Sustainable Strategies for the Conversion of Lignocellulosic Materials into Biohydrogen: Challenges and Solutions toward Carbon Neutrality

Abstract

The present review mainly discusses advanced pretreatment techniques for converting lignocellulosic biomass into hydrogen. The focus of this review is also to acquire knowledge concerning lignocellulosic biomass pretreatment processes and their impact on the efficiency of biohydrogen fermentation. The deconstruction of lignocellulosic biomass is presented using various pretreatment techniques albeit with several advantages and disadvantages, particularly about the interference due to the generated inhibitory compounds is toxic to microbes used for fermentation. The use of an appropriate pretreatment process can make the recalcitrant lignocellulosic biomass substrates amenable for further microbial fermentation to produce hydrogen. Although till date there is no ideal pretreatment step available to develop a cost-effective process for conversion of lignocellulosic materials into fermentable sugars, nanotechnology seem to be a more sustainable approach as compared to the traditional processes.

1. Introduction

The steady diminution of fossil fuels and increasing environmental issues have made us think about other renewable energy sources [1]. Considering these issues, biofuels are considered as a viable energy option as they are eco-friendly, carbon neutral fuels [2]. Among the available biofuels, biohydrogen has received considerable attention owing to its various advantages. The higher energy yield of hydrogen (122 kJ/g) than fossil fuels is one of the important properties of a fuel that is considered a promising energy carrier [3]. Hydrogen technologies sustained strong impetus in 2019 and have raised keen interest among policy makers. The fuel cell electric vehicle market has suddenly doubled, owing to outstanding expansion in most of the developed countries. However, low-carbon production capacity remained relatively constant and is still off track with the Sustainable Development Scenario (SDS). Hence, much attention is required for scaling up the hydrogen production process, which can be used in various applications.

Biohydrogen fermentation using lignocellulosic biomass (LCB) materials as feedstocks is better alternative to petroleum-based fuels due to its ecofriendly nature since there is no greenhouse gas (GHG) emissions during combustion. Hydrogen can be generated by gasification, steam reformation, water gas shift, and electrolysis of water [4]. Such types of processes utilize fossil fuels and are highly energy intensive, which disturbs green environment by carbon emission. LCB materials have been considered for biohydrogen production at large scale [4]. Thus, LCB can be a better substitute to generate biohydrogen energy in a sustainable manner to reduce emission to a certain extent [5]. Usually, hydrogen can be produced by various physical, chemical, biological, and physico-chemical processes [6]. However, traditional physico-chemical processes are not environmentally friendly and sustainable since fossil fuels serve as substrates. Contrastingly, biological methods appear to be better alternative as different organic waste materials can be employed as substrates for biohydrogen production using microbes under ambient conditions [7,8]. Moreover, the “food for fuel” debate has made us to think about other alternative options, such as LCB waste materials as substrates for biofuel production [9]. The limitation of LCB-derived biohydrogen through microbial fermentation is the generation of sugar intermediates during hydrolysis. Hence, it entails a proper LCB pretreatment in biohydrogen production. The purpose of the pretreatment process is to modify the rigid structure of biomass to upturn availability of range of hydrolytic reagents, e.g., enzymes, to disintegrate LCB substrates that can be further utilized for the microbial fermentation process. LCB materials are ecological as well as promising substrates for production of second generation (2G) biofuels, chiefly biohydrogen, bioethanol, biodiesel, biomethane, synthetic biofuels, biogas, etc. LCB, a renewable substrate, is an attractive option for petroleum resources as it encompasses huge amounts of energy and organic components. LCB materials have also been broadly utilized by food, cosmetics, pharmaceutical industries, and biorefineries [10]. LCB materials include mainly forestry and agricultural waste residues derived from plant wastes, such as rice straws, cotton stalks, corn cob, corn straw, potato haulms, etc. [11]. However, the recalcitrant nature of LCB materials makes their use difficult and hence pretreatment step is needed to make it amenable for further enzymatic action [11].

