SCI-expanded (since 1994), science technology (CPCI-S, since 1994), and social science (CPCI-SS, since 1994) databases in the ISI Web of Knowledge (Thomson Reuters) found that 2204 research articles were published on H
2, with a significant number of citations (46,723) and average citations per item (21.02), and a high H-index (92). As shown in
Figure 1, the literature linked to H
2 research increased sharply after 2003 and reached the maximum number of records of 230 in 2008 (total citations: 2883), which was followed by a sudden increase in 2012 (records, 335; citations, 8107). The average citations per year also increased year by year, which clearly indicated that the rapid and promising research continues to make the process of H
2 production technologically viable.
Figure 1.
Scientometric analysis of the research on H2 production. Published items (A) and citations (B) in each year.
Figure 1.
Scientometric analysis of the research on H2 production. Published items (A) and citations (B) in each year.
Different organisms yield H
2 under specific conditions, including microalgae that use light energy to split water molecules to produce H
2, and cyanobacteria that typically consume carbohydrates to store energy from photosynthesis to produce H
2 from water molecules [
11,
12]. Although there are striking advantages, the low production rates, low substrate conversion efficiencies, and production and accumulation of acid-rich intermediate metabolites from the acidogenic process are practical hindrances that must be overcome for the successful biological production of H
2. To overcome these limitations, many research projects on the biological production of H
2 are in progress, and numerous novel approaches are being studied to address some of the existing problems and to overcome these problems by increasing the efficiency of the process. To reach these goals, a number of advanced well-described technologies for high yields of molar H
2 use metabolic engineering to provide metabolic energy to exceed thermodynamic limitations, to reroute metabolic pathways to increase substrate utilization by the expression of heterologous proteins, and to improve the electron flux for H
+ reduction, among others [
11,
12]. In this review, we evaluate the biological pathways for the production of H
2 with respect to the factors that affect operations and potentially limit the production of H
2, and assess the efficiency and practical applicability of these technologies. Additionally, alternative options such as bioaugmentation, multiple process integration, and microbial electrolysis to improve process efficiency are discussed.
4. Economic Feasibility and Technical Challenges
During the last two decades, several efforts to make the H
2 production process economically more feasible were attempted [
46]. However, some key technical challenges remain, and if these challenges are overcome, the overall H
2 production efficiency will increase through the biological pathways described below (
Table 1).
Table 1.
Biological pathways for H2 production and the technical limitations.
Table 1.
Biological pathways for H2 production and the technical limitations.
Type of Bioprocess | Technical Challenges |
---|
Dark fermentation | low substrate conversion efficiency low H2 yield thermodynamic limitations mixture of H2 and CO2 gases as products, which require separation
|
Photofermentation | requirement of an external light source the process is limited by day and night cycles, with sunlight as the light source low H2 yield caused by extremely low light conversion efficiency
|
Direct biophotolysis | O2 generation caused by the activity of PS II requirement for customized photobioreactors low H2 yield caused by extremely low light conversion efficiency
|
Indirect biophotolysis | lower H2 yield caused by hydrogenase(s) requirement of an external light source total light conversion efficiency was very low
|
These challenges may be overcome with the efficient design of H
2 producing bioreactors, process modifications, selection of appropriate feedstocks, and with the selection of suitable and efficient microbial strains. In the metabolic pathways that produce H
2, the intermediate metabolites produced by the biocatalyst compete for the identical reductants as the H
2, and this redirection of the reductants toward soluble end metabolites reduces the H
2 yields. Hence, several researchers are attempting to reroute the metabolic pathways to reduce the production of the low-end metabolites. To overwhelm the stoichiometric limitation (4 mol H
2/mol glucose) of the dark fermentation process, a robust biocatalyst must be found that can be metabolically engineered. Thus, with a successful bioreactor design and the determination of the ideal process parameters, the yield of H
2 can be increased. However, low molar conversion rates affect the economics of the process, and therefore, research is underway to increase the H
2 yield above the 4 mol H
2/mol glucose limitation. Recently, several researchers focused on the development of suitable hybrid processes, such as the two-stage integration of the dark fermentation process, followed by the photofermentative process to produce H
2 [
18]. With this approach, the VFAs that are produced in the dark fermentation (first stage) are used as the substrate in the photofermentation (second stage). This approach might use efficiency to increase the theoretical limit of H
2 yield to 12 mol H
2/mol glucose [
14]. Similarly, hybrid processes that include subsequent methane production or electrofermentation are also being considered to increase the energy recovery of the process. Although there is an abundance of research in the past and at present, these specific areas must be investigated to further enhance the production of H
2 via the biological pathways [
37]. Additionally, the integration of the H
2 production process with a conventional wastewater treatment process has several advantages, such as waste remediation with simultaneous generation of clean energy. In the future, carbon-rich organic wastes may be targeted as suitable feedstocks for H
2 production because of their natural abundance. The use of cheaper raw material substrates would increase the H
2 yield from the biological processes, which would help significantly to make the process more economically viable and cost effective.
