TIBTEC 2068 No.

of Pages 15

Trends in
Biotechnology
Review

Food Waste: A Promising Source of

Sustainable Biohydrogen Fuel
Mahmoud M. Habashy , 1,* Ee Shen Ong, 1 Omar M. Abdeldayem, 1
Eslam G. Al-Sakkari, 2 and Eldon R. Rene 1

Annually, approximately 1.3 billion tons of food is lost worldwide, accounting Highlights
for one-third of annual food production. Therefore, turning food waste into Food waste and food-processing waste
energy is of enormous environmental significance because of its sustainable are important feedstocks for producing
biohydrogen that can be exploited to
nature. Nutrients and organic acids present in food waste can be used to
produce energy.
produce (bio)products such as biohydrogen through biological processes.
However, our understanding of the production of biohydrogen from food waste Biohydrogen has a higher energy con-
through photofermentation and dark fermentation is still restricted. This compre- tent per unit mass (34 Kcal/g-H2) than
fossil fuels.
hensive study aims to review the potential of food waste for biohydrogen produc-
tion using microbial mediators, including a brief overview of process parameters Microbial electrolysis is an emerging
that affect the (bio)hydrogen production pathway. technology that can achieve sustainable
and clean hydrogen production from a
wide range of renewable feedstocks.
Biohydrogen: An Alternative Clean Source of Energy
Demand for clean and sustainable energy is increasing whereas the limited reserve of fossil fuels Different operational parameters such as
is continually being depleted. It is estimated that, if the current trend of fossil fuel usage continues, pH, temperature, C:N ratio, hydraulic re-
tention time, and microbial community
fossil fuel resources may be consumed in 35, 107, and 37 years for oil, coal, and gas, respectively
proliferation influence the performance
[1]. Furthermore, the overuse of fossil fuels poses a significant threat to future generation because and efficiency of photofermentation and
toxic emissions from different combustion activities impose a strain on the environment, leading dark fermentation technologies.
to climate change [2]. For these reasons the exploration of new sustainable and eco-friendly
Pretreatment of food waste (e.g., by
replacements for fossil fuels to save the planet from environmental catastrophe has gained
physical, chemical, or enzymatic
significant attention among researchers, environmental scientists, and policymakers [2–4]. methods) is vital because this minimizes
downstream processing and enhances
Climate change is regarded as one of the defining challenges of our times; it is therefore not sur- biohydrogen yield.

prising that one of the sustainable development goals (SDGs) focuses on 'urgent action to com-
bat climate change and its impacts' [5]. Hydrogen is considered to be a promising source of clean
energy and fuel [6]. The production of (bio)hydrogen from renewable feedstocks or solid wastes
(e.g., food waste) is considered to be a green, sustainable, and eco-friendly approach.
Biohydrogen can play a vital role in reducing greenhouse gas (GHG; see Glossary) emissions
by the bioconversion of food waste, a carbon-neutral source, to (bio)hydrogen [7,8]. In the EU
post-coronavirus disease 2019 (COVID-19) economic recovery package, the hydrogen
economy is considered to be one of the priorities of the European Green Deal. 1
Department of Water Supply,
Sanitation, and Environmental
Moreover, biohydrogen has many features that make it more desirable than other biofuels that are Engineering, IHE Delft Institute for Water
Education, Westvest 7, 2611AX Delft,
presently being produced. These features include elevated gravimetric density, reduced emis-
The Netherlands
sions, and greater effectiveness in raw material (i.e., the feedstock) to energy conversion [9]. 2
Chemical Engineering Department,
Since it can be transformed directly into fuel cells instead of being combusted, fuel-cell vehicles Cairo University, Cairo University Road,
12613 Giza, Egypt
utilizing renewable hydrogen are recognized as life-cycle carbon-free options for the transporta-
tion sector (Figure 1). Moreover, hydrogen is highly efficient as an alternative fuel source com-
pared with other fuel types. It has the highest energy content per unit weight – 34 Kcal/g-H2,
compared with petroleum, paraffin, coal, castor oil, and wood which have energy contents of *Correspondence:
10.3–8.4 Kcal/g, 10.3–9.8 Kcal/g, 7.8 Kcal/g, 9.4 Kcal/g, and 4.2 Kcal/g, respectively [10]. mab007@un-ihe.org (M.M. Habashy).

Trends in Biotechnology, Month 2021, Vol. xx, No. xx https://doi.org/10.1016/j.tibtech.2021.04.001 1

