Figure: Lenguajes. Generaciones de lenguajes de programación.
VV DOH VD DV PH...V List of Figures Figure 1 A fuel cell car powered by hydrogen and sold by Hyundai...
Transcript of VV DOH VD DV PH...V List of Figures Figure 1 A fuel cell car powered by hydrogen and sold by Hyundai...
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Hydrogen production from organic wastes "clean energy production from low- value substrates”
Ahmed Hassan Salem Hassan
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Hydrogen production from organic wastes "clean energy production from low-
value substrates”
Von der Fakultät für Ingenieurwissenschaften, Abteilung Bauwissenschaften der Universität
Duisburg–Essen zur Erlangung des akademischen Grades Doktor- Ingenieur (Dr.-Ing.)
genehmigte Dissertation
vorgelegt von
Ahmed Hassan Salem Hassan M. Sc.
Geboren am 01. September 1987 in Kairo, Ägypten
Referent: Univ.-Prof. Dr.-Ing. Renatus Widmann
Koreferent: Univ.-Prof. Dr.-Ing. Anke Bockreis
Eingereicht: 17.10.2018
Mündliche Prüfung: 15.02. 2019
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Acknowledgement
First of all, I want to express my profound gratitude to my main supervisor, Professor Renatus
Widmann to give me the chance to join the research group. He took time out of his extremely
busy schedule to read my dissertation. In fact, the success of this research is due to his
guidance, thorough supervision, constructive criticisms, expert advice, encouragement and
support.
I like to thank Dr.-Ing. Ruth Brunstermann and Dr.-Ing. Thorsten Mietzel, my co- supervisors,
for the supervision and great inspiration, and push me ahead on the research works.
I would like to thank German Academic Exchange Service (DAAD) and department of Urban
Water and Waste Management (SiwAwi) for the financial support throughout this study.
I would like to thank all my colleagues in the department of Urban Water and Waste
Management for their continuous support.
Lastly, my special thanks go to my darling wife, for her patience and support, and for taking
good care of our kids, during my absence. I am also grateful to my mother and father for their
continuous supports and prayers. To my sisters, I appreciate you all for your endless love,
prayers, supports and encouragements.
Essen, 2018 Ahmed Hassan
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Content
Content .................................................................................................................................. I
List of Figures ........................................................................................................................ V
List of Tables ........................................................................................................................ VI
List of Abbreviations ............................................................................................................ VII
List of publication .................................................................................................................. IX
Abstract ............................................................................................................................... XII
1 Introduction .................................................................................................................... 1
1.1 Barriers to hydrogen energy..................................................................................... 2
1.2 Hydrogen production methods ................................................................................. 3
1.3 Biohydrogen production methods ............................................................................ 3
1.3.1 Direct photolysis ......................................................................................... 3
1.3.2 Indirect photolysis ...................................................................................... 4
1.3.3 Dark fermentation....................................................................................... 4
1.3.4 Photo-fermentation ..................................................................................... 5
1.3.5 Hybrid reactor system ................................................................................ 6
1.4 Cost of hydrogen production methods ..................................................................... 7
1.5 Technologies for hydrogen energy use .................................................................... 9
1.5.1 Internal Combustion Engines ..................................................................... 9
1.5.2 Fuel cells .................................................................................................... 9
1.6 Factors affecting fermentative hydrogen production ................................................10
1.6.1 Type of inoculum and pre-treatment ..........................................................10
1.6.2 Substrate type ...........................................................................................12
1.6.3 pH .............................................................................................................13
1.6.4 Organic loading rate (OLR) .......................................................................13
1.6.5 Hydraulic retention time (HRT) ..................................................................15
1.6.6 Temperature .............................................................................................16
1.6.7 Reactor configuration ................................................................................16
1.6.8 Nutrient concentration and metal ions .......................................................17
2 Research Objectives and strategies for improvement of biohydrogen production ..........19
2.1 Formation of granular sludge ..................................................................................19
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2.2 Use of biofilm carriers .............................................................................................19
2.3 Increasing the bioactivity of the hydrogenase enzyme ............................................20
2.4 Sequential systems .................................................................................................20
2.5 Pre-treatment of substrates ....................................................................................20
3 Materials and methods ..................................................................................................21
3.1 Sludge collection, characterization and pre-treatment .............................................21
3.2 Substrates for bio H2 production .............................................................................21
3.3 Pre-treatment of substrates ....................................................................................22
3.3.1 Pre-treatment of potatoes and bean wastes ..............................................22
3.3.2 Pre-treatment of TWW ..............................................................................22
3.4 Biohydrogen production experiments ......................................................................23
3.4.1 Batch H2-production experiments ..............................................................23
3.4.1.1 Biohydrogen production from potatoes and bean wastes ......................23
3.4.1.2 Biohydrogen production from TWW ......................................................24
3.4.2 Continuous H2 production..........................................................................24
3.4.2.1 Bioreactors construction, temperature and pH control and nutrient supply
.............................................................................................................24
3.4.2.2 Biohydrogen production from sucrose wastewater ................................24
3.4.2.3 Enhancement of biohydrogen production from sucrose wastewater .....25
3.4.2.4 Formation of hydrogen-producing granules and the impact of sucrose
concentration on the particle size .........................................................25
3.4.2.5 The use of biofilm and immobilized hematite NPs .................................25
3.4.2.6 Sequential continuous biohydrogen production ....................................25
3.4.2.7 Biohydrogen production from organic wastes .......................................27
3.4.3 Preparation of hematite NPs .....................................................................28
3.4.4 Analytical methods ....................................................................................28
3.4.4.1 Gaseous phase ....................................................................................28
3.4.4.2 Liquid phase .........................................................................................28
3.4.4.3 Calculations and data analysis .............................................................29
3.4.4.4 Bioreactor operation, performance, conversation efficiency and carbon
balance .................................................................................................30
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3.4.4.5 Calculations of hydraulic retention time (HRT) and organic loading rate
(OLR) ...................................................................................................30
3.4.4.6 Calculation of the hydrogen yield ..........................................................31
3.4.4.7 Carbon mass balance of the fermentation process ...............................31
3.4.4.8 Solid phase ...........................................................................................31
3.4.4.9 Characterization of the microbial species..............................................32
4 Results and discussion ..................................................................................................33