Many pretreatment methods have been reported but each with several advantages and disadvantages. No ideal pretreatment process is available till date for LCB conversion. These issues can be overcome by utilizing a nanobiotechnology approach, which offers a key to the presently available pretreatment methods. This novel nanotechnology method comprises the application of nanoparticles (NPs) that can be applied in various fields for the production of value-added products [12]. Owing to the smaller size of NPs, it can easily penetrate the cell wall of LCB materials, which amalgamates with the biomass components and alters the molecular and physical structures to liberate sugars [13]. NPs can also be used in both pretreatment and hydrolysis in combination with enzyme during LCB conversion. Nowadays, magnetic NPs have been used for conversion of LCB to biofuels, which can be recovered and reused easily for the next cycles and can aid in making the overall process economically feasible [14]. The application of nanocatalysts increases hydrolytic efficiency and enzyme stability. Hence, the use of nanocatalysts for LCB to biohydrogen conversion will aid in emerging the sustainable approach. In this review, we have discussed the use of nanocatalysts as a promising option for enhanced LCB conversion into biohydrogen production.

This review estimates nanobiotechnology application in the field of cellulosic biohydrogen production using microbial fermentation. The prospect of making the biofuel technology viable using nanomaterials from LCB waste materials has been presented here. We also highlighted various pretreatment methods, role of nanobiotechnology-based methods in the LCB conversion into biohydrogen and future prospects in the field of LCB to biohydrogen production.

2. Pretreatment of Lignocellulosic Biomass

The recalcitrant nature of LCB waste materials is mainly attributed to the rigid cell wall structure, crystalline cellular machinery, and lignin component, which makes lignocellulosic materials resistant toward chemical and biological actions. Hence, the pretreatment process is an obligatory step to make LCB materials accessible for the generation of sugar fractions after disintegration of biomass. Prior to the downstream processing of disintegrated complex LCB materials, appropriate pretreatment techniques can be used to decrease crystallinity and solubilization of hemicellulose moiety. Hence, the recalcitrant LCB materials become more accessible toward enzymes/microbial attack, which also enhances the activity of enzymes on the biomass surface [15]. Pretreatment processes mainly propose the disintegration of cellulose, hemicellulose, and lignin moieties of lignocellulosic materials, which results in diminution in the size of the particles of LCB materials. Hence, there is a mode to augment surface areas for proficient enzyme action, which can bring about effective degradation of the complex LCB polysaccharides into simple sugars. These fermentable sugars can be further utilized by microbes to generate biohydrogen [15,16]. Several pretreatment processes have been used for divergent LCB materials but each with some pros and cons. Several kinds of pretreatment techniques are available (Figure 1), including physical, chemical, physico-chemical, biological, and nanotechnology-based processes [17,18]. Every pretreatment process trails its own specific experimental settings to disrupt complicated LCB structure to generate value-added products including various chemicals and biofuels.

2.1. LCB Pretreatment by Physical Process

LCB pretreatment by physical methods augments hydrolytic efficiency and decomposition of LCB materials into biofuels and value-added products [19]. Various physical methods for treating LCB materials mainly include mechanical milling, steam explosion, ammonia fiber expansion (AFEX), pyrolysis, microwave irradiation method, etc. Mechanical processes include shredding, grinding, milling of LCB materials, which can break LCB fibers and thus decreases the required time to utilize LCB materials for subsequent treatments to obtain fermentative bioethanol [20]. The shredded materials after fractionation become finer, which makes their hydrolysis effective. This method can offer green pretreatment without generation of any other inhibitory compounds, which can be used directly for conversion into simple sugars [21]. The only problem is that this is highly energy requiring method and hence it can be used for limited applications. Pyrolysis is another technique for LCB pretreatment [20,21], which mainly comprises of thermochemical breakdown of LCB materials initiating at approximately 200 °C. Microwave is one of the new options for pretreatment of biomass materials to disintegrate complex 3D structure of LCB materials, which can make them accessible for further sugar generation [20]. The microwave irradiation treatment on sugarcane bagasse using phosphoric acid along with glycerol exhibited the release of lignin (5.4%) and xylan (11.3%) fractions. Further, to obtain higher yields, sugarcane bagasse can be treated with microwave irradiation along with enzyme hydrolysis of cellulose/hemicellulose fractions [20]. Ultrasonication radiation (of approximately 20–40 kHz) can be applied for biomass pretreatment, which leads to degradation of LCB structures by dint of rupturing and loosening fibrils through breakage of inter-chain hydrogen linkages [22], which can be used for biohydrogen production.