© 2021 Elsevier Ltd. All rights reserved.
 Trends in Biotechnology

Glossary
Chemical oxygen demand (COD):
the number of oxygen equivalents
consumed during the chemical oxidation
of organic matter.
Greenhouse gases (GHGs): gases
that trap heat in the atmosphere. The
primary GHGs are water vapor (H2O),
carbon dioxide (CO2), methane (CH4),
nitrous oxide (N2O), and ozone (O3).
Green non-sulfur bacteria (GNSB):
oxygenic phototrophic and
nonphototrophic bacteria that grow
under facultative aerobic conditions.
Green sulfur bacteria (GSB):
anoxygenic phototrophic bacteria that
grow under strictly anoxic conditions.
Hydraulic retention time (HRT): the
average time duration that soluble
compounds remain in a bioreactor. It is
usually measured as the ratio of the
reactor volume to the flow rate.
Light-emitting diode (LED): a
semiconductor light source that emits
light when current passes through it.
Metal–organic frameworks (MOFs):
organic–inorganic hybrid crystalline
Trends in Biotechnology
materials with high porosity and a regular
Figure 1. Biohydrogen Passes through Different Stages, Starting from Food Production in the Agricultural array of positively charged metal ions
Sector to Its End Use by the Consumer. After the food is consumed, waste is produced. The food waste can be surrounded by organic linker molecules.
transported by trucks to the collection and processing industries for use as feedstock for bioreactors operating by either Polyhydroxyalkanoate (PHA): a
photofermentation or dark fermentation. Biohydrogen is produced and distributed for use as a clean fuel in the biopolyester produced by various
transportation sector. Transport vehicles produce water vapor that will condense and help in forming clouds. These microorganisms that is used as an
clouds will produce precipitation to grow food again, which will be consumed by people, and this cycle will be repeated. energy storage material within the cells.
Purple non-sulfur bacteria (PNSB):
anoxygenic photosynthetic bacteria that
Compared with hydrocarbon fuel, hydrogen has a 2.75-fold higher energy content, making it an utilize organic compounds as electron
ideal energy source [11]. donors.
Purple sulfur bacteria (PSB):
anoxygenic photosynthetic bacteria
However, hydrogen utilization as a fuel source remains challenging because of its unavailability utilize sulfide and hydrogen as electron
as a ready-to-use source and the relatively high-price of its production chain from natural gas. donors.
Conventionally, commercial-grade hydrogen is produced by steam reforming of methane Pyruvate-ferredoxin oxidoreductase
(PFOR): an enzyme that catalyzes the
(SRM), steam reforming of other hydrocarbons (SRH), noncatalytic partial oxidation of fossil
conversion of pyruvate, CoA, and
fuels (POX), and autothermal reforming that combines SRM, POX, and coal gasification (Box 1) oxidized ferredoxin into acetyl-CoA,
[7,12]. However, these methods use a nonrenewable source of feed/raw material and are there- CO2, and reduced ferredoxin.
fore not sustainable. Alternatively, hydrogen can also be produced by splitting water, which is Pyruvate-formate lyase (PFL): an
enzyme that catalyzes pyruvate and
considered to be the cleanest technology for hydrogen production (Box 1) [13]. Nevertheless, CoA conversion into formate and
high cost remains a significant limitation for all of these conventional technologies because all acetyl-CoA.

Box 1. Mechanisms to Produce Hydrogen

Steam reforming of methane (SRM) CH4 + H2O ⇌ CO + 3H2 [I]
Steam reforming of other hydrocarbons (SRH) Cn Hm þ nH2 O ⇌ nCO þ ðm2 þ nÞ H2 [II]
1
Partial oxidation of fossil fuels (POX) CH4 þ 2 O2 ⇌ CO þ 2H2 [III]
1 1
Autothermal reforming Cn Hm þ 2 nH2 O þ 4 nO2 ⇌ nCO þ ðmþn
2 Þ H2 [IV]

Water–gas shift CO + H2O ⇌ CO2 + H2 [V]

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these processes have a high thermal requirement or electricity requirement [14]. Hence, generat-
ing clean hydrogen fuel from polluting and nonrenewable feedstock is a contradictory approach.

On account of the current global crisis on climate change, the benefits of hydrogen can be
effectively exploited if cost-effective and efficient hydrogen-production technologies can be
developed. As an alternative to physicochemical methods, commercial-scale hydrogen produc-
tion can be achieved using a biological approach from biorenewable sources [15]. Hydrogen that
is produced using this approach is commonly known as biohydrogen [16]. Biohydrogen can be
produced by direct or indirect biophotolysis, photofermentation, dark fermentation, or a combi-
nation of these processes (Figure 2) [17]. Microbe-mediated biological processes have a signifi-
cant advantage in terms of their operating temperature and pressure, and the ability of
microorganisms to utilize a broad range of substrates. Therefore, such an approach is more prac-
tical and relevant for (bio)hydrogen production because the costs are much lower than those of
conventional physicochemical processes [18,19]. Physicochemical processes require high tem-
peratures (150–850°C) and complex machinery to produce hydrogen, which consumes more
energy and hence, incurs more operational costs.

Although previous studies have been published on biohydrogen production, there is a knowledge
gap in its sustainable production using food waste as the feedstock [20]. Food waste in the context
of this article refers to the organic fraction of the waste derived from agricultural production/resi-
dues, post-harvest handling and storage, processing and packing, distribution, consumption,
and discarded food. In this article we provide a comprehensive review of the potential of using
food waste, including industrial food-processing waste, for biohydrogen production using micro-
bial mediators. We provide a brief overview of process parameters that affect hydrogen production,
and strategies are described to improve technical performance and enhance biohydrogen yield.

Biomass to hydrogen producon routes

Physico-
Biological
chemical

Direct/indirect Photo Dark

Pyrolysis
biophotolysis fermentaon fermentaon

Microbial
electrolysis cell
(MEC)

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Figure 2. Hydrogen Can be Produced by Two Different Methods – Physicochemical and Biological. The
physicochemical method focuses on physical and chemical ways that use food waste to produce hydrogen, such as
pyrolysis and gasification. The biological method depends on microorganisms that produce biohydrogen from food waste.
There are three biological routes to produce biohydrogen – direct/indirect biophotolysis, photofermentation, and dark
fermentation. Moreover, a recent technology combines the physicochemical and biological methods to produce
biohydrogen in a device known as a microbial electrolysis cell. This technology requires further research at the pilot and
semi-industrial scales before it can be commercialized.

