51

Hydrogen production from organic wastes "clean energy production from low- value substrates”

Ahmed Hassan Salem Hassan

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

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

I

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

II

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

III

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

IV

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

V

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

VI

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

VII

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

VIII

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

IX

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.

X

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).

XI

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.

XII

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

XIII

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.

XIV

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.

2

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).

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).

4

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).

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.

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.