2.2. LCB Pretreatment by Chemical Process

Usually, chemical pretreatment methods are used extensively as compared to other biological or physical methods due to their effectiveness toward degradation of complex LCB materials. In the acid pretreatment process, LCB materials can be treated by organic/inorganic acids, such as HNO₃, HCl, H₂SO₄, formic acid, phosphoric acid, etc., to breakdown hydrogen and glycosidic bonds present in cellulose-hemicellulose fractions to liberate simple sugar units [10,23]. Mostly, during the acid pretreatment process, concentrated acids (30–70% at lower temperature) or dilute acids (0–10% v/v at 120–250 °C) are added to the LCB materials [24]. Dilute acid pretreatment can be used for pretreatment of various substrates, such as, poplar, corn stover, corn cob, switch grass, etc. The concentrated acid pretreatment can increase the rate of sugar release, but acids are more toxic in nature. The use of acid during pretreatment of biomass generates a large amount of inhibitory compounds, such as phenolic acids, aldehydes, furfurals, and 5–hydroxymethyl furfural (5-HMF). Further treatment is also required to recover acids after hydrolysis [25]. Thus, dilute acid treatment is usually preferred to hydrolyze LCB materials into sugar with the generation of lower amount of inhibitor products, which is an economical and environmentally friendly option [23]. The dilute acid, i.e., H₂SO₄ (0.4%) pretreatment on wild rice grass exhibited the release of 163 mg sugar per gram of substrate [26]. Alkaline pretreatment of LCB materials encompasses the application of bases (e.g., NaOH, NH₄OH, etc.) resulting into increase in surface area and decline in crystallinity through breakage of ester and other bonds among lignin, hemicellulose and others [10,25]. Thus, after pretreatment, the obtained cellulose/hemicellulose fractions exhibit higher hydrolysis to generate simple fermentable sugars through enzymes/microbial actions. Usually, LCB sources, including agricultural wastes, hardwood, low lignin containing plants are suitable for alkali treatment. Nonetheless, the extreme use of alkali inhibits the anaerobic methanogenesis processes and it may also lead to soil salination and water pollution [27]. The pretreatment of wheat straw with NaOH at 100 °C for 6 h exhibited dissolution efficiency of 86.7% [28]. Various studies have been reported for enhanced biofuel production titers, using alkali treated LCB materials, including corn stover, corn cob, wheat straw, etc. [29,30]. The advantage of using alkali for biomass pretreatment is that it can remove lignin/hemicellulose moieties effectively to enhance biomass surface area and make it more amenable for further hydrolysis.

Another promising approach for LCB pretreatment is organosolv method which can be conducted using organic solvents, such as phenol, acetone, alcohols, ethylene glycol, etc., along with the addition of some inorganic acids to stimulate the pretreatment efficiency of LCB materials under specific conditions [10,31]. This organosolv pretreatment can absolutely remove hemicellulose fractions in LCB, without affecting cellulose moiety, which provides a larger surface area and pore volume of cellulose. Hence, lignin moieties are dissolved in the liquid solvent phase and cellulose can be isolated in solid form [32]. The ethanosolv pretreatment along with sulphuric acid of poplar biomass exhibited 78% hydrolysis [33]. The organosolv pretreatment method has several advantages over other methods, such as easier solvent recovery, environmentally safer, and obtaining superlative lignin to generate value-added products.