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Food Waste, a Potential Feedstock for Biohydrogen Production

Food waste is a global problem. It is of high importance for promoting a circular economy because
utilizing it can increase the income, improve food security, and provide energy in the world's poorest
countries. Food waste has a high impact on food security, food quality, and economic develop-
ment, and exploiting food waste has a beneficial role in preserving the natural environment [21].

Furthermore, global energy demand is rapidly growing in parallel with population and economic
growth [22]. To cope with the rapid increase, the use of renewable resources such as food and
food-processing wastes for biohydrogen production can be an innovative approach to replace
fossil fuels and solve municipal solid-waste disposal problems [22].

Food waste is commonly used as the feedstock/raw material in anaerobic digestion systems to
produce energy because of its dependable physicochemical and biological characteristics,
such as moisture content (75–90%), high substrate concentration [chemical oxygen demand
(COD): 19–346 g/l, carbohydrate content: 25.5–143 g/l], and high carbon to nitrogen (C:N) ratio
(14–37) [23]. The physicochemical characteristics of food waste can affect the production and
yield of (bio)hydrogen. The main parameters that can be controlled are the pretreatment condi-
tions for food waste (e.g., physical, chemical, or enzymatic), temperature, pH, and the hydrogen
partial pressure [24]. In addition, conditions such as the moisture content, nutrient content, vola-
tile solid composition, particle size, and biodegradability of food waste need to be considered to
maximize biohydrogen production at the industrial scale [25].

There are several ongoing research on biohydrogen production from food waste using mixed cul-
tures from anaerobic sludge, manure, and compost in batch, semi-continuous, and continuous
bioreactors. This research suggests that the availability of endogenous microorganisms and
the high carbon content in food waste make it an excellent candidate for use as a feedstock for
biohydrogen production under non-sterile conditions [26]. The use of a pure culture inoculum
for biohydrogen production from food waste, and maintaining the purity of the culture during
long-term reactor operation, have not been reported in the literature [26].

Hydrogen fuel cell technologies have been recognized as one of the technologies to reduce atmo-
spheric GHGs. The current global trend in hydrogen usage in the transportation sector further
increases demand and will ensure that hydrogen has adequate market potential over the next
10 years [27]. Globally, the entire food production and supply chains are responsible for resource
and environmental burdens – food waste causes adverse environmental effects and unnecessary
use of resources. Given the EU projections for future increases in food-waste generation,
biohydrogen from food waste can be a potential solution by transforming a linear economy into a
modern circular (bio)economy, which is also in line with the EU Circular Economy Action Plan [28].

This article also overviews current trends in biohydrogen production from food waste through
methods such as photofermentation and dark fermentation, as well as of the factors influencing the
production efficiency of each of these methods. This article gives in-depth technical knowledge re-
garding the industrial applications, challenges, and economics of photofermentation and dark fer-
mentation for optimal bioconversion of food waste to biohydrogen. It also suggests different
methods to overcome the processing, production, and storage problems of biohydrogen for use in
practice.

Only a limited number of studies have been performed using non-sterile food waste without
adding any further inoculum. Interestingly, untreated food wastes produce only a low yield of
biohydrogen compared with heat-pretreated food waste because of problems related to

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hydrogen-consuming bacteria [29]. It is therefore evident that food waste pretreatment can
enhance biohydrogen production efficiency, and this is considered to be a vital parameter that in-
fluences the operational efficiency of the process [30]. Food and food-processing wastes can
also be utilized to produce biohydrogen that can generate electricity for food-processing
industries.

Pretreatment as the First Step in Biohydrogen Production

Unlike typical anaerobic digestion processes, bioconversion by photofermentation and dark
fermentation technologies is designed to overcome the methanogen barrier to avoid further
conversion of hydrogen to methane. This can typically be achieved by adding a suitable methane
inhibitor and/or by altering the reactor operating conditions such as retention time, pH, and vol-
atile solid content. One of the commonly adopted approaches in laboratory scale studies is the
heat-based pretreatment of the inoculum. However, evidence proves that these methods have
only short-term effects on hydrogen production/yield and are not considered a practical
(industrial) option for full-scale fermentative biohydrogen production [31].

Generally, the main components of food products or food waste are carbohydrates, proteins,
and lipids [32]. Carbohydrate is readily biodegradable, except for the lignocellulose fraction
because the highly crystalline configurations of cellulose with hemicellulose and lignin make
them resistant to hydrolysis [33]. The common practice of pretreatment includes preheating
and hydrolysis assisted by acid, alkali, and ultrasonics. However, their economic feasibility
remains unclear in full-scale applications because they incur high costs for energy and chemicals.
Kuang and colleagues [34] tested one approach that uses potassium ferrate, a strong oxidant, on
catering food waste. The results showed a significant enhancement of biohydrogen production
from food waste. From an economic point of view, the use of potassium ferrate will increase
the operational cost; nevertheless, the enhancement of biohydrogen production may compen-
sate for the additional expense.