4.1 Biohydrogen production from sucrose wastewater ..................................................33
4.1.1 Effect of sucrose concentration on biohydrogen production and yield .......33
4.1.2 Enhancement of biohydrogen production from sucrose .............................36
4.1.2.1 Formation of hydrogen producing granules (HPGs) ..............................37
4.1.2.2 Characterization of hydrogen producing granules .................................40
4.1.2.3 Effect of immobilized hematite NPs and supporting materials ...............41
4.1.2.4 Hydrogen production efficiency and carbon balance .............................44
4.1.2.5 The multi-stage integrated method .......................................................45
4.1.2.6 Production of VFAs in the combined system .........................................48
4.1.2.7 Characterization of the dark and phozo-fermentative bacterial species 50
4.2 Biohydrogen production from agricultural residues .................................................51
4.2.1 Effect of HRT and type of substrate on continuous biohydrogen production .
..................................................................................................................51
4.2.2 Pre-treatment of potatoes and bean wastes ..............................................54
4.2.2.1 Effect of pre-treatment on the chemical composition of the substrates .54
4.2.2.2 Effect of pre-treatments on biohydrogen production in batch tests ........55
4.2.2.3 Hydrogen yield, kinetic parameters and production of VFAs .................59
4.2.2.4 Continuous biohydrogen production from pre-treated potatoes and bean
wastes ..................................................................................................61
4.2.2.5 Production of VFAs and SEM characterization .....................................63
4.3 Biohydrogen production from starch-containing textile wastewater .........................64
4.3.1 Biohydrogen production in the blanc tests .................................................65
4.3.2 Biohydrogen production from pre-treated TWW ........................................65
4.3.2.1 Biohydrogen production photocatalytic degradation pre-treated TWW ..65
4.3.2.2 Biohydrogen production from Fenton oxidation pre-treated TWW .........69
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4.3.2.3 Hydrogen yield, kinetic parameters and production of VFAs .................72
4.3.2.4 Photocatalytic degradation vs Fenton oxidation pre-treatment ..............73
4.3.2.5 Characterization of the microbial species..............................................73
5 Summary and conclusion ..............................................................................................75
6 Outline and future studies ..............................................................................................79
References ...........................................................................................................................83
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List of Figures
Figure 1 A fuel cell car powered by hydrogen and sold by Hyundai (Tenca, 2010/2011) ..10
Figure 2 Effect of substrate concentration on biohydrogen production from sucrose
wastewater: (a) HPR, and (b) H2 yield ................................................................36
Figure 3 Progress of the granulation process with increasing sucrose concentration .......38
Figure 4 Biohydrogen production versus sucrose concentration in granular based CSTR
bioreactor: (a) HPR and (b) H2 yield ...................................................................40
Figure 5 Scanning electron microscopy of (a) hydrogen producing granule, (b) sporeforming
rod shape bacteria and (c) fusiform bacilli ..........................................................41
Figure 6 Effect of cell immobilization and hematite NPs versus control reactor for
biohydrogen production from sucrose wastewater ..............................................44
Figure 7 Biohydrogen production: (a) HPR and (b) H2 yield in second dark and photo-
fermentation stage .............................................................................................48
Figure 8 Variation of pH and VFAs concentration in (a) second dark fermentative production
and (b) photo-fermentative stage........................................................................49
Figure 9 SEM characterization of dark and photo-fermentative bacteria ...........................50
Figure 10 Effect of HRT on biohydrogen production from sucrose, potatoes and bean wastes:
(a) HPR and (b) H2 yield .....................................................................................53
Figure 11 TVS concentrations in the raw and pre-treated wastes .......................................55
Figure 12 Effect of pre-treatments on biohydrogen production from (a) potatoes wastes and
(b) bean wastes ..................................................................................................58
Figure 13 Effect of pre-treatments on hydrogen yield in continuous biohydrogen production
from potatoes wastes .........................................................................................62
Figure 14 Effect of pre-treatment on hydrogen yield in continuous biohydrogen production
from bean wastes ...............................................................................................63
Figure 15 SEM characterization of hydrogen-producing bacteria sampled from CSTR
bioreactor operated using (a) potatoes wastes and (b) bean wastes ..................64
Figure 16 SEM of H2-producing bacteria collected from start-up tests as (a) sa,ples pre-
treated using photocatalytic degradation and (b) samples pre-treated using Fenton
oxidation .............................................................................................................73
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List of Tables
Table 1 Avarage properties of Hydrogen (Krupp, 2007) .................................................... 2
Table 2 Comparison of hydrogen production costs with different processes (Pandu and
Joseph, 2012) ........................................................................................................ 8
Table 3 Comparison of the hydrogen yields depending on the operating conditions ..........18
Table 4 Average characteristics of the sludge ...................................................................21
Table 5 Average properties of the substrates ....................................................................22
Table 6 Performance of the CSTR bioreactors at different sucrose concentrations ...........35
Table 7 Four reaction modes: hydrogen yield efficiency and fate of carbon .......................45
Table 8 Biohydrogen production and effluent characteristics in the first stage ...................46
Table 9 Composition of potatoes and bean wastes before and after pre-treatment ...........55
Table 10 Hydrogen yield and kinetic parameters using various pre-treatments methods .....60
Table 11 Biohydrogen production in the reference tests ......................................................65
Table 12 Biohydrogen production performance from TWW using combined photocatalytic
degradation pre-treatment and anaerobic fermentation ........................................68
Table 13 Biohydrogen production from Fenton oxidation pre-treated TWW .........................71
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List of Abbreviations
ABR Anaerobic baffled reactor
AD Anaerobic digestion
AGSBR Agitated granular sludge bed reactor
AOPs Advanced Oxidation Processes
C/N Carbon nitrogen ratio
C/P Carbon phosphorous ratio
CH4 Methane
CHP Combined heat and power
CIGSB Carrier-induced granular sludge bed reactor
CO Carbon monoxide
CO2 Carbon dioxide
COD Chemical oxygen demand
CSTR Continuous stirring tank reactor
d Day
Eq. Equation
FBR Fixed bed reactor
FC Fuel cell
g Gram
h Hour
H2 Hydrogen
H2O Water
H2O2 Hydrogen peroxide
H2S Hydrogen sulphide
H2 yield Hydrogen yield
HAc Acetic acid
HCl Hydrochloric acid
hPa Hectopascal
HPGs Hydrogen-producing granules
HPR Hydrogen production rate
HRT Hydraulic retention time
ICE Internal combustion engine
K Kelvin
KOH Potassium hydroxide
kW, kWh Kilowatt, Kilowatt hour
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L Litre
MBTU Mega British Thermal Unit
MJ/kg Mega Joule/Kilogram
min Minute
N Nitrogen
N2 Nitrogen gas
NaOH Sodium hydroxide
NL Litre at standard pressure and temperature (1013 hPa
and 0 ⁰C)
Nm3 Cubic meter at standard pressure and temperature
(1013 hPa and 0 ⁰C)
NmL Millilitre at standard pressure and temperature
(1013 hPa and 0 ⁰C)
NOx Nitrogen oxides
NPs Nanoparticles
O2 Oxygen
OLR Organic loading rate
OMW Olive mill wastewaters
P Phosphorous (phosphate)
PBBR Pack bed biofilm reactor
PNSB Purple non-sulphur bacteria
POME Palm oil mill effluent
SC Sucrose concentration
SLM Soluble liquid metabolites
TCD Thermal conductivity detector
TS Total solids
TSS Total suspended solids
TVS Total volatile solids
TWW Textile wastewater
UASB Up-flow anaerobic sludge blanket
UK United Kingdom
US United States
US$ United States dollar
VFAs Volatile fatty acids
VS Volatile solids
W/V Weight to Volume ratio
WWTP Wastewater Treatment Plant
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List of publication
The thesis is founded on the results presented in the following articles:
Paper I:
Ahmed H. Salem, Thorsten Mietzel, Ruth Brunstermann and Renatus Widmann. Effect of cell
immobilization, hematite nanoparticles and formation of hydrogen- producing granules on
biohydrogen production from sucrose wastewater. International Journal of Hydrogen Energy,
42 (2017) 25225–25233.
Abstract
This study investigated the effect of granules formation, hematite nanoparticles and biofilm
carriers on biohydrogen production from sucrose wastewater in continuous stirring tank
reactors operated at 12 h HRT, pH of 5.5 and 35 °C. Granular-based bioreactor was
subjected to acid incubation period for 24 h by shifting the pH from 5.5 to 3. Before
application of the acid incubation, hydrogen-producing granules (HPGs) diameter and
hydrogen production rate (HPR) of 0.5 mm and 4.3 L/L.d, respectively were measured at 10
g-sucrose/L. Application of acid incubation enhanced the granulation process, where the
particle size increased to 2.8 mm and higher HPR of 7.8 L/L.d was obtained. Higher sucrose
concentration (15-30 g\L) enhanced HPGs diameter and increased the HPR. At 10 g-
sucrose/L, addition of hematite nanoparticles increased the HPR to 5.9 L/L.d higher than 3.87
L/L.d measured in control reactor. Biofilm-based reactor showed HPR of 2.48 L/L.d lower than
the control reactor.
Paper II:
Ahmed H. Salem, Ruth Brunstermann, Thorsten Mietzel and Renatus Widmann. Effect of pre-
treatment and hydraulic retention time on biohydrogen production from organic wastes.
International Journal of Hydrogen Energy, 43 (2018) 4856–4865.
Abstract
This study investigated the effect of pre-treatment and hydraulic retention time (HRT) on
biohydrogen production from organic wastes. Various pre-treatments including thermal, base,
acid, ultrasonication, and hydrogen peroxide were applied alone or in combination to enhance
biohydrogen production from potato and bean wastewater in batch tests. All the pre-treated
samples showed higher hydrogen production than the control tests.
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Hydrogen peroxide pre-treatment achieved the best results of 939.7 and 470 mL for potato
and bean wastewater, respectively. Continuous biohydrogen production from sucrose, potato
and bean wastewater was significantly influenced by reducing the HRT as 24, 18 and 12 h.
Sucrose and potato showed similar behavior, where the hydrogen production rate (HPR)
increased with decreasing the HRT. Optimum hydrogen yield results of 320 mL-H2/g-VS
(sucrose) and 150 mL-H2/g-VS (potato) were achieved at HRT of 18 h. Bean wastewater
showed optimum HPR of 0.65 L/L.d with hydrogen yield of 80 mL-H2/g-VS at 24 h HRT.
Paper III:
Ahmed H. Salem., Thorsten Mietzel, Ruth Brunstermann and Renatus Widmann. Two-stage
anaerobic fermentation process for bio-hydrogen and biomethane production from pre-treated
organic wastes. Bioresource technology, 265 (2018) 399–406.
Abstract
In this study, the effect of pre-treatments including alkaline, acid and hydrogen peroxide on
continuous hydrogen and methane production was investigated. Two different substrates as
potatoes and bean wastes were used. Pre-treatment showed positive effect on bio-hydrogen
and bio-methane production; higher bio-hydrogen and bio-methane production results using
pre-treated samples than the control bioreactors (without pre-treatment), were recorded. In
case of potatoes wastes, the hydrogen yield ranged between 126.4 and 252.7 mL-H2/g-TVS
using pre-treated samples compared to 58.7 mL-H2/g-TVS observed in the reference test.
Pre-treated bean wastes showed hydrogen yield of 93.0–152.1 mL-H2/g-TVS higher than
53.3 mL-H2/g-TVS measured in the control test. In the second stage, average methane yield
results of 322.9–507.1 and 284.3–462.6 mL-CH4/g-TVS higher than 198.6 and 124.3 mL-
CH4/g-TVS measured for potatoes and bean wastes control bioreactors, respectively. The
best results were observed using H2O2 pre-treatment. The energy production efficiency was
improved by combining H2 and CH4 bioreactors.
Paper IV:
Ahmed H.S. Hassan, Sebastian Schmuck, Ruth Brunstermann, Thorsten Mietzel and
Renatus Widmann. Improving the biohydrogen recovery from sucrose wastewater using
combined two-stage (dark/dark or dark/photo) fermentation process. (Submitted to
International Journal of Hydrogen Energy).