Ozonolysis can be considered as a powerful method to pretreat LCB materials using ozone (O₃), which degrades hemicellulose and lignin moieties by keeping cellulose intact [22]. During ozonolysis, lignin is oxidized into low molecular weight compounds, such as acetic acid, formic acid, etc. This pretreatment exhibited more accessibility toward enzymatic hydrolysis, which improved sugar release significantly using various LCB materials, such as corn stalks, wheat straw, rye straw, etc. [34,35]. The best thing about using this pretreatment is that there is no generation of toxic inhibitory products, but the overall process is more expensive than others.

Ionic liquids (ILs) can be another better option for disintegration of various LCB feedstocks. ILs are thermostable organic salts constituted of cations and anions in liquid form with lower melting point and vapor pressure [23]. During LCB pretreatment using ILs, both cations and anions form a strong interaction with surface –OH groups present on carbohydrate components through hydrogen bonding. Consequently, lignin is dissolved in ILs and the extent of cellulose suspension increases in the presence of electron-donating groups of IL cations, which can be obtained in the form of precipitate [36].

Several ILs, such as, 1–butyl–3–methylimidazoliumchloride, 1–butyl–3–methylimidazoliummethylsulfate, 1–benzyl– 3–methylimidazoliumchloride, 1,3–dimethylimidazolium groups, etc., have been broadly applied for pretreating different substrates, such as rice straw, rice husk, poplar wood, wheat straw, and pine [22,37]. Several studies reported hydrolysis and fermentation using LCB materials through different ILs such as choliniumlysinate and ethanolamine acetate produced glucose (25–85%) and xylose (15–80%) [38,39]. Although all methods described here are effective to a certain extent for biomass disintegration, they have some flaws concerning environmental aspects, generation of inhibitory compounds, and final yields of individual components.

Table 1. Advantages and disadvantages of various chemical pretreatment methods.

Pretreatment Process	Advantages	Limitations and Disadvantages	References
Ionic liquids (ILs)	Environmentally friendly Nonderivatizing, nonvolatile, thermostable single component solvent for cellulose with potential applications incellulose fractionation and dissolution	High cost Poor biodegradability Toxic to micro-organisms	[40]
Ozonolysis	Reduces lignin content Does not produce toxic residues	Large amount of ozone required Expensive	[41]
Acid hydrolysis	Hydrolyzes hemicellulose to xylose and other sugars to alter lignin structure	High cost Equipment corrosion Formation of toxic substances	[42]
Alkaline hydrolysis	Removes hemicellulose and lignin Increases accessible surface area	Long residence times required Irrecoverable salts formed and incorporated into biomass	[43]
Organosolv	Organosolv lignin is sulfur free with high purity and low molecular weight Can be used as fuel to power pretreatment plant or further purified to obtain high quality lignin, which is used as a substitute for polymeric materials Very effective for the pretreatment of high-lignin lignocellulose materials	Solvents need to be drained from the reactor, evaporated, condensed, and recycled High cost Generation of compounds inhibitory to micro-organisms	[44]
Pyrolysis	Produces gas and liquid products	High temperature Ash production	[45]

presents several advantages and disadvantages associated with different chemical methods.

2.3. LCB Pretreatment by Physico-Chemical Process

Amongst the known physicochemical methods, hot water, steam explosion, and ammonia fiber explosion (AFEX) are promising processes to disintegrate the obstinate structure of LCB materials. Steam explosion and hot water pretreatment processes generate high amounts of degradation products, such as furfural, 5-HMF, phenolic compounds, formic acid, etc. which can be inhibitory to microbial strains [46]. In the case of steam explosion, LCB materials are treated at high steam pressure of approximately 0.6–4.9 MPa and a temperature ranging from 160–210 °C for different time. At very high pressure/temperature conditions, both steam explosion and AFEX can certainly breakdown the rigid components of LCB materials to generate fermentable sugars [47]. The major flaw of using the steam explosion method is the partial hemicellulose degradation, which thus divert into generation of toxic byproducts. The application of the steam explosion method for corn stover treatment was used to generate 113.5 million liters of butanol annually. The higher release of glucose was achieved using olive tree prunes, which was up to 86% at high temperature, and pressure conditions for 15 min [48]. Hot water pretreatment in the presence of chemicals is an ultimate method for LCB substrates, which can result in the effective utilization of biomass. This process is somewhat similar to steam explosion pretreatment method, which does not need chemicals, and hence there is no generation of inhibitory compounds [49]. Further, AFEX is an effective and novel process for breaking down LCB components into simple sugars. This method can act effectively on low lignin containing LCB substrates, such as corn stover, miscanthus, switch grass, etc., exhibiting approximately 90% of glucose generation during hydrolysis process.