In the photofermentation process, photofermentative bacteria are highly dependent on light for
the generation of energy required for their cellular growth and maintenance, and hence for gener-
ating hydrogen from the substrates supplied [35]. Therefore, light penetration through the me-
dium and its intensity is vital for the recovery of hydrogen via photofermentation. In addition, the
use of food waste as a substrate without pretreatments such as filtration and dilution is likely to
affect the biohydrogen production rate because the dark color and high turbidity of the substrate
will reduce the depth of light penetration.

Photofermentation
Photofermentation is a biological method that produces hydrogen from organic substrates with
the help of photosynthetic bacteria (Figure 3A). Generally, two types of bacteria are responsible
for hydrogen production – the purple non-sulfur bacteria (PNSB) and the green sulfur
bacteria (GSB). These bacteria include Rhodobacter sphaeroides, Rhodopseudomonas
palustris, Rhodobacter capsulatus, and Rhodospirillum rubrum [18]. They utilize sunlight to con-
vert the carbon sources (i.e., simple sugars or different volatile fatty acids) into hydrogen and CO2,
as shown in Equation 1 [36].

C6 H12 O6 þ 6H2 O þ 0 sun light0 → 12H2 þ 6CO2 ½1

Furthermore, although photosynthetic bacteria do not have sufficient energy to break down water
molecules, they can use simple organic acids as electron donors under anaerobic conditions.
Therefore, photofermentation uses nitrogenase enzymes to produce biohydrogen. As shown in

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Trends in Biotechnology

Figure 3. Biohydrogen Can Be Produced by Two Main Biological Methods, Photofermentation and Dark Fermentation. (A) In photofermentation,
photosynthetic bacteria utilize sunlight to convert the carbon-based substances in the food waste that act as electron donors to produce biohydrogen via nitrogenase
enzymes. (B) In dark fermentation, anaerobic bacteria (in the absence of light) catalyze a series of biochemical reactions that involve the conversion of carbon sources
in the food waste into biohydrogen via hydrogenase enzymes. Microorganisms use glycolysis to produce pyruvate, which produces acetyl-CoA that further breaks
down into lactate, acetate, and butyrate. Abbreviations: Fd(ox), oxidized ferredoxin; Fd(red), reduced ferredoxin.

Equation 2, one mole of nitrogen produces one mole of hydrogen and two moles of ammonia with
the help of nitrogenases [36]. However, more hydrogen can be produced in the absence of nitro-
gen, as shown in Equation 3 [36].

N2 þ 8Hþ þ 8e− þ 16ATP → 2NH3 þ 16ADP þ 16Pi ½2

8Hþ þ 8e− þ 16ATP → 4H2 þ 16ADP þ 16Pi ½3

Many factors can affect the performance and yield of photofermentation (Table 1). These in-
clude waste diversity, light efficiency, intensity, and wavelength, culture medium, and ammonia
formation. Ammonia can restrict the enzymatic activity of some phototrophic microorganisms;
it is therefore essential to remove ammonia to enhance hydrogen production from the
carbohydrate/sugar substrates in the medium. Mirza and colleagues [37] reported that
PNSB can improve hydrogen yields from food waste by eliminating polyhydroxyalkanoate
(PHA) synthesis.

Factors Affecting Hydrogen Production by Photofermentation

pH and Temperature
Two of the crucial parameters that can affect hydrogen production efficiency are pH and temper-
ature. In a recent study it was found that the best temperature for photofermentation was in the
range 28–32°C (mesophilic conditions), while the optimum pH was around neutrality (7.0) [36].
Although some microorganisms can function beyond 35°C, the use of thermophilic microorgan-
isms for photofermentation has not been reported [36].

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Table 1. Food Waste and Hydrogen Generation through Photofermentation

Substrate Reactor Microorganism pH Temperature Light Pretreatment Hydrogen Refs
type (°C) yield
Maize straw Batch Rhodobacter 7.1–7.8 30 4000 ± 200 lx Added 5% HCl at 4.6 mol-H2/ [62]
sphaeroides 118°C for 30 minutes mol-reducing
sugar
Cornstalk Batch Mixed culture 7.0 30 2000 lx by Added cellulase enzyme 2.6 mol-H2/ [63]
pith filament lamps mol-sugar
consumed
Corn stover Batch Mixed culture 6.5 30 3500 lx Air-dried, ground by a 141.4 ml-H2/ [44]
superfine pulverizer, g-TS
and then stored in
sealed bags at room
temperature
Dark Batch Rhodopseudomonas 6.8 34 8.5 W/m2 by a Supplemented with 755.0 ml-H2/ [64]
fermentation tungsten lamp growth medium l-waste
effluent of
sugarcane
bagasse
Sugar beet Batch Rhodopseudomonas 7.4–7.5 30 114 W/m2 by a Added buffer solution, 19.0 mol-H2/ [65]
molasses palustris tungsten lamp adjusted pH, and mol-sucrose
applied sterilization
Rotten apple Batch Mixed culture 7.1 30.5 3030 lx Crushed and screened 112.0 ml-H2/ [38]
with a 40-mesh sieve g-TS
Dark Continuous Mixed culture 7.0 30 3000 lx by solar Adjusted pH to 7.0 and 4.7 m3-H2/ [66]
fermentation and LED C:N ratio to 10:1 m3/d
effluent of bundles
corn stover

Lu and coworkers [38] studied the effects of different initial pH and temperature on hydrogen pro-
duction yield using apple waste as the substrate in a stirred batch reactor. The yield of hydrogen
was found to increase remarkably when the initial pH was increased from 4.0 to 7.0, and the
highest hydrogen yield (97 ml-H2/g-total solids, TS) was produced at an initial pH of 7.0. The
photofermentative bacteria usually produce organic acids during the hydrogen production pro-
cess, and these could inhibit hydrogen production at low initial pH levels. However, alkalinity
can also inhibit hydrogen production and slow down the acidification of the liquid medium.