5. Strategies to Enhance the Efficiency of the Process
The major deterrents to the conventional biological H
2 production from any of the processes described above are the low substrate conversion efficiency and the accumulation of VFAs. Because of these deterrents, the overall yield of H
2 is far too low for the process to be economically feasible and commercially applicable [
38,
79]. In particular, although the theoretical H
2 production could reach 12 mol of H
2/mol glucose, the dark fermentative H
2 production is metabolically limited to 4 mol H
2/mol glucose, which is a major technical hurdle for practical applications [
86]. Additionally, after dark fermentation, significant amounts of residual organic substances such as VFAs or solvent remain in the effluent. Thus, additional treatments are necessary before disposal into the environment. The reuse of the residual carbon fraction of the fermentative effluents for further energy generation together with proper environmental treatment would be wise considering the environmental and economic factors [
87]. Moreover, the design and fabrication of photobioreactors that use the internal light supply efficiently remains a challenge in photofermentation [
88,
89].
5.1. Integration of Approaches
Recently, many integrated approaches were proposed to overcome the limitations of several processes to increase the production of H
2 in dark fermentation. The use of the residual acid-rich organic substances from the fermentation effluents as carbon-rich substrates for further energy recovery is a viable and novel idea, particularly when in the form of an integrated two-stage energy producing process (
Table 2 and
Figure 3). Numerous secondary processes, including methanogenesis for methane, acidogenic fermentation for H
2, photobiological processes for H
2 [
90,
91,
92], MECs for H
2 [
44], anoxygenic nutrient-limiting processes for bioplastics, cultivation of heterotrophic algae for lipids, and MFCs for bioelectricity generation, were integrated with the primary dark fermentative process of H
2 production. With these integrated approaches, the primary process uses these further substrates for additional energy production, and therefore, the entire process is more economically viable and practically applicable than without the integration.
Table 2.
A list of the processes integrated with the production of H2 from dark fermentation (DF, dark fermentation; PF, photofermentation; MEC, microbial electrolysis cell; BEH, bio-electrohydrolysis).
Table 2.
A list of the processes integrated with the production of H2 from dark fermentation (DF, dark fermentation; PF, photofermentation; MEC, microbial electrolysis cell; BEH, bio-electrohydrolysis).
Substrate | First Stage | Second Stage | Reference |
---|
Process Type | Yield | Process Type | Yield |
---|
Cornstalks | Hydrogen (DF) | 58.0 mL/g | Methane (DF) | 200.9 mL/g | [93] |
Rice straw | Hydrogen (DF) | 20 mL/g | Methane (DF) | 260 mL/g | [94] |
Water hyacinth | Hydrogen (DF) | 38.2 mmol H2/L/day | Methane (DF) | 29 mmol CH4/L/d | [95] |
Water hyacinth | Hydrogen (DF) | 51.7 mL of H2/g of TVS | Methane (DF) | 43.4 mL of CH4/g of TVS | [96] |
Laminaria japonica | Hydrogen (DF) | 115.2 mL of H2/g | Methane (DF) | 329.8 mL of CH4/g | [97] |
Cassava wastewater | Hydrogen (DF) | 54.22 mL of H2/g | Methane (DF) | 164.87 mL of CH4/g | [98] |
Microalgal biomass | Hydrogen (DF) | 135 ± 3.11 mL of H2/g/VS | Methane (DF) | 414 ± 2.45 mL of CH4/g/VS | [99] |
Glucose | Hydrogen (DF) | 1.20 mmol | Hydrogen (PF) | 5.22 mmol | [100] |
Cheese whey wastewater | Hydrogen (DF) | 2.04 mol | Hydrogen (PF) | 2.69 mol | [101] |
Vegetable waste | Hydrogen (DF) | 12.61 mmol H2/day | Electricity (DF) | 111.76 mW/m2 | [87] |
Fruit juice industry wastewater | Hydrogen (DF) | 1.4 mol H2/mol hexose | Electricity (DF) | 0.55 W/m2 | [102] |
Corn stover lignocellulose | Hydrogen (DF) | 1.67 mol H2/mol glucose | Hydrogen (MEC) | 1.00 L/L-d | [103] |
Cellobiose | Hydrogen (DF) | 1.64 mol H2/mol glucose | Hydrogen (MEC) | 0.96 L/L-d | [104] |
Distillery spent wash | Hydrogen (DF) | 39.8 L | Bioplastic | 40% dry cell weight | [105] |
Food waste | Hydrogen (DF) | 3.18 L | Bioplastic | 36% dry cell weight | [106] |
Pea shells | Hydrogen (DF) | 5.2 L of H2 from 4 L | Bioplastic | 1685 mg of PHB/L | [107] |
Food waste | Hydrogen (DF) | 69.94 mmol | Lipid | 26.4% dry cell weight | [108] |
Olive oil mill wastewater | Hydrogen (DF) | 196.2 mL/g | Biopolymer | 8.9% dry cell weight | [109] |
Molasses wastewater | Hydrogen (DF) | 130.57 mmol | Ethanol | 379.3 mg/L | [110] |