2.4. LCB Pretreatment by Biological Process

Usually, biological pretreatment methods are better than other chemical and physical methods [40] due to several benefits, such as a low energy requirement, no generation of toxic compounds, etc. However, biological processes show a lower hydrolysis rate, which is the major drawback of these processes. In biological pretreatment of LCB substrates, different microbes (e.g., bacteria, fungi, etc.) and enzymes have been employed which plays a significant role in pretreating biomass [50]. Usually, fungal strains, such as soft rot, brown, and white fungi have been broadly used to deconstruct LCB materials to be used for fermentation [50]. White rot fungus is able to degrade the hard layer of lignin present in biomass, due to the action of lignin degrading enzymes, such as laccase, peroxidase, etc. Brown rot fungi attack specifically cellulose moiety, whereas white and soft rot fungi mainly act on both lignin and cellulose components of LCB. The widely used white rot fungi include Pleurotus ostreatus, Pycnoporus cinnabarinus, Ceriporiopsis subvermispora, Cyathus stercolerus, Cyathus cinnabarinus, etc., which can degrade lignin as these strains can secrete lignin degrading enzymes, such as ligninase, peroxidase, laccases, etc. [46]. The pretreatment of bamboo using lignin degrading Punctularia sp. exhibited an increase in the sugar concentration of almost 60% by reducing lignin content [51].

2.5. LCB Pretreatment Using Nanotechnology

Pretreatment of LCB materials using a nanotechnology approach is one of the significant methodologies to generate biofuels. The application of NPs can display catalytic actions for LCB processing almost similar to using chemical methods [34]. Mostly, magnetic nanoparticles (MNPs) have been used widely for the pretreatment of LCB materials since they can be reused for subsequent cycles which ultimately reduces the overall process cost [19]. Due to the nano size of particles, NPs enter the cell wall of LCB materials, thereby interacting with biomass components to generate fermentable sugars [25]. Acid-functionalized MNPs possess higher affinity for hydrolyzed LCB materials, which are known as solid acid nanocatalysts. Owing to the strong magnetic nature, the reusability of MNPs has added a beneficial role in various applications. Such acid-functionalized MNPs exhibit better hydrolytic ability for biofuel production [14,52]. The treatment of wheat straw with perfluoroalkylsufonic and alkylsufonic acid-functionalized NPs liberated almost 31% higher sugar concentration as compared to the control reaction [14]. In addition, NPs are required in small quantities and can be reused for the next cycle of the process. Recently, the effect of magnetic iron oxide (Fe₃O₄) NPs on LCB pretreatment and further biogas production has been studied which exhibited the remarkable improvement in biogas production in the presence of Fe₃O₄ MNPs [53]. Nonetheless, till date there is not much information available on LCB pretreatment using NPs. Thus, more efforts are needed to search for possible ways to make the process viable at commercial scale. In recent years, an inventive improvement has been made in nanotechnology field, where enzyme/biocatalyst can be immobilized on MNPs. The immobilization of enzymes on MNPs is a budding method that can increase the enzyme’s catalytic efficiency [13]. Owing to the magnetic nature of MNPs, the immobilized enzymes can be recycled and reused for several cycles of biomass hydrolysis. Enzyme immobilization using NPs is called ‘nanobiocatalyst’, which is a depiction of emerging developments in the field of nanobiotechnology. The different aspects of enzymatic hydrolysis of LCB substrates have already been described by Singhvi et al. [15].