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Paper V:
Based on an official Invitation from the Review-commissioning Editor for the World Journal
of Microbiology and Biotechnology (Prof. Ian Maddox), published by Springer, a review article
has been submitted to the journal, and the manuscript has been sent back from the journal for
revision.
Paper VI:
Ahmed H.S. Hassan, Ruth Brunstermann, Thorsten Mietzel, Sebastian Schmuck and Renatus
Widmann. Enhancement of biohydrogen production from starch-containing textile wastewater
using advanced oxidation processes pretreatment. (To be submitted to Chemical Engineering
Journal).
Statement of Contributions
Ahmed Hassan’s contributions to each of the above publications are: Responsible for part of
the experimental work, data analyses, manuscript writing, submitting the manuscript and
revising the manuscript based on the reviewer’s comments.
Congress communications
A. H. Salem, R. Brunstermann, T. Mietzel, R. Widmann (2017). Anaerobic biohydrogen
production with concurrent wastewater treatment: influence of substrate concentration,
hydraulic retention time and type of Substrate. International Conference "Progress in Biogas
IV Biogas production from agricultural biomass and organic residues" 8-11 March 2017,
Stuttgart, Germany., Oral Poster presentation.
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Abstract
Recently, the use of hydrogen as a clean energy source in industry has been increased.
Compared to conventional hydrogen production methods, biological hydrogen production
methods are characterized by being less energy intensive, non- polluting and low-value
substrates (waste substrates) can be used for the bio-H2 production.
Among different biological methods, dark fermentation has gained more attention because the
process can be conducted in absence of light and a variety of organic substrates can be
utilized. The main drawback of the dark fermentation process is the low H2 yield; this occurs
because of the biomass washout in case of using CSTR bioreactor and low biodegradation
substrates such as food waste, agricultural residues and/or industrial wastewaters that contain
high complex pollutants e.g. textile wastewater, pesticides wastewater, etc.
In order obtain high H2 yields, the efficiency of the fermentation process must be enhanced by
maintaining high biomass concentration in the bioreactor and improving the biodegradation of
the used organic wastes.
The cell density increased in the bioreactor by formation of granular sludge using sucrose
wastewater. Different sucrose concentrations of 10–30 g/L were studied, with 5 g/L
increment. Although the HPR increased with increasing sucrose concertation in the feed,
optimum hydrogen yield of 361.1 NmL-H2/g-sucrose was obtained at 10 g/L and the H2 yields
decreased at higher sucrose concentrations.
The hydrogen yield from sucrose wastewater was enhanced by using two-stage process such
as dark/dark and/or dark/photo-fermentation systems. In case of dark/dark combined system,
the hydrogen yield increased from 2.14 (one-stage) to 4.20 mol-H2/mol-sucrose (two-stage).
Likewise, the hydrogen yield increased from 2.64 mol-H2/mol-sucrose in one-stage (dark
fermentation) to 4.84 mol-H2/mol- sucrose when dark/photo-fermentation system was used.
In case of agricultural residues, several pre-treatment methods including heat, ultrasonication,
alkaline, acid, hydrogen peroxide were applied alone or in combination to enhance the
biohydrogen production from potatoes and bean wastes in batch and continuous experiments.
The H2 yields were higher using pre-treated samples than the corresponding yields achieved
using the raw substrates e.g. potatoes and bean wastes.
For both substrates, the best H2 yields were observed using H2O2 pre-treated wastes, while
low H2 yields were measured in case of heat and ultrasonic pre-treatments. The biohydrogen
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production from starch-containing textile wastewater (TWW) was enhanced by application of
photocatalytic degradation and Fenton oxidation pre- treatments. The H2 yield increased from
157.9 NmL-H2/g-VS using the raw TWW to 169.4–284.0 and 186.9–304.1 NmL-H2/g-VS using
photocatalytic degradation and Fenton oxidation pre-treatments, respectively.
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Introduction 1
1 Introduction
Nowadays, increasing the global energy requirements, reduction of the fossil fuels resources
and their serious negative impact on the environment due to CO2 emission are the major
challenges in the future and are driven the researchers to find new sustainable energy
sources that could substitute fossil fuels (Kapdan and Kargi, 2006). For these reasons, a
great attention has been given to biofuel-based energy as alternative energy sources for the
fossil fuels. At the same time, the waste generation has been drastically increased due to
increasing the global world population and their high food consumption. In the recent years,
the term bio-waste- to-energy has introduced especially in developing countries in that the
produced waste is utilized for bioenergy production (Arimi et al., 2015). Production of energy
from waste substrates can not only produce valuable products such as biohydrogen,
biomethane, biodiesel, bioethanol, etc., but also it can improve the waste stabilization, reduce
the pollution, odours and dieses (Angelidaki, 2002). Among several bioenergy candidates,
hydrogen has attracted more interest as a promising alternative source of energy because it
is carbon free fuel with zero greenhouse emissions compared to those from petroleum fuels.
In addition, hydrogen has high mass-based energy yield which is about 2.75 times higher than
that of hydrocarbon fuels (Lin et al., 2012). In addition, hydrogen can be directly combusted in
an internal combustion engine or to produce electricity via fuel cell (Kotay and Das, 2008).
Hydrogen is colourless, odourless, non-metallic, tasteless, highly flammable diatomic and
non-toxic gas with the molecular formula H2, with an atomic number of 1 and atomic
weight of 1.00794. Hydrogen is the most abundant element in the universe representing
around 75 %. Hydrogen is the lightest element with density of 0.084 g/L at 1013 hPa (normal
conditions), and 70.99 g/L at temperature range of -253 and -259 ⁰C. Based on these
characteristics, hydrogen has the highest energy to weight ratio compared to other gases
(Krupp, 2007). The average properties of hydrogen are summarized in Table 1.
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Table 1 Avarage properties of Hydrogen (Krupp, 2007)
Parameter Unit Value
Density at normal conditions g/L 0.08
Density in liquid phase (-253 ⁰C) g/L 70.99
Ignition temperature in air ⁰C 530
Ignition limit in air Vol.-% 4.1–72.5
Lower heating value MJ/kg 119.97
kWh/kg 33.330
MJ/Nm3 10.783
kWh/Nm3 2.995
Higher heating value MJ/kg 141.89
kWh/kg 39.41
MJ/Nm3 12.745
kWh/Nm3 3.509
Demand on hydrogen is not limited to utilization as a source of energy. Hydrogen gas is a
widely used feedstock to produce chemicals, hydrogenation of fats and oils in food industry,
production of electronic devices, processing steel and for desulfurization and re-formulation
of gasoline in refineries. It has been reported that 50 million tonnes of hydrogen are traded
annually worldwide with a growth rate of nearly 10 % per year for the time being and the
contribution of hydrogen to total energy market will be 8–10 % by 2025. Due to increasing the
need for hydrogen energy, development of cost-effective and efficient hydrogen production
technologies has gained significant attention in recent years (Kapdan and Kargi, 2006).
1.1 Barriers to hydrogen energy
Technical challenges in achieving a hydrogen economy include how to lower the cost of
hydrogen production, transportation, storage, conversion, and applications. Although
hydrogen is the most abundant element in the universe, it is produced from high-cost
processes, which depend mainly on fossil fuels, biomass or water. These methods require
high extreme operating conditions such as high temperature and/or high pressure as well as
these processes are not environmentally friendly because greenhouse gases are mostly
produced. Therefore, hydrogen must be produced from other renewable substrates such as
wastes or wastewaters. Production of bioenergy (biohydrogen) from waste substrate can
achieve the dual goal of bioenergy production and waste stabilization (Momirlan and Veziroglu,
2002).
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Introduction 3
1.2 Hydrogen production methods
Hydrogen production methods can be divided into two broad categories: conventional and
alternative methods. Conventional hydrogen gas production methods are steam reforming of
methane, and other hydrocarbons, non-catalytic partial oxidation of fossil fuels and
autothermal reforming which combines steam reforming of methane and oxidation of fossil
fuels. Those Methods involve the usage of fossil fuels and they are all energy intensive
processes requiring high temperatures. Alternative methods of hydrogen generation from
organic waste materials include electrolysis of water, biophotolysis and fermentation
processes. Among all the novel processes, biological hydrogen production has two main
advantages over the conventional methods; it generates less greenhouse gases and couples
the metabolic activity of hydrogen- producing microorganisms with the simultaneous disposal
of wastes rich in organics. Waste is generated everywhere in the form of solid, liquid or gas
(Kapdan and Kargi, 2006; Kothari et al., 2012).
1.3 Biohydrogen production methods
Biological hydrogen production is a viable alternative to the conventional methods for hydrogen
gas production. According to sustainable development and waste stabilization issues,
biohydrogen gas production from renewable sources, also known as “green technology” has
received a considerable attention in recent years. Several methods are used for the
biohydrogen production such as direct photolysis, indirect photolysis, dark fermentation,
photo-fermentation and sequential dark and photo- fermentation (Kapdan and Kargi, 2006),
as given below. In each process, specific microbes are used for biohydrogen production (Arimi
et al., 2015).