Food waste | Bioelectricity | 85.2 mW/m2 | Hydrogen (DF) | 0.91 L | [39] |
Starch hydrolysate | Hydrogen (DF) | 5.40 mmol H2/g of COD | Hydrogen (PF) | 10.72 mmol H2/g of COD | [111] |
Sucrose | Hydrogen (DF) | 0.98 ± 0.32 mol H2/mol | Hydrogen (PF) | 4.48 ± 0.23 mol H2/mol | [112] |
Glucose:xylose (9:1); Microalgae biomass | Hydrogen (DF) | 250 mL/L/h; 2.78 mol H2/mol | Mixotropic microalgae cultivation | 205 mL/L/h; 1.12 g of biomass/g of COD | [113] |
5.2. Photobiological Process
The photosynthetic bacteria readily consumed the residual organic fraction (VFAs) [
100,
101]. Because dark fermentative metabolic intermediates can be effectively used by some PNS bacteria, the integration of the anoxygenic photofermentation process with the dark fermentation process will have the dual advantages of increased H
2 production with simultaneous removal of the substrates [
114]. Chandra and Venkata Mohan [
100] investigated the composition and the survivability of mixed microalgal populations during their growth and the production of photofermentative H
2 using glucose and acid-rich effluents generated from the process of dark fermentation. Photofermentation with the acid-rich effluents of glucose had a higher efficiency of H
2 production (5.22 mmol H
2) than dark fermentation (1.21 mmol H
2) with glucose as the carbon source. Green algae such as
Chlorella also use carbon-rich organic acid intermediates from dark fermentation to produce H
2, particularly when acetate is a viable substrate (Equations (6) and (7)) [
115].
Dark fermentation (Stage I):
Photoheterotrophy (Stage II):
The Equations (6) and (7) define an ideal condition in which all the carbon in the form of substrate is processed in the suitable metabolic pathways and none of the carbon is routed to the formation of biomass or alternative metabolites. However, the photofermentation of acid-rich effluents from the H
2 production process is more complex than dark fermentation with respect to the efficiency of processing because of poor light penetration, nutritional requirements of the biocatalyst, maintenance of environmental conditions, inhibition of substrates, and contamination obstacles [
116,
117]. To overcome these limitations, appropriate light arrangements must be either inside or outside of the bioreactor, sufficient nutrients must be supplemented, optimum temperatures and substrate concentrations must be maintained, and the bioreactors must be enclosed systems for ease of sterilization.
5.3. Biodegradable Plastics
The VFA-rich effluents generated from dark fermentation are a potential substrate for the production of bioplastics, such as polyhydroxyalkanoates (PHA) and polyhydroxybutyrates (PHB). The PHAs are a biodegradable biopolyester of hydroxyalkanoates that are produced under extra carbon and nutrient-deprived circumstances and that accumulate as cellular reserve storage material [
105,
106]. The biopolyesters are deposited as water-insoluble, cytoplasmic micro-sized inclusions in bacterial cells when excess carbon is available and when other nutrients are growth limiting. In general, the PHAs are produced using pure microbial cultures with synthetic substrates (e.g., acetate and butyrate, among others), which is not a cost-effective method of production. The VFAs are simple substrates with a low number of carbon atoms, and the synthesis of PHA requires fewer metabolic enzymes than those of the glycolysis and β-oxidation pathways [
106]. The production of PHBs from individual fatty acids (e.g., acetate and butyrate, among others) and acid-rich effluents from dark fermentation was reported for an anoxic microenvironment that used a mixed culture as the biocatalyst [
118]. Reddy
et al. [
106] investigated the production of bioplastics (PHA) using
B. tequilensis in aerobic conditions with synthetic acids (SA) and acid-rich effluents of food waste (AFW) as substrates, which were collected from bioreactors producing H
2 with dark fermentation. The synthesis of PHAs was higher with SA (59% dry cell weight) than with AFW (36% dry cell weight). They also reported on the presence of a copolymer (P(3HB-
co-3HV)) with varying amounts of hydroxy butyrate (HB, 80%–90%) and hydroxy valerate (HV, 10%–15%) for both substrates. Accordingly, the use of the acid-rich effluents from H
2 producing reactors as substrates contributed to a significant reduction in the production costs of both the H
2 and the PHA embedded with the waste valorization. The entire process was more economically viable when the production of bioplastics was coupled with the production of H
2 and their effluents were used for methanogenesis [
107].