3. Biohydrogen Production by Fermentation

Generally, hydrogen production could be achieved by biological, physical, and physio-chemical processes [7,54]. Physico-chemical processes are not environmentally friendly and sustainable due to the involvement of fossil fuels as substrate. Biological processes seem to be more attractive as fermentation processes can be conducted under ambient conditions [8]. In particular, dark fermentative hydrogen production is regarded as a more feasible commercial process since it could achieve high hydrogen production rate without the limitation of light. The maximum theoretical yield of biohydrogen from hexose fermentation is 4 mol H₂ per mol of consumed hexose as in Equation (1) [55].

C₆H₁₂O₆ + 4H₂O → 2CH₃COO⁻ + 2HCO₃⁻ + 4H⁺ + 4H₂

(1)

A wide variety of microorganisms are able to form hydrogen in dark fermentations, including strict anaerobes, facultative anaerobes, aerobes, and co- and mixed cultures that produce hydrogen gas during the exponential growth phase. The highest hydrogen yields have been obtained by thermophilic anaerobic bacteria, i.e., Clostridia, and hence they are preferred for hydrogen production. The bioconversion of LCB materials under anaerobic conditions can evolve hydrogen as a by-product via hydrogenase enzymes and protons, acting as an electron sink to dispose of the excess electrons [55] (Figure 2).

Till date, commercial scale/pilot scale microbial hydrogen production technologies have not yet been established. Much research focus needs to be directed toward making the technology shift from bioethanol and biobutanol toward hydrogen. The employment of LCB as a renewable substrate for hydrogen fermentation is challenged by its recalcitrant nature and hence pretreatment is needed to obtain fermentable sugars from LCB components. Nanobiotechnology can be a key to the currently available pretreatment processes. The use of MNPs enables easy recovery of enzymes. Hence, the exploitation of nanomaterials for LCB to hydrogen generation will aid in developing a sustainable approach. With this perspective, we assume that the use of nanomaterials in LCB pretreatment and hydrolysis can replace cellulase/hemicellulase enzymes and aid in providing the commercially viable technology. Figure 3 illustrates the various steps in LCB processing, including pretreatment and hydrolysis using NPs and further the conversion of released sugars into hydrogen along with the generation of useful byproducts.

Role of Nanotechnology in Hydrogen Production

Currently, the production of biofuels using nanomaterials is attracting significant attention in the biofuel industry [50]. In such cases, reactions can be performed using metal NPs, such as nickel (Ni), iron (Fe), cobalt (Co) materials [15,23,25,53]. During hydrogen fermentation, these NPs can also act as cofactors for hydrogenase enzymes, which can reduce the exchange of protons (H⁺) [50]. The function of the hydrogenase enzymes present in microbes has been affected by NPs concentration yielding biohydrogen. Thus, the inclusion of nanotechnology advances the electron transfer rate, which ultimately improves the metabolic activities of microbial strains [15]. The major shortcoming of using NPs-associated approach is a slower reaction rate and a low hydrogen yield. Moreover, in the case of LCB substrates, only approximately 35% of LCB components can be converted into hydrogen and the rest of residues generate some other byproducts [56]. Hence, there is a need to improve overall technique for commercial production of biohydrogen using LCB substrates through microbial strain improvement as well as by synthesizing efficient and suitable nanomaterials.

During the hydrogen fermentation process, hydrogenase enzyme present in hydrogen producer strains plays a significant role in the reduction of proton exchange. Mainly, Fe and Ni are present as cofactors in hydrogenase enzyme’s active site [57], which demonstrated their influence in the hydrogen production pathway during fermentation. Since the catalytic efficiency of hydrogenase enzyme depends on the occurrence of Fe/Ni cofactors, the concentration of hydrogen production is majorly affected by the presence or absence of metal cofactors [56]. The supplementation of higher concentration of NPs can inhibit the hydrogenase enzyme activity, thus microbial metabolism and ultimately biohydrogen productivities will be affected [57]. An employment of nanomaterials is a novel technology to improve metabolic activities during biohydrogen fermentation. The use of NPs has shown their potential in ameliorating oxidoreductase activities by accelerating the electron transfer rate, which is consistent with catalytic efficiencies [58].