1.3.1 Direct photolysis
For the direct photolysis process, the green algae utilize the solar energy to produce hydrogen
from water under anaerobic conditions using hydrogenase enzyme as shown in the following
Eq. 1:
2𝐻2𝑂𝑙𝑖𝑔ℎ𝑡 𝑒𝑛𝑒𝑟𝑔𝑦→ 2𝐻2 + 𝑂2
(1)
The process is characterized by being simple, the substrate is cheap and there is no emission
of greenhouse gases, but the main drawbacks of the direct photolysis are the sensitivity of the
enzyme to oxygen and low light energy conversion (Levin et al., 2004; Nath et al., 2008).
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1.3.2 Indirect photolysis
Biohydrogen can be also produced by cyanobacteria (also known as blue-green algae) in two
separate stages of photosynthesis and fermentation. These processes involve fixation of CO2
into carbohydrates (starch in green algae, glycogen in cyanobacteria), followed by their
conversion to H2 by the reversible hydrogenase (Eq. 2 and 3) (Arimi et al., 2015):
6𝐻2𝑂 + 6𝐶𝑂2𝑙𝑖𝑔ℎ𝑡 𝑒𝑛𝑒𝑟𝑔𝑦→ 𝐶6𝐻12𝑂6 + 6𝑂2
(2)
𝐶6𝐻12𝑂6 + 6𝐻2𝑂𝑙𝑖𝑔ℎ𝑡 𝑒𝑛𝑒𝑟𝑔𝑦→ 12𝐻2 + 6𝐶𝑂2
(3)
The process is simple and cheap substrates can be used, however, the process still has some
drawbacks such as expensive bioreactors are required, the sensitivity of the enzyme to O2,
low production rate and H2 consumption by uptake of hydrogenase (Nath et al., 2008).
1.3.3 Dark fermentation
Dark fermentation is the conversion of organic substrate to biohydrogen. Fermentative
anaerobic microorganisms utilize organic materials to produce hydrogen in the absence of
light. The anaerobic digestion (AD) is divided into four main steps, as follows:
• Hydrolysis: conversion of non-soluble biopolymers to soluble organic compounds.
• Acidogenesis: conversion of soluble organic compounds to VFAs and CO2.
• Acetogenesis: conversion of volatile fatty acids to acetate, H2 and CO2.
• Methanogenesis: conversion of acetate and H2 to CH4 and CO2.
Diverse group of bacterial species of Enterobacter, Bacillus, and Clostridium can produce
hydrogen from organic wastes. Fermentative/hydrolytic microorganisms hydrolyze complex
organic polymers to monomers which further converted to a mixture of lower molecular weight
organic acids and alcohols by necessary H2 producing acidogenic bacteria (Chandrasekhar
et al., 2015). Utilization of wastewater as a potential substrate for biohydrogen production has
been drawing a considerable interest in recent years especially in dark fermentation process.
When acetate is the end-product, 4 moles hydrogen per mole glucose (Eq. 4), can be achieved:
𝐶6𝐻12𝑂6 + 2𝐻2𝑂 → 2𝐶𝐻3𝐶𝑂𝑂𝐻 + 4𝐻2 + 2𝐶𝑂2 (4)
When butyric acid is the end-product (butyric acid fermentation type), maximum theoretical
hydrogen yield of 2 moles hydrogen per mole glucose (Eq. 5) can be obtained:
𝐶6𝐻12𝑂6 → 𝐶𝐻3𝐶𝐻2𝐶𝐻2𝐶𝑂𝑂𝐻 + 2𝐻2 + 2𝐶𝑂2 (5)
-
Introduction 5
In case of mixed acetic/butyric acids fermentation type, the theoretical hydrogen yield is 2.5
moles hydrogen per mole glucose (Eq. 6) (Brunstermann, 2010):
4𝐶6𝐻12𝑂6 + 2𝐻2𝑂 → 2𝐶𝐻3𝐶𝑂𝑂𝐻 + 3𝐶𝐻3𝐶𝐻2𝐶𝐻2𝐶𝑂𝑂𝐻 + 10𝐻2 + 8𝐶𝑂2 (6)
However, hydrogen is not produced (or consumed) in case of other fermentation pathways
such as propionic acid, ethanol, malic acid fermentation type (Eq. 7-9):
For propionic acid fermentation type:
𝐶6𝐻12𝑂6 + 2𝐻2 → 2𝐶𝐻3𝐶𝐻2𝐶𝑂𝑂𝐻 + 2𝐻2𝑂 (7)
For ethanol fermentation type:
𝐶6𝐻12𝑂6 → 2𝐶𝐻3𝐶𝐻2𝑂𝐻 + 2𝐶𝑂2 (8)
For malic acid pathway:
𝐶6𝐻12𝑂6 + 2𝐻2 → 2𝐶𝑂𝑂𝐻𝐶𝐻2𝐶𝐻2𝐶𝑂𝑂𝐻 + 𝐶𝑂2 (9)
In dark fermentative hydrogen production, the Gibbs free energy is a negative value, which
implies that the energy is evolved, and the process can by carried out without an additional
energy source. The main advantages of dark fermentation over than other biological hydrogen
production methods are low energy requirements because it can be conducted in absence of
light, different substrates can be used, simple bioreactors can be used and production of
valuable by-products such as acetic acid, butyric acid, etc., which can be further used for
methane production or photo-fermentation, etc. (Nath and Das, 2004). On the other hand, the
main drawbacks of this process are the low hydrogen yield as maximum 33 % of the electrons
in the substrate can be converted to H2, with 66 % of the substrate electrons are consumed to
form soluble liquid metabolites (SLM) such as VFAs, alcohol, etc. High VFAs (organic content)
concentrations in the final effluent means that the process has low efficiency (low organic
content e.g. COD, VS, etc.,) and the H2 fermented effluent must be post-treated before being
discharged (Akinbomi et al., 2015).
1.3.4 Photo-fermentation
In photo-fermentation, anaerobic bacteria utilize VFAs such as acetic, butyric acid, etc., to
produce hydrogen gas in presence of light. These volatile acids are used by the microbes as
a carbon source for their metabolism thereby releasing hydrogen as a by-product. The volatile
acid substrates should be produced in a separate process e.g. dark fermentation. For the
process of photo-fermentation, purple non-sulphur photosynthetic bacteria (PNSB), including
Rhodobacter species, are used to convert organic acids such as acetate, lactate, and butyrate
to H2 and CO2 in anaerobic conditions.
-
6
In photo-fermentation, theoretical hydrogen yield of 4 moles hydrogen per mole glucose can
be achieved using the dark fermentation effluent rich in VFAs Species (Eq.10):
𝐶𝐻3𝐶𝑂𝑂𝐻 + 2𝐻2𝑂𝑙𝑖𝑔ℎ𝑡 𝑠𝑜𝑢𝑟𝑐𝑒→ 4𝐻2 + 2𝐶𝑂2
(10)
The photo-fermentation process has a positive Gibbs free energy, indicating that the process
needs external energy source to be carried out. The photo-fermentation process has the
advantages of high conversion rate of organic acids and it can be applied as a second stage
or post-treatment for the dark fermentation effluent. However, the process has some
drawbacks such as high energy requirements due to the need for external light source, only
dark fermentation effluent (VFAs) can be used which means that limited substrates can be
used in photo-fermentation, the photo- fermenters are expensive compared to the bioreactors
used in dark fermentation, activation of some methanogenic activity can compete with the
photo-fermentative bacteria (Arimi et al., 2015).
1.3.5 Hybrid reactor system
The sequential dark and photo-fermentation system can be used to improve the low hydrogen
yield achieved in the dark fermentation as well as reduce the organic content of the first stage
effluent by using organic acids as substrate to produce hydrogen as shown in Eq. 11 (Arimi et
al., 2015; Nath and Das, 2004).
𝐶6𝐻12𝑂6 + 6𝐻2𝑂 → 12𝐻2 + 6𝐶𝑂2 (11)
In this case initial substrate is fed into the dark fermentative bioreactor to produce hydrogen
gas and soluble metabolic products in absence of light. The liquid effluent of the dark
fermentation stage is rich in valuable products e.g. organic acids, which can be used by the
microorganisms in the presence of an external energy source. However, few works have
studied the combined dark and phot-fermentation system. For example, Nath et al. (2008)
studied coupled dark and photo-fermentation system; in their study, they observed hydrogen
yield of 3.31 mol-H2/mol-glucose in the first dark fermentation stage and acetate was the
main volatile product of using Enterobacter cloacae. A subsequent step of photo-fermentation
by Rhodobacter sphaeroides resulted in an additional hydrogen yield of 1.50–1.72 mol-
H2/mol-acetic acid. In another study, very high hydrogen yield in the range of 3.8–10 mol-
H2/mol- sucrose has been reported by coupling photo-fermentation with dark biohydrogen
fermentation using the species Clostridium pasteurianum and sucrose substrate (Chen et al.,
2008b). The study also reported high hydrogen contents in biogas (75–89 %) as well as the
COD removal efficiency was very high (90 %). A sequential fermentation was also
demonstrated by another study using cassava and food wastes where photo-fermentation
indicated higher hydrogen yield than the initial phase of dark fermentation.
-
Introduction 7
The combined (sequential) process can achieve higher production rate, hydrogen yields,
efficiency for the removal of COD and lower VFAs concentration in the final effluent than single
step process (dark fermentation). However, the high operational costs are higher due to
use of external light source and complex set-up because the two stages must be separated
(Arimi et al., 2015).