Moreover, the results showed that the optimum temperature for hydrogen production was 30°C
(105 ml-H2/g-TS). There was a slight increase in hydrogen yield as the temperature increased
from 25 to 30°C, whereas the yield decreased when the temperature was increased from 30 to
40°C. These results indicate that temperature variations may influence the activity of essential en-
zymes such as nitrogenases, and could thus significantly impact on the hydrogen produced by
the photofermentative bacteria. Notably, higher temperatures may cause denaturation of these
enzymes, resulting in less hydrogen production.

C:N Ratio
Another essential factor that affects the efficiency of hydrogen production from PNSB is the C:N
ratio as nitrogen is vital for the growth of microorganisms. Utilizing R. capsulatus, Androga and
colleagues [39] reported that a low C:N ratio produced a low concentration of biomass and
hence a low hydrogen yield, whereas a high C:N ratio produced a high biomass concentration
but a lower hydrogen yield because the high cell concentration prevented light penetration into
the photobioreactor. In that study it was found that the optimum C:N ratio was 25, leading to
the production of 0.44 mmol-H2/l/h.

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Furthermore, the concentration of free ammonia can also affect the efficiency of
photofermentation. Seifert and coworkers [40] reported that an ammonia concentration above
20 mM in the solution/fermentation broth may inhibit the available nitrogenases. Consequently,
it is crucial to adjust the operating conditions to the desired C:N ratio and ammonia concentration
during photofermentation to produce a high hydrogen yield.

Hydraulic Retention Time

The hydraulic retention time (HRT) is an essential parameter for the continuous operation of a
bioreactor and to accomplish an optimum balance between the hydrogen production rate, sub-
strate conversion efficiency, and light energy in the photofermentation process. Researchers
have found that the HRT of photofermentation is longer than that of dark fermentation because
of the slower metabolic rate of the photosynthetic bacteria relative to dark fermentation bacteria,
and can take up to 5 days.

Furthermore, a short HRT can cause washing-out of the bacteria from the system leading to low
hydrogen yield, whereas a long HRT can lead to inhibition of some bacteria, resulting in a low
hydrogen yield. However, there is so far no definitive range or optimum HRT for hydrogen produc-
tion using photofermentative bacteria because of the diverse operating/testing conditions of the
bioreactors used in each study.

Light Efficiency
Unlike dark fermentation, photofermentation relies on light to provide energy for microorganism
growth [41]. Hence, studying the effects of light efficiency, including different intensities, sources,
and wavelengths, on photofermentation is vital for achieving a high light conversion efficiency [42].
Previous studies have used various light sources for photofermentation, including solar and
artificial light [36]. However, one of the main limitations of using solar light is its instability and
constantly changing light intensity (i.e., diurnal variation) as a function of the time of day, weather,
and the season [43]. In addition, the light utilization efficiency of sunlight is lower than for an opti-
mized artificial illumination system [36].

Other than solar light, the different light sources which have been used recently include light-
emitting diodes (LEDs), mercury-tungsten lamps, and halogen lamps [44]. LEDs have a
longer lifetime and a higher price than tungsten lamps that have a wider wavelength but a shorter
lifetime [45].

Lu and colleagues [38] studied the effects of different light intensities of incandescent lamps
using apple waste as the substrate in a stirred batch reactor. The maximum hydrogen yield
(100 ml-H2/g-TS) was observed at a light intensity of 3000 lux (lx). A further increase in light in-
tensity resulted in a sudden decrease in the hydrogen yield from 100 to 33 ml-H2/g-TS at a light
intensity of 5000 lx. This suggests that low light intensities can promote hydrogen production
by photosynthetic bacteria, but higher light intensities can suppress hydrogen production.

Microbial Communities
Pure bacterial strains have recently been used for photofermentation, whereas mixed culture
strains have been used in other studies [36]. There are four main types of anoxygenic
photosynthetic microorganisms: purple sulfur bacteria (PSB), GSB, PNSB, and green
non-sulfur bacteria (GNSB) [46]. Of all these four species, the PNSB is the most commonly
used photosynthetic hydrogen-producing bacteria for photofermentation. The most widely
used strains are Rhodobacter spp., Rhodopseudomonas spp., and Rhodobacter sphaetoide
spp. [47].

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Dark Fermentation
Another well-known technology, dark fermentation, has been developed to produce hydrogen
using anaerobic bacteria grown in the dark on carbohydrate-rich substrates [48]. In this process,
different microorganisms are involved in converting multiple biochemical substrates such as
complex sugars or polysaccharides into biohydrogen and other products, as shown in
Equations 4, 5, and 6 [49].