Fe has substantiated the fact of being the principal cofactor of hydrogenase enzymes, therefore Fe-based NPs have been vastly applied during biohydrogen production. Apart from Fe and Ni NPs, various other NPs, e.g., Co, Ag, Au, etc., have been used for increased hydrogen production rate and yield during fermentation conditions [59,60]. A higher concentration of hydrogen was produced with a production rate of 80.7 mL/h in the presence of Fe₂O₃ NPs under acidic pH conditions. Further, kinetic studies exhibited that increased substrate concentration causes a decrease in the level of biohydrogen production [61]. The employment of α-Fe₂O₃ (hematite) and NiO (nickel oxide) NPs elevated hydrogenase enzyme activities, which showed almost 4.5-fold enhancement in hydrogen production levels [62]. It has been observed that metal NPs can upturn 4.5 times of biohydrogen production as compared to control conditions. The addition of Fe₃O₄ NPs exhibited almost a 33% enhancement in biohydrogen production in sucrose containing media [63]. The supplementation of TiO₂ NPs along with diluted sulphuric acid in the fermentation media containing sugarcane bagasse liberated an approximately 260% higher concentration of sugar, which ultimately elevated the levels of H₂ production by 127% [64]. Moreover, the use of NiNPs (0.05 wt%) during hydrogen fermentation demonstrated 22.71% higher hydrogen yields, with a production of 2.54 mol of H₂/mol of glucose [65].

The influence of different nanomaterials on biohydrogen production levels has been studied using various substrates, as displayed in

Table 2. Influence of various nanoparticles on hydrogen production using different substrates.

Sr. No.	Nanoparticles	Microbial Strains	Substrates	Improvement in Hydrogen Production (%)	References
1.	NiO,	Bacillus anthracis	Palm oil mill effluent	151	[68]
	CoO			167
2.	FeO	Enterobacter sp., Clostridium sp.	Grass	73.1	[59]
3.	Fe3O4	Anaerobic sludge	Sugarcane bagasse	69	[69]
4.	FeNPs	Mesophilic culture	Starch	200	[70]
5.	Fe2O3	Enterobacter aerogenes	Cassava starch	92	[71]
6.	NiO	Anaerobic sludge containing H2 producing bacteria	Molasses waste	24	[62]
	Fe2O3			43
7.	TiO2	Anaerobic sludge	Sugarcane bagasse	127	[64]
8.	Fe2O3	Thermophillic anaerobic mixed culture	Glucose	53.6	[72]
9.	Fe2O3-Fe3O4/carbon nanocomposite	Anaerobic mixed bacteria	Glucose	33.7	[73]
10.	FeNPs	Enterobacter cloacae	Glucose	130	[74]
11.	Nano activated carbon	Anaerobic sludge	Sucrose	70	[75]
12.	Nano activated carbon	Anaerobic sludge	Sucrose	70	[76]
13.	Pd, Ag, Cu, and Fe encapsulated SiO2 NPs	Clostridium butyricum	Glucose	38	[66]
14.	Caron nanotube	Anaerobic sludge	Glucose	50	[20]
15.	Activated carbon	Anaerobic sludge	Sucrose	62.5	[77]
16.	CeFe3O4	Clostridium cellulovorans	Corn cob	149	[78]
17.	Fe3O4	Parageobacillus thermoglucosidasius	Potato peel	315	[79]

. The inclusion of various metal oxide NPs, such as Ni, Cu, Ag, Fe, Pd, etc., showed significant improvements in hydrogen production levels using pure sugar substrates by Clostridium sp. under dark fermentation. Amongst those NPs, Fe₂O₃ generated higher titres of hydrogen (~40%) as compared to the control [66]. The impact of all NPs on hydrogen production levels depends on the sort of interaction between enzymes and nanomaterials to boost the rate of electron transfer, which will eventually raise yields of hydrogen. It is well known that the nano size of NPs can improve the rates of reaction immensely, and hence the use of NPs will aid in removing extra oxygen from broth, which increases hydrogenase enzyme activity [67]. The green synthesized NPs also exhibited enhancement in hydrogen production rate [60], with a yield of 2.35 mol of hydrogen/mol of carbon feedstock. Table 2 presents the various studies conducted using different nanomaterials and their impact on hydrogen yields.