1.4 Cost of hydrogen production methods
Compared to other energy fuels, hydrogen is more expensive than other fuel options, so it is
expected that hydrogen will play a major role in the economy in the long run. However, a major
obstacle of commercialization of this technology is the high cost of the conventional production
methods. Production of hydrogen from low-value substrates e.g. waste substrates via
biological hydrogen production methods represent a potential solution to lower some of the
economic drawbacks and provide new energy sources. The use of sugar-rich waste substrates
such as sugarcane juice molasses, distillery wastewater represents a promising method to
reduce the energy costs of biohydrogen (Pandu and Joseph, 2012).
Table 2 presents the cost of H2 generated from biological processes compared to those of the
conventional processes. Only the costs of the pyrolysis are lower than those of the biological
hydrogen production methods. Therefore, the efficiency of the biological hydrogen production
methods must be enhanced to increase the net energy gain and decrease the cost of these
processes to facilitate their commercialization. From the data in Table 2, it can be seen that
the costs of hydrogen production by conventional methods are too high except in the case
pyrolysis. Hence, it was shown that despite of its high energy content; the cost of biological
hydrogen production was still not a cost effective when compared to the existing pyrolysis
method (conventional hydrogen production). More future research in the biological hydrogen
methods are recommended to increase the efficiency of the biological methods to
overcome or to replace the available conventional processes with the cost-effective methods
to improve the technical feasibility and scalability of the hydrogen production based on
renewable energy, higher carbon emission, large investment growth in renewable energies,
etc., could make cost parities to be reached in the near future (Nath and Das, 2004).
-
8
Table 2 Comparison of hydrogen production costs with different processes (Pandu and Joseph, 2012)
Production process Raw materials
Energy content
Unit cost of energy content
of the fuel
(MJ/kg) (US$/MBTU)
Photo-biological hydrogen H2O, organic acids 142 10
Fermentative hydrogen Molasses - 10
Pyrolysis for hydrogen production Coal, biomass - 4
H2 from advanced electrolysis H2O - 11
H2 from steam-reforming CH4 - 12.5
H2 from Nuclear Energy Electrolysis and water splitting - 12–19
H2 by biomass gasification Biomass - 44–82
H2 from Wind Energy Wind mill - 34
H2 from Photovoltaic power station Solar energy - 42
H2 from thermal decomposition of steam H2O - 13
H2 from photochemical Organic acids - 21
Gasoline Crude petroleum 43.1 6
Fermentative ethanol Molasses 26.9 31.5
Biodiesel Jatropha seeds 37 0.4
Natural gas Raw natural gas 33-50 10
-
Introduction 9
1.5 Technologies for hydrogen energy use
Hydrogen can be combusted directly in internal combustion engines or it can be used to
produce electricity using fuel cells.
1.5.1 Internal Combustion Engines
An internal combustion engine (ICE) is an engine in which the combustion of a fuel (from fossil
fuels - petroleum and carbon - to biofuels and hydrogen) can be carried out with an oxidizer
(usually air) in a combustion chamber. In an ICE, mechanical energy can be produced by
application of high temperature and high-pressure gases to some component in the engines
(such as pistons, turbine blades, nozzle). There are different designs of ICEs; each one has
advantages and disadvantages. Gasoline, Diesel, Wankel engines and open gas turbines
are all examples of internal combustion engines. Hydrogen is expected to substitute
conventional fossil fuels in traditional ICE (Yamin et al., 2000).
1.5.2 Fuel cells
A fuel cell (FC) is an electrochemical device that produces electrical energy (and heat) from
chemical energy of gaseous (e.g. hydrogen, natural gas, and biomass derived gas) or solid
(coal syngas mixture) fuels via an electrochemical process with high conversion efficiency. The
basic FC consists of an electrolyte layer, a porous anode and cathode. A fuel, such as
hydrogen, is fed to the anode, where negatively charged electrons are produced and separated
from positively charged ions. The electrons flow from the anode through an electrolyte an ionic
current toward the cathode, where protons combine with oxygen or air, producing water.
Simultaneously, the excess electrons flow through an external electric circuit, generating an
electric current (Edwards et al., 2007). The fuel cells can be classified according to the type of
electrolyte used, the operating conditions, the power density range, applications and
advantages and disadvantages (Larminie and Dicks, 2000). According to the type of electrolyte
used in the fuel cells, they can be proton exchange membrane fuel cell (PEMFC), phosphoric
acid fuel cell (PAFC), molten carbonate fuel cell (MCFC) and solid oxide fuel cell (SOFC)
(Baaske and Trogisch, 2004). The amount of energy produced in the FC by hydrogen oxidation
reaction depends on the FC type and its conversion efficiency. The conversion efficiencies of
the fuel cells are much higher than (nearly the double) those of the internal combustion engines
because fuel cells are not subject to the intrinsic limitations of the Carnot cycle. In
transportations, hydrogen fuel cell engines operate at an efficiency of up to 65 %, compared
to 25–30 % for current oil-fuelled car engines. Because the reaction occurs in the FC is
exothermic one, heat is generated in fuel cells; this heat can be used in combined heat and
power (CHP) systems, with high efficiency of 85 % or more (Dutton, 2002).
-
10
From the environmental point of view the fuel cells seem to be better than ICEs because they
can be operated at low temperature compared to ICEs, only water is produced and no harmful
pollutants (NOx) are released. If the hydrogen fuel could be produced from renewable routes
i.e. waste substrates and not from hydrocarbon-based fuel, real zero emission will be reached
by hydrogen-powered fuel cell vehicles Figure 1.
Figure 1 A fuel cell car powered by hydrogen and sold by Hyundai (Tenca, 2010/2011)
1.6 Factors affecting fermentative hydrogen production
Table 3 shows variation of the hydrogen production rates (yields) depending on the operating
conditions of the fermentation process. The hydrogen yield and conversion rate of the
substrates by hydrogen-producing bacteria in dark fermentation are highly dependent on
several factors. These factors must be optimized to maximize the hydrogen yield as follow.
1.6.1 Type of inoculum and pre-treatment
The inoculum used for bio-H2 production can be pure or mixed cultures. The pure cultures
have the advantages of being highly efficient in the degradation of carbohydrate-rich
substrates i.e. simple sugars such as glucose, sucrose, xylose, etc., into hydrogen, carbon,
acetic acid, butyric acid, and organic solvent such as ethanol, methanol, etc. However, the
hydrogen yields are low when complex substrates such as wastewaters, agricultural wastes,
etc., are used and the extraction process is another obstacle for the use of pure cultures.
Mixed cultures can be considered more effective when substrates with complex compositions
are used for biohydrogen production, where, the presence of a variety of microbial species
gives the advantage of degradation of different substrates; the culture is simple to operate and
-
Introduction 11
easy to control. However, the main disadvantage of mixed culture is that the fermentation
process may be shifted from the target of hydrogen production into formation of other
products such as methane. Therefore, pre-treatment of the sludge before inoculation is
recommended to inhibit any non-hydrogen-producing bacteria. Several inoculum pre-
treatments have been reported such as thermal pre-treatment (heat-shock), load-shock pre-
treatment, acid/base pre- treatment, chemical pre-treatment and combined pre-treatment (Lin
et al., 2012, Liu and Shen, 2004).
Heat-shock pre-treatment inhibits the bioactivity of non-spore forming species such as
methanogens and other hydrogen-consuming species and activates the growth of spore-
forming species such as Clostridium species, which are very important for the biohydrogen
production. The inoculum thermal pre-treatment can be conducted at temperatures in the rage
of 70–105 ⁰C for 15–120 min. These pre-treatment operating conditions have been reported
by researchers as effective conditions to suppress the methanogenic bioactivity and protect
the spore-forming bacteria (Alibardi et al., 2016; Bakonyi et al., 2014; Brunstermann and
Widmann, 2010; Cai et al., 2004; Salem et al., 2018a).
Methanogenic bacterial species show activity in a pH range of 6.8–7.2. Therefore, operating
the biohydrogen-producing reactor at low pH value (5.5) can suppress the activity of
methanogens. Authors reported that acid treatment is an efficient technique to inhibit the
bioactivity of H2 consuming-bacteria and acidic pH (5.0-5.5) has been reported as ideal for
effective H2 production (Fang et al., 2002; Fang and Liu, 2002). Other researchers reported
that optimum acid treatment can be carried out at pH 2–3 for exposure period of 24 h (Chang
et al., 2002).
Alkaline pre-treatment using NaOH at pH 8.5–12 for 24 h has been reported as an effective
method to supress partially the bioactivity of methanogens so the hydrogen yield is quite low
in case of using alkaline pre-treated sludge compared to other pre- treatment methods such
as heat-shock and/or acid pre-treatments (Mohan, 2008a).
The hydrogen-consuming bacteria can be eliminated from the mixed cultures by using
inhibiting chemicals such as iodopropane, or acetylene and 2- bromoethanesulfonic acid.
Chemical pre-treatment using 2-bromoethanesulfonic acid can inhibit the methanogenic
bioactivity without disturbing the activity of hydrogen- producing bacteria. Chemical pre-
treatment may also inhibit the acetate-producing species (Kotsopoulos et al., 2006).
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12
Other pre-treatment methods such as aeration, freezing and thawing, application of infrared
radiations have been used by authors to deactivate the methanogenic bioactivity.