C6 H12 O6 þ 2H2 O → 4H2 þ 2CH3 COOH þ 2CO2 ½4

C6 H12 O6 þ 2H2 O → 2H2 þ 2CH3 CH2 OH þ 2CO2 ½5

C6 H12 O6 → 2H2 þ CH3 ðCH2 Þ2 COOH þ 2CO2 ½6

Furthermore, anaerobic metabolism of pyruvate is a significant reaction for most microbe-

mediated hydrogen production during the catabolism of various substrates (Figure 3B). Two
types of enzymes can catalyze pyruvate metabolism – pyruvate-formate lyase (PFL) and
pyruvate-ferredoxin oxidoreductase (PFOR) – as shown in Equations 7 and 8, respectively
[50], where Fd(ox) and Fd(red) are oxidized and reduced ferredoxin, respectively.

PFL : pyruvate þ CoA → acetyl−CoA þ formate ½7

PFOR : pyruvate þ CoA þ 2FdðoxÞ → acetyl−CoA þ CO2 þ 2FdðredÞ ½8

The advantages of using dark fermentation are continuous production of biohydrogen in the
absence of light, high biohydrogen production efficiency, and yield. Moreover, it requires less
energy input and uses organic and inorganic waste feedstocks and stable hydrogen-evolving
enzymes [51]. Likewise, dark fermentation does not produce or consume oxygen during its
biochemical reactions; hence, it reduces the likelihood of inactivation of (Fe-Fe)- and (Ni-Fe)-
hydrogenases. Thus, both enzymes can be utilized to produce hydrogen using a pure culture
or a mixture of microorganisms during dark fermentation [18,52].

Hydrogen production from dark fermentation can be classified into four different types based on
the various byproducts formed during fermentation: ethanol-type fermentation, propionate-type
fermentation, butyrate-type fermentation, and mixed-type fermentation [36]. Moreover, many
factors have been investigated that affect the hydrogen yield during dark fermentation (Table 2).
These factors are different feedstocks, reactor configuration, operational parameters (e.g., pH,
temperature, and HRT), and the microbial community [36].

Factors Affecting Hydrogen Production in Dark Fermentation

Temperature
Dark fermentation depends on diverse microorganisms, and because the temperature is a key
factor that influences microbial growth, it also affects the hydrogen yield and efficiency. Generally,
bacteria can live in a range of temperatures from −20°C to 122°C [53]. Nevertheless, mesophilic
(25–45°C) and thermophilic (45–65°C) conditions were most commonly reported in the literature
[36].

In general, the use of higher temperatures in dark fermentation leads to increased hydrogen
yield and efficiency. However, in some cases the opposite may occur, and the hydrogen
yield can decrease at a higher temperature that is associated with increased production of
volatile fatty acids, depending on the characteristics of microorganisms used in the fermenta-
tion process [36].

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Table 2. Food Waste and Hydrogen Generation through Dark Fermentation

Substrate Reactor Microorganism pH Temperature (°C) Pretreatment Hydrogen yielda Refs
type
Food waste Batch Mixed culture 4.0–4.6 37 Food waste pretreated by 219.9 ml-H2/g-VS [49]
hydrolysate glucoamylase and protease
Food waste Batch Mixed culture 35 ± 1 Shredded in a kitchen mill 180.0 ml-H2/g-VS [67]
and crude
glycerol
Food waste, Batch Mixed culture 5.5 35 ± 1 Sewage sludge pretreated at 179.3 ml-H2/g-VS [68]
sewage 100°C for 30 minutes
sludge, and
crude glycerol
Wheat waste Continuous Mixed culture 5.0–6.0 37 Shredded into 70 μm particles, 645.7 ml-H2/g-TS [59]
hydrolysate sulfuric acid was added,
and autoclaved at 90°C for
15 minutes
Garden and Batch Caldicellulosiruptor 7.0–7.2 35 Crushed and shredded into a 46.0 ± 1 [69]
food waste saccharolyticus size less than 5 mm ml-H2/g-VS
Fruit and Continuous Clostridium, 5.5–6.75 37 ± 1 Crushed into a size >5 mm and 23.5 ml-H2/g-VS [70]
vegetable Bifidobacterium, autoclaved at 120°C for 20
wastes and Lactobacillus minutes
Food waste Batch Mixed culture 3.76 ± 37 Crushed into a size <5 mm using 169.0 ml-H2/g-VS [71]
taken from a 0.44 a grinder
university
canteen

a
Abbreviation: VS, volatile solids.

Rangel and coworkers [54] studied the effect of different temperatures (45°C and 55°C) on hydro-
gen production from agroindustry waste in a stirred batch reactor. They reported that microor-
ganisms work better under mesophilic conditions, leading to enhanced hydrogenase enzyme
activity and a higher hydrogen yield (465.5 ml-H2/g-COD) than under thermophilic conditions
(356.7 ml-H2/g-COD).

pH
The operating pH is another key factor that can affect hydrogen yield and the efficiency of dark
fermentation. A change in pH can affect the activity and biological function of the microorganisms,
their metabolic pathways, cell morphology, and intracellular pH [36]. Therefore, it is vital to study
the effect of pH on dark fermentation. Generally, the optimum pH level used in dark fermentation
is in the range of 5.0–7.0, depending on the characteristics of the microorganisms and the feed-
stock used in the process [17].