4. Future Perspectives and Directions

Recently, research on biohydrogen production has attracted tremendous attention worldwide. LCB comprises the largest waste produced by agro-industrial (e.g., agricultural by-products and paper mill sludge) and other (urban solid waste) activities. Unfortunately, available technologies for cellulose and hemicellulose (i.e., 2nd generation) biorefinery are yet too expensive since they employ commercial cellulase and hemicellulase cocktails (for biomass hydrolysis), which represent a main economic constraint because of their price. Twenty-year attempts to render cellulase production cheaper were substantially unsuccessful since commercial enzyme cost has basically remained the same since the 1990s. Third generation biofuels and chemicals, such as hydrogen, ethanol/butanol, etc. through cellulose/hemicellulose consolidated bioprocessing (CBP) can help to solve these problems and can sustainably supply a larger proportion of global fuel supply.

Today, hydrogen is mainly produced from natural gas, but its production is more sustainable when using renewable resources, such as biomass as raw materials of the process. Fermentative hydrogen production processes have several advantages over other processes. Considering the problem of less hydrogen productivities using the microbial fermentation process, our efforts should be toward improving the ability of the strains to degrade LCB components.

Our main emphasis should be on the utilization of both cellulose and hemicellulose fractions of biomass after delignification of LCB materials. Accordingly, protocols and technologies can be optimized to find their potential for further industrial implementation. In addition, hyperproducing recombinant strains can be developed, which can degrade cellulose and hemicellulose fractions of waste LCB materials by secreting required enzymes for enhanced production of biohydrogen. The metabolic and biochemical characterization of this pathway will therefore be of fundamental academic interest. Metabolic engineering approach can be exploited to manipulate the cellulose and hemicellulose degradation process, which is of great interest in industrial biotechnology. Moreover, this will be an original application of metabolic engineering route on the new biomass degradation pathway in microbes, which may boost future projects considering the break-down of both cellulose and hemicellulose components for hydrogen production. In addition, new concept of LCB-to-biohydrogen will also be boosted through the detailed study of the evaluation of novel bioproducts generated that will provide alternatives for the available conventional energy sources. The final aim is to achieve biohydrogen with a high final titer and/or yield and/or productivity required by industrial processes. Further study will provide a novel biocatalytic route to biohydrogen production from a renewable feedstock and highlight the opportunity for bioconversion of cellulose and hemicellulose into biohydrogen using biotechnology. Realistic previsions of the economic advantage of CBP over current technologies estimate a cost reduction of approximately 50–75%. Attaining the present goal will have a dramatic reduction effect on the cost of renewable LCB derived fuels and can help to solve problems of lignoprocessing with greater environmental benefits.

5. Conclusions

Bioconversion of LCB materials into biohydrogen is the most sustainable option toward economic development and carbon neutrality. Suitable pretreatment processes are required for making LCB materials more accessible toward enzyme attack. The utilization of lignin-based molecules and other value-added products, i.e., lignin derived molecules are highly recommended. The cost of these enzyme and their application in biofuel production are the most challenging because of the complex structure and the diverse nature of LCB waste. Work effort and progress toward development of an efficient and economical enzyme system need critical and in-depth evaluation of LCB substrate and a sound bioprocess to achieve a more sustainable and economical process to develop carbon neutrality and industrial production of biofuels, such as hydrogen. Additionally, applications of different microbial engineering tools are expected to accelerate toward sustainable developments in this area.