Methanogens may be also removed from the mixed cultures by operating the bioreactor at
short HRT (2–10 h), since the H2-producing species grow faster than the methanogens.
The main disadvantage of this method is the low hydrogen yields compared to the yields that
can be achieved in case of heat-shock (Wang et al., 2003; Zhu and Beland, 2006).
Combination of different pre-treatment methods showed a positive effect on inhibition of
methanogens, activation of hydrogen-producers and increasing the hydrogen yield. Combined
pre-treatments such as heat-shock (100 ⁰C for 2 hours) and acid pre-treatment (pH 3 for 24
h), and heat-shock, acid and chemical pre-treatments have been reported to suppress
the methanogenic activity as well as enhance the hydrogen productivity (Mohan et al.,
2008b).
1.6.2 Substrate type
The composition (biodegradation) of the used substrate determines the metabolic pathway,
the fermentation type (the volatile fatty acids produced), and the hydrogen yields and
conversion efficiency of the fermentation process. High organic-rich substrates show high
potential for biohydrogen production via dark fermentation. The cost and availability of the
substrate are the main factors that must be considered to achieve high efficient biohydrogen
production process. Waste substrates such as simple sugars, food waste materials, and
industrial wastewaters have been studied for biohydrogen production. Carbohydrates-rich
substrates can be considered as ideal feedstocks for biohydrogen production because they
can be easily digested by bacteria. These substrates contain high COD content.
Carbohydrates-rich sugars such as glucose (Beckers et al., 2013; Liu and Fang, 2002;
Kotsopoulos et al., 2006; Morimoto et al., 2004; Mullai et al., 2013), starch (Liu and Shen,
2004), sucrose (Chen et al., 2008; Fang et al., 2002; Keskin et al., 2012; Lee et al., 2003;
Lee et al., 2004; Lin and Lay, 2004; Salem et al., 2017), and xylose (Kongjan and Angelidaki,
2009; Lin et al., 2008) were used to produce biohydrogen production. Olive wastewaters have
been used widely for biohydrogen production either via dark fermentation or photo-
fermentation (Scoma et al., 2013; Singh et al., 2013); the authors reported that these
substrates have the advantages of containing high volatile fatty acids concentration.
These substrates have the advantage of high hydrogen yields, but, the main disadvantage of
these substrates is high use in food and health sectors (Arimi et al., 2015).
-
Introduction 13
Lignocellulosic materials so far have highest abundance on the earth but their application in
biohydrogen production is limited. The main obstacle of using these substrates for
biohydrogen production is the low hydrogen yields achieved because of the low
solubilizations of the substrates. They recommended application of additional pre-treatment
step before conduction of the anaerobic fermentation (Han et al., 2012; Hendriks et al.,
2009). Other substrates such as dairy products e.g. cheese (Azbar et al., 2009), tapioca
wastewater (Thanwised et al., 2012), TWW (Lay et al., 2014; Lin et al., 2017a and 2017b)
were used to produce bio-H2 production and different hydrogen yields were reported.
1.6.3 pH
pH is another important factor that influences the activities of hydrogen-producing bacteria and
the fermentative hydrogen production because it may affect the metabolism pathway,
therefore, the biohydrogen production must be conducted under pH control. When the pH of
the fermentation medium is too low due to production of high VFAs concentration, the
bioactivity of the hydrogen-producing bacterial population would be inhibited, or metabolic
pathway would be switched resulting in cessation of biohydrogen generation (Lin et al., 2012).
The authors found that the H2-producing bioreactor should be conducted pH lower than 7. In
some studies, it has been reported that maximum HPR and H2 yield were achieved at pH
range of 5.5–6.0. For instance, optimum hydrogen yields of 492.8 NmL-H2/ /g-COD in a CSTR
system fed with cheese processing wastewater at a pH of 5.5 (Azbar et al., 2009), and the
highest HPR values of 8.3–8.6 L/L/d were obtained at an initial pH of 6.05 using brewery
wastewater (Shi et al., 2010). Although, pH in the range of 5.5–6.0 has been reported to be
optimal values for operating H2-producing bioreactors as well as to achieve maximum
hydrogen yields, the optimal pH for biohydrogen production can be varied depending on the
substrate used in the fermentation process.
1.6.4 Organic loading rate (OLR)
The OLR describes the amount of organic material per unit reactor volume which is subjected
to digestion in the reactor in a certain time (increasing the substrate concentration at constant
HRT). OLR is an important factor that has a great impact on biohydrogen production. High
substrate concentrations could enhance biohydrogen production efficiency, but substrate or
product inhibitions would occur when the organic loading (substrate concentration) exceeds a
threshold level. However, there is no OLR that can be considered as an optimal value to
achieve maximum hydrogen production (yield) so far.
Several studies have considered the effect of substrate concentration on biohydrogen
production from waste substrates (wastewaters). In a previous study, authors found that
efficient hydrogen yield from preserved fruits soaking solution increased from 0.59 mol-
-
14
H2/mol-hexose at OLR of 0.44 g - COD/L.d to 2.64 mol-H2/mol-hexose when the OLR
increased to 2.19 g - COD/L.d, then the yield decreased to 1.38 mol-H2/mol-hexose at OLR
of 1.31 g- COD/L.d (Lay et al., 2010).
Salem et al. (2017) found that continuous biohydrogen production rate (HPR) increased with
increasing sucrose concentration from 10 up to 30 g/L, with 5 g/L increments, but the hydrogen
yield decreased at high sucrose concentrations. In the above study, the authors reported
optimum hydrogen yield of 390 mL-H2/g-sucrose at sucrose concertation of 10 g/L. In other
work, the effect of substrate concentration on bio-H2 production was studied in batch assays,
the authors found that increasing the substrate (xylose) concentration from 0.5 up to 4.0 g/L,
decreased the hydrogen yield with maximum hydrogen yield of 1.62 mol-H2/mol-xylosedegraded
at initial xylose concentration of 0.5 g/L (input xylose of 10 mg with 9.54 mg xylose
consumed). While, the hydrogen yields were in the range of 0.45–1.46 mol-H2/mol-
xylosedegraded at xylose concentrations of 1.0–4.0 g/L (input xylose of 20–80 mg with 18.85-
35.28 mg degraded) (Kongjan et al., 2009).
The optimal OLR for biohydrogen production depends on several factors such as sludge
loading rate, pH, substrate type and concentration, temperature, reactor type, etc. (Arimi et al.,
2015).
Mohan, (2008a) found that the optimal OLR for biohydrogen production may be higher
in case of simple sugars such as glucose, sucrose, xylose than that in case of industrial
wastewaters. They explained this behaviour by inhibition of the hydrogen- producing bacteria
due to accumulation of recalcitrant pollutants in the bioreactors at high substrate
concentrations.
High OLR results in low H2 yields probably because the fermentation reaction would produce
solvents rather than hydrogen gas, which is unfavourable for biohydrogen production (Lay
et al., 2010). Wu et al. (2006) reported that the fermentation reaction may be shifted from
hydrogen production to the formation of propionate and ethanol species, which inhibit the
biohydrogen production at high OLR. The low H2 yield achieved may be attributed to the
inhibitory effect of high hydrogen partial pressure at high OLR (Kongjan et al., 2009; Wu et
al., 2006).
At high substrate concentrations, the concentration of VFAs would be high; this has a negative
effect on the bioactivity of the hydrogen-producing microorganisms and the hydrogenase
enzyme (Fang and Liu, 2002).
-
Introduction 15
1.6.5 Hydraulic retention time (HRT)
HRT refers to the mean time that a defined volume element of substrate remains in a
combined reactor system before being discharged (Arimi et al., 2015). HRT is one of the
most important control parameters affecting continuous production of hydrogen. HRT control
can avoid the hydrogen utilization by hydrogen-consumers like methanogens (Kothari et al.,
2012).
Lin et al. (2008) reported that increasing the HRT from 2 to 4 hours increased the hydrogen
yield with maximum yield of 3.2 mol-H2/mol-glucose at HRT of 4 h, the hydrogen yield was the
same when the HRT increased to 8 h. They explained low hydrogen yields at short HRT by
the fact that the cell washout increases as the HRT decreases. In other study, Salem et al.
(2018a) found that the optimal HRT was dependent on the waste composition; the authors
found that optimum hydrogen yields of 320 and 150 mL-H2/g-VS were achieved at HRT
of 18 h for sucrose and potatoes wastewater, respectively, while, in case of bean wastes
optimum yield of 80 mL-H2/g-VS was observed at HRT of 24.
Thanwised et al. (2012) found that the optimal hydrogen productivity was observed at HRT of
6 h, while low productivities were achieved at short and/or long HRT in an anaerobic baffled
reactor used for biohydrogen production from tapioca wastewater.
Therefore, it can be concluded that the optimal HRT for biohydrogen production ranges
between few hours and one day; longer HRT is required for methane production than those
required for biohydrogen production systems. However, the optimal HRT is influenced by
many factors including type and concentration of substrate, temperature, biomass
concentration and composition (pure or mixed culture), etc. (Scoma et al., 2013). H2-producing
bioreactor must be operated at optimum HRT to limit the biomass washout at short HRT as
well as avoid the activation of methanogenic bioactivity at long HRT.
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16
1.6.6 Temperature
Temperature is one of the most important factors that influences on the activities of hydrogen-
producing bacteria and the fermentative hydrogen production.