5.4. Electrically Driven Biohydrogenesis from Acid-Rich Effluents
In recent years, the integration of MECs with other bioprocesses has also received considerable attention [
103,
119]. As an alternative electrically driven process of H
2 production, the MECs facilitate the transformation of biodegradable materials into H
2 with an external voltage applied. Indeed, the MEC process was feasible to generate H
2 in association with simultaneous wastewater treatment for a wide variety of soluble organic substances [
104,
120]. A two-stage process was used to convert the acid-rich dark fermentation effluents into substrates for additional H
2 production (
Figure 5) [
103,
104]. Babu
et al. [
104] investigated the feasibility of integrating the MEC process with the dark fermentation process to use the acid-rich effluents for additional H
2 recovery. For this integration, the MECs were operated with a small range of varying applied potential (0.2, 0.5, 0.6, 0.8, and 1.0 V) and with acid-rich effluents (concentration of 3000 mg/L) using an anaerobic mixed consortium as a biocatalyst. The maximum hydrogen production rate (HPR) and the cumulative hydrogen production (CHP) were 0.53 mmol/h and 3.6 mmol, respectively, with 49.8% of the VFAs utilized at 0.6 V. With a high substrate conversion efficiency (90%), a two-stage approach,
i.e., MECs integrated with dark fermentation, could be a viable option to achieve higher substrate conversion efficiency and H
2 yield [
44].
5.5. Bioaugmentation
Many biotic and abiotic factors (e.g., microbial physiology and concentration and composition of substrates) also affect the overall yield of the dark fermentative H
2 production process with mixed cultures. With the reactor in operation, higher substrate concentrations lead to an accumulation of VFAs and a decrease in system pH (˂4.0), which results in the inhibition of the H
2 production process. In an effort to improve the process capability with higher substrate conversion efficiencies, the addition of the desired microbial strains to a native microbial community would be a practical option to overcome the inhibition in the process [
121]. These bioaugmentation strategies used single or mixed native microflora with the acidogenic consortia [
122], fermentative H
2 producing bacteria [
123], and
C. acetobutylicum [
124] to increase H
2 production efficiency. Moreover, similar studies were performed to recover the start-up of a bioreactor [
125], to boost reactor performance [
126], and to protect the native microbial community against problems in the process [
121], which indicated that the bioaugmentation strategy was effective to increase H
2 production. In some cases, however, the bioaugmented microbial flora might fail to compete with the native population, most likely because of inappropriate operating conditions, failure of substrate utilization, and type and/or diversity of the native microbial population in the system [
127,
128,
129].
Figure 5.
Schematic illustration of microbial electrolysis cells (MECs) integrated with the dark fermentation process for higher H2 yield (A: anode; C: cathode; Biofilm: electrochemically active mixed microbial population). Green, orange, brown, and blue symbols represent a mixed microbial population. In stage 1, initially, complex substrates were used for H2 production in dark fermentation, and in stage 2, acid-rich effluents were used as substrates in MECs for further H2 production.
Figure 5.
Schematic illustration of microbial electrolysis cells (MECs) integrated with the dark fermentation process for higher H2 yield (A: anode; C: cathode; Biofilm: electrochemically active mixed microbial population). Green, orange, brown, and blue symbols represent a mixed microbial population. In stage 1, initially, complex substrates were used for H2 production in dark fermentation, and in stage 2, acid-rich effluents were used as substrates in MECs for further H2 production.