Wongthanate and colleagues [55] studied the use of coconut milk waste as a substrate at dif-
ferent initial pH levels in a stirred batch reactor. They reported that the highest hydrogen pro-
duction (0.28 l-H2/l) was at an initial pH of 6.5. The authors suggested maintaining the
operating pH close to 6.0 to achieve higher fermentative conversion of this substrate to
biohydrogen.

C:N Ratio
Maintaining an optimum C:N ratio is a crucial factor that can affect the stability of dark fermenta-
tion [56]. It is therefore a vital parameter for hydrogen producers because their proteins, nucleic
acids, and enzymes depend on an appropriate the C:N ratio. Rangel and coworkers [54] also re-
ported that maximum hydrogen production (465.5 ml-H2/g-COD) from a Colombian agroindustry

10 Trends in Biotechnology, Month 2021, Vol. xx, No. xx

Trends in Biotechnology

waste in a stirred batch reactor was achieved at an optimum C:N ratio of 45. They suggested that Outstanding Questions
the presence of excess carbon in the substrate is beneficial for microorganism production of Is it possible to have a practical and
hydrogen. cost-effective process for biohydrogen
production from food waste within the
next 10 years?
HRT
This is another key parameter that can affect the efficiency of dark fermentation and determine the Can biohydrogen-producing systems
duration of fermentation [57]. HRT depends mainly on the characteristics of the substrate used, from food waste be integrated with hy-
drogen fuel cell technologies to gener-
especially its biodegradability [58]. The easier the degradation of substrate, the lower HRT is
ate electricity at the semi-industrial and
needed. Furthermore, the HRT cannot be too low, and it should be longer than the bacterial dou- commercial scales?
bling time to ensure bacterial proliferation [36].
Are the biohydrogen-producing sys-
tems from food waste economically
In addition, when dark fermentation is compared with other biological energy recovery technol- efficient, and do they have sufficient
ogies such as anaerobic methane production and bioethanol fermentation, dark fermentation capacity to be integrated with hydro-
usually has a short HRT (in hours) whereas the other technologies have a longer HRT gen fuel cell technologies?
(in days) [36,59].
Can a more versatile bioreactor
configuration be developed that is
Gorgec and coworkers [59] studied the effect of varying HRT on hydrogen production yield resilient to harsh operating conditions
using waste wheat as a substrate in a continuous stirred tank reactor. Decreasing the HRT and be commercialized in developing
countries that generate a large amount
from 8 h to 3 h enhanced hydrogen yield from 20 to 646 ml-H2/g-TS. However, decreasing
of food waste?
the HRT further to 1 h had a negative impact on hydrogen yield because the yield decreased
from 646 to 116 ml-H2/g-TS. Hydrogen production performance is affected by low HRTs Is the idea of using food waste as a
(<3 h) because the microorganisms are washed out from the system as a result of shear feedstock to produce biohydrogen
applicable to all countries, or is it only
forces during the high linear velocity at low HRTs or high feeding/loading rates. Another applicable in countries that generate
possible reason could be that substrate inhibition occurred because of the high organic large quantities of food waste?
loading rate.
Will it be safe and economically viable to
transport the biohydrogen produced
Microbial Communities from food waste to nearby regions?
Different consortia of microorganisms, for example, hydrogen-producing bacterial strains
and mixed cultures, can be used for dark fermentation [60]. The hydrogen-producing Will it be possible to design hydrogen-
impermeable tanks that are resistant
microorganisms can be classified into three different categories based on their optimum
to high pressures for storing and dis-
temperature [61]. These categories are psychrophiles (0–25°C), mesophiles (25–45°C), tributing biohydrogen produced from
and thermophiles (45–65°C), and mesophiles and thermophiles are most commonly used food waste?
for dark fermentation [36]. Hydrogen-producing microorganisms can also be categorized
Is it possible to use a mixed culture of
based on their hydrogen tolerance, such as strict anaerobes and facultative anaerobes [36]. microorganisms and algae to produce
biohydrogen from food waste using the
The complex nature of the microbial community involves the coexistence of different microorgan- techniques mentioned in this article?
isms, including beneficial bacteria (e.g., bacteria that help to hydrolyze polymers), unwanted hy-
Is it possible to mix food waste with
drogen consumers (e.g., methanogenic archaea and homoacetogenic bacteria), and other different feedstocks to obtain higher
microbes that compete with hydrogen-producing bacteria for the different nutrients [36]. As a re- yields of biohydrogen? If yes, what will
sult, various pretreatment methods are recommended to support the hydrogen-producing bac- be the optimal operating conditions of
the fermenter and the mixing ratio of
teria and eliminate hydrogen consumers, namely heating, freezing, adding chemical inhibitors, the feedstocks?
and using microwave irradiation, among others [17].
What are the major challenges facing
biohydrogen production, its storage,
Concluding Remarks and Future Perspectives and utilization? Are hydrogen energy-
Many researchers have provided promising solutions to issues that are commonly confronted based governmental policies well articu-
by other renewable energy systems, such as the aspects related to policies, governance, en- lated in developed and developing
nations?
ergy storage, and distribution, production costs and energy yield. Although the proposed
methods, techniques, and technologies demonstrate the potential of biohydrogen production
from food waste, there are still barriers to the commercialization of this waste-to-energy ap-
proach. The lack of two-way communication between researchers and engineers is the

Trends in Biotechnology, Month 2021, Vol. xx, No. xx 11

 Trends in Biotechnology

primary barrier to commercialization because of societal and economic limitations (Table 3).
Researchers need to understand the perspective of the industrial sector; they must also con-
sider the economic and technical points of view of the companies in supporting the concept
of biorefineries.