It has been demonstrated that in an appropriate range, increasing temperature could increase
the ability of hydrogen-producing bacteria to produce hydrogen during fermentative hydrogen
production, but temperature at much higher levels could decrease it with increasing levels
(Wang and Wan, 2008). Most of the biohydrogen production studies have been performed at
ambient (15–30 ⁰C), mesophilic (32–40 ⁰C) and thermophilic (50–60 ⁰C) conditions, while few
studies have investigated biohydrogen production under extreme thermophilic (65–75 ⁰C)
conditions (Lin et al., 2012). However, due to the big differences in bioreactor, substrate, seed
sludge and other process conditions, there is no agreement on the optimal temperature that
can achieve maximum hydrogen yields.
The optimum yields were 266 mL-H2/g-hexose for 15–30 ⁰C (Fang et al., 2002), 333 mL-H2/g-
hexose for 32–39 ⁰C (Van Ginkel et al., 2001), and 327 mL-H2/g-hexose for 50–64 ⁰C (Ueno
et al., 1995). However, it is possible to increase hydrogen productivity from wastewater at
high temperatures; this can be due to elimination of the competing mesophilic methanogens,
but very high temperatures may result in low hydrogen yields due to denaturation of the
of microbial enzymes (Arimi et al., 2015). For textile industry effluent, a high temperature
around 70–80 ⁰C is needed to operate the biohydrogen production system (Lin et al., 2012).
While developing biohydrogen production technology, it is very important to operate the
bioreactors at low temperatures to achieve positive energy gain as well as ensure safety during
maintenance and monitoring (Lin et al., 2012).
1.6.7 Reactor configuration
Several types of reactors such as CSTR, UASB, CIGSB, AGSBR, FBR etc., have been
studied to generate biohydrogen efficiently; each reactor type has its own benefits and
drawbacks (Lin et al., 2012). UASB reactor is effective in treating organic wastes and
converting them into biohydrogen, but the main drawbacks of this reactor are low hydrogen
yields due to the low mass transfer and the possibility of formation of hydrogen-consuming
species e.g. methanogens (Chang and Lin, 2004). FBR bioreactors have been reported to
produce biohydrogen efficiently, but there are several problems associated with these
reactors such as localized populations differing over the length of the reactor, channelling due
to inefficient mixing, and incomplete conversion of substrates due to poor mass transfer
efficiency (Chang et al., 2002). CIGSB and AGSBR bioreactors can achieve high production
rates due to maintain high cell density due the formation of granular sludge in the bioreactor
at a high dilution rate, but still there is need to overcome the problems related to inefficient
-
Introduction 17
mixing and stability of functional granule (Lee et al., 2004). CSTR bioreactor is the most
common bioreactor in biohydrogen production because high hydrogen yields can be achieved
in CSTR due to high mass transfer rate, but the main disadvantage CSTR is the low biomass
concentration due to the washout of the bacterial species (Salem et al., 2017).
1.6.8 Nutrient concentration and metal ions
Hydrogen production requires nutrients for bacterial metabolism, growth and activity. The
nutrients include nitrogen (N), phosphate (P) and some trace elements. However, hydrogen
production may be inhibited when the nutrients concentration exceeds the optimal values.
Nitrogen is one of the most essential nutrients needed for growth. Several researchers have
studied the effect of nitrogen concentration on biohydrogen production. For instance, Liu and
Shen (2004) investigated the effect of increasing ammonium bicarbonate (NH4HCO3)
concentration as N source from 0.1 to 2.0 g/L, corresponding to C/N ratios of 67–3.3 on the
batch biohydrogen production from starch. Results showed that the maximum hydrogen
yield of 175 mL-H2/g-hexose was achieved at 1.0 g-N/L or C/N ratio of 6.7. Optimum hydrogen
yield of 170 mL- H2/g-hexose was achieved at 0.4 g-N/L concentration or at C/N ratio of 10
using glucose (carbon source) and yeast extract as N source at three concentrations, i.e.
0.2, 0.4 and 0.8 g-N/L, corresponding to C/N ratios of 20, 10 and 5 (Morimoto et al., 2004),
and 327 mL-H2/g-hexose from sucrose at C/N ratio of 47 higher than the corresponding yields
achieved at C/N ratios of 130, 98, and 40 (Lin and Lay, 2004).
Phosphate (phosphorous source) is needed in hydrogen production for its nutritional purpose
as well as for buffering capacity. Argun et al. (2008) found that optimum hydrogen yield of
281.0 mL-H2/g-starch from wheat powder was achieved at C/P ratio of 1000, and O-Thong
et al. (2008) achieved maximum hydrogen yield of 6.33 L- H2/L-substrate from palm oil mill
effluent at C/P ratio of 559.
Trace metals such as iron, nickel, magnesium, zinc, sodium, etc., are also important in
hydrogen production. Magnesium ion is an important co-factor that activates almost 10
enzymes including hexokinase, phosphofructokinase and phosphoglycerate kinase during
glycolysis process (Voet et al., 1999), and the presence of other metals such as iron and nickel
is essential for hydrogenase (Mullai et al., 2013; Salem et al., 2017).
Optimum concentrations of the metal ions must be used because higher concentrations of
metal ions than the optimum doses lead to reduction of the biohydrogen production as they
have toxicity effect in that the metal ions penetrate and disrupt the cell wall as well as high
-
18
concentrations of metal ions can cause oxidative stress on the bacteria which may have a
negative impact on the biohydrogen production (Mullai et al., 2013; Salem et al., 2017).
Table 3 Comparison of the hydrogen yields depending on the operating conditions
OLR
HR
TT
g-C
OD
/L.d
h⁰C
Wheat
pow
der
Mix
ed
Batc
h7.0
NA
NA
37.0
5–1000
20–200
74.4–281 m
L-H
2 /g-
sta
rch
Arg
un e
t al. (2
008)
Cheese
Mix
ed
CS
TR
5.5
21.0–47.0
24–84
NA
NA
NA
3–22 m
mol-H
2 /g-
CO
DA
zbar e
t al. (2
009)
Corn
sta
lkP
ure
Batc
h4.5–7.0
NA
-36.0
NA
NA
20-1
76 m
L-H
2 /g-T
SF
an e
t al. (2
008)
Sucro
se
Mix
ed
Batc
hN
AN
AN
A35.0
40–98
NA
2.6
4–4.8
0 m
ol-
H2 /m
ol- s
ucro
se
Lin
and L
ay (2
004)
TW
WM
ixed
CS
TR
6.8
30–60
4–8
35.0
NA
NA
0.6
4–1.5
2 m
ol-
H2 /m
ol- h
exose
Lin
et a
l. (2017b)
Sucro
se
Mix
ed
CS
TR
5.5
22.4–67.6
12
35.0
NA
NA
0.2
0–0.3
9 L
-H2 /g
-
sucro
se
Sale
m e
t al. (2
017)
OM
WM
ixed
PB
BR
75.5–38.8
24–168
35.0
NA
NA
NA
Scom
a e
t al. (2
013)
PO
MW
Pure
UA
SB
5.5
NA
8–32
37.0
NA
NA
0.2
3–0.3
5 L
-H2 /g
-
CO
Dadded
Sin
gh e
t al. (2
013)
Tapio
ca
waste
wate
rM
ixed
AB
R9.0
16.1
5–130.8
23–24
32.3
NA
NA
10.1
8–18.7
0 m
L-H
2 /g-
CO
DThanw
ised e
t al. (2
012)
NA
0.1
9–0.2
7 L
-H2 /g
-
sucro
se
NA
: no
t ava
ilab
le
Liu
and F
ang (2
002)
H2 y
ield
Refe
rences
Sucro
se
Mix
ed
CS
TR
5.5
25
4.6–28.6
26.0
NA
Reacto
rC
ultu
reR
eacto
rpH
C/N
C/P
-
Research Objectives and strategies for improvement of biohydrogen production 19
2 Research Objectives and strategies for improvement of biohydrogen production
Although dark fermentation process has been reported as a cheap process compared to
other processes, the low hydrogen yield represents a major challenge that must be studied to
achieve higher yields and increase the net energy gain from the process by stimulating the
conversion of the substrate to hydrogen gas rather than formation of VFAs. To achieve high
hydrogen yields from dark fermentation as well as improve the efficiency of the dark
fermentation process, some strategies have been proposed by authors as follow. Depending
on the solubilization of the used substrate in the fermentation process, the enhancement
method can be decided. The main objectives are enhancement of biohydrogen production
from waste substrates as discussed below:
2.1 Formation of granular sludge
Carbohydrate-rich substrates such as sucrose, glucose etc., are easily biodegradable
substrates that can be hydrolysed and form biohydrogen and organic acids. To improve the
hydrogen yields from such substrates, it is necessary to keep high biomass density in the
bioreactor, while operating the bioreactor at short HRT.
The main drawback of the CSTR bioreactor, the most common reactor used in biohydrogen
production process, is the washout of the biomass. Keeping high cell density inside the
bioreactor is an important factor to achieve high hydrogen yields. One possibility to maintain
high cell density in the bio-fermenter is to produce (or use) granular sludge.