5.6. Utilization of Organic Wastes as a Fermentable Substrate
With rapid urbanization and industrialization, waste management is at the forefront as a major human health and environmental concern [
42], and improper waste management increases GHG emissions, which contribute to climate change. Moreover, because many effluents and wastes from foods and food processing and industries that use paper, dairy, cellulosic, and glycerol require a high chemical and biological oxygen demand [
130], they potentially threaten the aquatic fauna [
131]. For practical and economical aspects, the use of carbon-rich wastes/wastewater as fermentable substrates is an attractive and promising approach for H
2 production, which may solve the dual purpose of waste disposal and clean energy generation [
132]. This approach would greatly reduce the H
2 production processing costs, when compared with chemical and electrolytic processes [
133]. The fermentable waste contains biodegradable organic materials to yield a net positive energy, which remains valid for thermophilic fermentative H
2 production, although this process requires additional energy for the heating of the substrates and the reactor [
16]. Moreover, bioreactors may be installed at or proximal to waste generation sites, which further increases the economic viability of the process [
62]. Therefore, a variety of wastes could be used as potentially fermentable substrates for H
2 production.
5.7. Pretreatment of Substrates and Biocatalysts
Complex substrates/feedstocks are not the preferred substrates for H
2 producing biocatalysts and are a challenging bottleneck to the development of biological pathways for H
2 production. To transform nonutilizable substances into fermentable substrates, many pretreatment processes were assessed to make complex substrates into simple ones, based on the types of substrate available [
134]. Primarily, pretreatment methods are classified into four major groups: physical (mechanical pretreatment, extrusion, and pyrolysis), physicochemical (steam explosion, ammonia fiber explosion, CO
2 explosion, liquid hot water, wet oxidation, sonification, and microwave-based pretreatment), chemical (ozonolysis, acid hydrolysis, alkaline hydrolysis, oxidative delignification, organosolvation, and ionic liquids), and biological (enzymatic hydrolysis) pretreatments. Among these pretreatments, the physicochemical and chemical treatments are the most efficient [
135].
The feasibility of H
2 production using a mixed microbial population is likely to be restricted because of H
2 consumption by methanogens. Therefore, pretreatment of the biocatalyst parent culture may be beneficial for shifting the metabolic pathways to increase acidogenesis, and to inhibiting methanogenesis to improve the H
2 production yield with prevention of competitive growth and coexistence of other H
2 consuming microorganisms [
60,
79]. The different biocatalyst pretreatment methods include heat shock (temperature, >80 °C), chemical methods (to inhibit specific metabolic functions; 2-bromoethanesulfonic acid, acetylene, Na
2SO
4, fluvastatin, chloroform, and iodopropane), acid shock (pH < 4), alkaline shock (pH > 9), an oxygen shock method (oxygen/air), load shock (higher substrate concentration), infrared irradiation, and freezing and thawing (−25 °C for 24 h, followed by a 5 h thaw at 30 °C). The different functional properties of ozone (ozone bubbles) and microwave irradiation methods were evaluated. Indeed, the pretreatments applied to the parent inoculum facilitated the selective enrichment of acidogenic bacteria capable of producing H
2 as the end product with the simultaneous prevention of hydrogenotrophic methanogens [
136]. Because of the physiological differences between the H
2 producing acidogenic bacteria and the H
2 consuming bacteria (methanogens), the pretreatment of biocatalysts can also provide a fundamental basis for the development of a H
2 production system [
60,
137].
6. Conclusions
The multidisciplinary fermentation processes used for the production of H2 were numerous, and a variety of substrates were examined. The individual processes possess their own inherent limitations, such as low substrate conversion efficiency, accumulation of VFAs as carbon-rich acid intermediates, and change in system redox conditions and buffering capacity. Thus, to overcome the potential limiting factors and to improve the efficiency of the H2 production process, an understanding of the mechanisms of H+ reduction, functional roles of membrane components, composition of the communities, development of cultures, and design and development of competent bioreactors are the critical areas for both photo- and dark fermentation processes. The initial research on H2 was typically confined to the use of pure cultures as a biocatalyst, and the selection of the biocatalyst depends primarily on the type of fermentable substrates. There is strong consensus that using a mixed microbial population as a biocatalyst is a favorable and practical choice to scale up the technology of H2 production, primarily with wastewater as the substrate (carbon source). Additionally, mixed cultures are typically preferred because of the operational ease, stability, diverse biochemical functions, and probability of using a wide variety of substrates. The optimization of the process parameters is clearly necessary to scale up the technology. The residual organic fraction as a soluble fermentation product after acidogenesis is one of the key limiting factors that requires considerable attention. The approaches with integration that use the acid-rich reactor effluents with the simultaneous recovery of energy must be efficient and completely established for the commercialization of the process to be economically feasible.
In conclusion, the basic and applied research on H2 production provides additional insight into the process for a better understanding to establish an optimized environment. Although various novel approaches are anticipated in future years to overcome some of the persistent problems, biological H2 production technology requires a multidisciplinary approach for the process to be eco-friendly and economically feasible.