However, several technical challenges and management aspects need to be systematically

tested and assessed in the future to enable the full-scale application of these biological ap-
proaches. First, the optimum design of systems, whether it is an integrated system or a single-
stage system, and the optimal operating conditions for high biohydrogen yield must be
established. At the laboratory scale, optimization of several process parameters can be achieved
by studying a wide range of values for each factor (i.e., at low, intermediate, and high levels) and
by considering more than one factor at the same time to elucidate their interactions. Such exper-
iments can be designed statistically using full-factorial, response surface, mixture, Taguchi array,
and split-plot designs. Artificial intelligence (AI) techniques can also be used in conjunction with
nature-inspired optimization algorithms to optimize parameters and predict the hydrogen yield
in different bioreactor configurations.

Second, there is an urgent need to assess the potential outcome at the industrial scale, such as
defining the benchmarking criteria for scaling up the process and the economic feasibility of pre-
treatment methods for the bioconversion of food waste to biohydrogen. In some cases, large-
scale implementation strategies (i.e., from the semi-industrial to the full scale) might provide eco-
nomic benefits in the longer term. There is a possibility that high-cost methods at the laboratory
scale might be economically realizable in full-scale applications. However, it is evident that a high
yield of hydrogen might not be achieved immediately after starting up the bioreactor, and the use
of large residence times would require large reactor volumes and thus additional investment
costs. Furthermore, a slow process with a low hydrogen yield will affect the feasibility of practically
applying the technology at large scale. One solution is to create new genetically engineered
biocatalysts that can thrive under harsh operating conditions of pH, temperature, and organic
loads. Such genetically engineered microorganisms may adapt more quickly to the substrate
(s) and thus reduce the residence time – leading to a high rate of substrate conversion to
biohydrogen, improved operational stability, and decreased bioreactor size requirements.

Table 3. Overall Comparison of Hydrogen Production Technologies

Process Advantages Limitations
Photofermentation High hydrogen yield Depends on photosynthetic
High COD removal bacteria to produce H2
rate Requires light to produce H2
Low hydrogen-production rate
Requires waste pretreatment
Complex design configuration
Dark fermentation A wide variety of Low hydrogen yield
different waste High biochemical oxygen
sources demand (BOD) in the effluent
High Requires waste pretreatment
hydrogen-production (e.g., lignocellulose and
rate inoculation of a microbial
Simple design consortium)
configuration The partial pressure of H2
must be monitored
Methanogens might oxidize H2
Inability to produce H2
continuously
Produces several intermediates
Requires separation of CO2
Future perspectives
Optimization of light efficiency
Ability to be used in genetic
engineering applications
Requires more high-quality
life-cycle assessment studies
Ability to be used in genetic
engineering applications
Requires more high-quality
life-cycle assessment studies
Ability to be coupled with other
technologies
(e.g., photofermentation, anaer-
obic digestion, and microbial
electrolysis cells)

12 Trends in Biotechnology, Month 2021, Vol. xx, No. xx

Trends in Biotechnology

Third, there is a need for a comprehensive economic, environmental assessment, and market
analysis study covering all the socio- and techno-economic factors involved in the
generation/collection of food wastes and the commercial-scale production of hydrogen (see
Outstanding Questions). Finally, a safety and risk assessment analysis should be considered
during the scaling-up stage because stored hydrogen is prone to leakage in composite
cylinders/tanks. Therefore, designing these reservoirs with pressure-relief devices to ensure
high safety standards is a practical challenge. Another challenge with hydrogen storage that
makes these tank reservoirs unfeasible is their economics. The costs of a hydrogen storage
tank depend on several factors and characteristics, including the on-board fuel system gravi-
metric capacity, the volumetric capacity, and the cost of tank materials, indirect materials, util-
ities, and safety accessories. Hence, there is a need to search for a new method to store
hydrogen at near ambient pressure and temperature. One promising approach will be the
use of synthetic adsorbents to capture hydrogen in a highly porous and active structure,
which is inspired by the effectiveness of metal–organic frameworks (MOFs) in capturing
CO2. These adsorbents could offer a high capacity for hydrogen at near ambient conditions.
An advantage of this approach is that the pressure swing desorption technique could be
used to free/release the adsorbed hydrogen for its utilization.

As a concluding remark, if biohydrogen research is pursued in a unified approach by involving

technological, industrial, and governmental stakeholders, the desired level of commercial-scale
biohydrogen can be realized within a short period.

Acknowledgments
M.M.H., E.S.O., and O.M.A. acknowledge the Erasmus+ Programme for providing scholarships to pursue an International
Master of Science in Environmental Technology and Engineering (IMETE) at the University of Chemistry and Technology,
Prague – VŠCHT Praha (Czech Republic), IHE Delft Institute for Water Education (The Netherlands), and Ghent University
(Belgium).

Declaration of Interests
The authors declare no conflicts of interest.

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