For this purpose, HPGs species were produced in the CSTR bioreactor using sucrose
wastewater at 10 g-sucrose/L, by application of acid incubation time at pH 3 for 24 h. The
biohydrogen production was compared before and after the formation of HPGs. Because the
granulation process is highly dependent on the substrate concentration (OLR), the sucrose
concentration increased up to 30 g/L, with 5 g/L increments.
2.2 Use of biofilm carriers
Based on the findings of the previous experiment, optimum hydrogen yield was achieved at
sucrose concentration of 10 g/L, therefore, this concentration (10 g/L) was used in next
investigations.
Cell immobilization was also studied as another possibility to avoid the bacterial species
washout and keep high cell density. Wheel-shaped plastic carriers were used at packing
-
20
ratio of 11.1 % (V/V), at operating conditions of 12 h (HRT), 10 g- sucrose/L (sucrose
concentration), and 5.5 (pH).
2.3 Increasing the bioactivity of the hydrogenase enzyme
The effect of hematite nanoparticles (NPs) on biohydrogen production from sucrose
wastewater was studied. The operating conditions were kept at 10 g-sucrose/L, 12 h (HRT),
5.5 (pH). Because the CSTR was operated at continuous mode and to limit the washout of
the NPs, they were immobilized into supporting carriers (silicone carriers), after preparation,
then they were fed into the CSTR bioreactor.
2.4 Sequential systems
Because the dark fermentation effluent is rich in VFAs species, it is a promising process to
use this effluent in a second stage. With the of aim improving the energy recovery from the
initial feed, two combined systems were studied as dark and dark system, and dark and photo
fermentation. The first stage CSTR bioreactors were operated at the same conditions of 10 g-
sucrose/L, 12 h (HRT), and 5.5 (pH). For the second stage, two different bioreactors as
continuously mixed fermenter (photo- fermentation), and UASB (dark fermentation) were used.
2.5 Pre-treatment of substrates
Because of the low solubilisation of some organic wastes especially the agricultural wastes,
application of pre-treatment methods before the conduction of the biohydrogen production
methods is necessary to achieve high hydrogen yields. For potatoes and bean wastes, it
was observed that the hydrogen yields were too low. In order to improve the solubilization and
increase the hydrogen yields, several pre- treatment methods including heat, ultrasonication,
alkaline, acid, H2O2 pre-treatments alone or in combination were applied on the waste
substrates and biohydrogen production was studied in batch assays.
Industrial wastewaters such as TWW, pharmatheutical wastewater, pesticides wastewater,
etc., need strong pre-treatment methods such as chemical oxidation, activated carbon, etc.
Previous works studied some pre-treatment methods such as adsorption using activated
carbon, cation resin (Li et al., 2012), chemical coagulation-flocculation pre-treatment (Lin et
al., 2017a, 2017b), etc., but the authors reported that the hydrogen yields were still low.
Biohydrogen production was studied using AOPs pre-treated TWW. Two different processes
as photocatalytic degradation and Fenton oxidation were investigated. The operating
conditions of the AOPs pre- treatment were changed to obtain the doses that can achieve
optimum hydrogen yields keeping the cost of the pre-treatment methods as low as possible.
-
Materials and methods 21
3 Materials and methods
3.1 Sludge collection, characterization and pre-treatment
The sludge samples were collected from two sources as Kasslerfeld wastewater treatment
plant (WWTP) in Duisburg and Bottrop wastewater treatment plant. The collected sludge was
sieved using a mesh (2 mm) to remove waste big materials. Before inoculation, the harvested
sludge was subjected to heat-shock pre-treatment to activate the hydrogen-producing
bacteria such as Clostridium species and to deactivate the hydrogen-consuming species e.g.
methanogens and other non H2- producing microorganisms in the sludge, and then the pre-
treated sludge was transported into the bioreactor. The pre-treatment was conducted by
heating the sludge at 105 ⁰C for two hours in an oven. This pre-treatment was chosen
because the heat-shock is characterized by being fast, simple and effective process (Bakonyi
et al., 2014). The sludge was characterized for the following parameters pH, COD, total solids
(TS), volatile solids (VS) as presented in Table 4.
Table 4 Average characteristics of the sludge
Parameter Unit Kasslerfeld WWTP Bottrop WWTP
pH - 7.56 7.25
TS g/L 31.6 18.1
VS g/L 18.15 9.41
Solid content % 36.4 52.6
Water content % 63.6 47.4
3.2 Substrates for bio H2 production
Different substrates including sucrose, potato, bean and textile wastewater were tested.
Sucrose wastewater was prepared by dissolving specific amount of sucrose in tap water.
Potatoes wastes were prepared using commercialized potatoes. The potatoes (without
washing and peeling) were cut using knife and homogenized using a blender for 5 min with
proper addition of water at a 1:4 ratio (w/w). Bean wastes were prepared by mixing bean
with tap water at a 1:2 ratio (w/w), and homogenized using a blender for 5 min. The resulting
liquid mixtures (potatoes and bean wastes) were screened to remove the large particles and
then the solutions were diluted to have nearly equal total solid (TS) concentrations.
-
22
Textile wastewater was prepared using three dyes as deep blue, ruby red and deep brown
(DEKA-Textilfarbe GmbH Serie “L”, Germany) and carbohydrate-rich substrate (starch). The
stock TWW solution was prepared by dissolving 0.75 g dyes (0.25 g for each dye) with 1 g-
starch in 1 L tap water. The average properties of the substrates are given in Table 5.
Table 5 Average properties of the substrates
Parameter Unit Sucrose WW Potatoes WW Bean WW Textile WW pH - 7.47 7.25 6.25 7.49 SCOD g/L 12.1 25.6 21.8 1.133 TS g/L 9.31 22.6 19.60 1.816 TVS g/L 9.1 17.4 13.82 1.076 VFAs mg/L 1100 409 206 32.0
3.3 Pre-treatment of substrates
3.3.1 Pre-treatment of potatoes and bean wastes
To increase the solubilization of potatoes and bean wastes as well as enhance the
biohydrogen production, various pre-treatment methods such as heat, acid, alkaline,
ultrasonication, and hydrogen peroxide were applied alone or in combination on substrates
before conduction of biohydrogen fermentation tests. For heat pre- treatment, the waste
substrates were heated at 100 °C for 30 min. Acid and base pre-treatments were performed
by adding aqueous 5% HCl and NaOH (2 N) solutions to pH values of 4 and 10, respectively,
and these conditions were lasted for 30 min with proper mixing. The waste materials were
also treated using an ultrasonic processor (Fritsch, Laborgeräte, Idar-Oberstein, W.-Germany)
for 30 min. Hydrogen peroxide pre-treatment was performed by mixing 1 L of wastewater with
3 mL H2O2. For combined heat/acid pre-treatment, the wastewater was boiled for 30 min at
100°C and mixed with 5 % HCl (pH=4). The mixtures were then neutralized to pH 7.0 by
addition of dilute NaOH and/or HCl aqueous solution.
3.3.2 Pre-treatment of TWW
The pre-treatment experiments using TWW were performed in glass reactor (active volume =
0.75 L) with an inner diameter of 12 cm. The reactor containing reaction solution was placed
on a magnetic stirrer to provide appropriate mixing. The light source was placed 18 cm above
the reaction surface. Temperature of the solution was maintained constant by circulating
cooled water around the reactor.
The photocatalytic degradation reaction was studied under operating parameters of 0.25–1
g-TiO2/L, 0.1–0.4 g-dye/L, contact time (1–4 h), pH (4–10), temperature (20–40 ⁰C) and two
light sources (UV- and visible light lamp). The reaction was started-up with conditions: 0.25
-
Materials and methods 23
g-TiO2/L, pH 7, 0.25 g-dye/L, reaction time of 2 h, 30 ⁰C and UV-radiation. The reaction was
started when the light source was turned on.
The Fenton oxidation pre-treatment was tested under variable conditions including Fe2+
concentration (0.5–1 g/L), reaction time (20–60 min), dye concentration (0.1–0.4 g/L), H2O2
dose (1–3 mL/L), temperature (20–40 ⁰C) and light sources (UV- and visible light). For the
start-up, the reaction was carried out at 0.5 g- Fe2+/L, 0.25 g- dye/L, pH 4, 40 min, 1 mL-
H2O2/L and 30 ⁰C. The reaction was started when the H2O2 was added.
In the experiments set-up, one parameter was variable, while, the others were maintained
constants (as in the start-up). After pre-treatment of the wastewater, the pH of the liquid was
adjusted to pH 7, filtered through a Whatman filter paper no. 47 to separate the (photo)
catalyst and transferred to the biological reactor for hydrogen production.
The conditions, used in the start-up experiments, were collected from the literature and were
reported as the optimum concentrations to achieve high efficient removals process and better
biodegradation.
The pre-treatment methods were performed to determine the optimum operating conditions of
the pre-treatment to increase the biodegradation of the substrate as well as to maximize
the biohydrogen production.
3.4 Biohydrogen production experiments
3.4.1 Batch H2-production experiments
3.4.1.1 Biohydrogen production from potatoes and bean wastes
Batch experiments were conducted in 500 mL glass bottles with effective volume of 250 mL.
The fermentation liquid in each bottle consisted of 210 mL pre-treated substrate, 30 mL sludge,
and 10 mL nutrient solution. The food to microorganisms (F/M) ratio was maintained at 0.