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HYDROGEN PRODUCTION BY ENRICHMENT GRANULES IN
AN ACIDOGENIC FERMENTATION PROCESS
SHIVA SADAT SHAYEGAN SALEK
NATIONAL UNIVERSITY OF SINGAPORE
2007
HYDROGEN PRODUCTION BY ENRICHMENT GRANULES IN
AN ACIDOGENIC FERMENTATION PROCESS
SHIVA SADAT SHAYEGAN SALEK
(B.Sc, Tehran University)
A THESIS SUBMITTED
FOR THE DEGREE OF MASTER OF ENGINEERING
DIVISION OF ENVIRONMENTAL SCIENCE AND ENGINEERING
NATIONAL UNIVERSITY OF SINGAPORE
2007
ACKNOWLEDGEMENTS
Sincerest appreciation is extended to Prof. NG Wun Jern, my professor, for his
patience, encouragement and support during all phases of the project resulting in the
completion of this thesis. I would also like to express my gratefulness to my current
supervisor Dr HE Jianzhong for her guidance, support and kindness during last year of
this project.
My special thanks to my senior MUN Cheok Hong for patiently training me laboratory
skills, and most importantly to think critically and scientifically. CHENG Dan, TO
Priscilla, and FAN Caian have also helped me in many ways. Their friendship and help
will always be remembered.
Finally, I express my genuine and deepest gratitude to my parents and brothers for
their boundless love, encouragement and moral support.
I
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ..................................................................................................................... I
SUMMARY ............................................................................................................................................ IV
LIST OF TABLES ................................................................................................................................. VI
CHAPTER 1: INTRODUCTION .......................................................................................................... 1
1.1 INTRODUCTION ............................................................................................................................. 1
1.1.1 RESEARCH OBJECTIVES ................................................................................................................ 5
1.1.2 HYPOTHESES................................................................................................................................. 6
1.1.3 CHAPTERS IN BRIEF ...................................................................................................................... 7
CHAPTER 2: LITERATURE REVIEW ............................................................................................ 10
2.1 ENERGY SUPPLY AND ENVIRONMENTAL PROTECTION ............................................... 10
2.2 THE TRANSITION TO THE RENEWABLE ENERGY ECONOMY ..................................... 11
2.2.1 HYDROGEN PRODUCTION FROM RENEWABLE ENERGY .............................................................. 11
2.2.2 RENEWABLE ENERGY THROUGH ANAEROBIC TREATMENT TECHNOLOGY.................................. 12
2.2.3 HYDROGEN PRODUCTION FEASIBILITY ....................................................................................... 12
2.3 ANAEROBIC WASTEWATER TREATMENT .......................................................................... 13
2.3.1 IMPORTANT PARAMETERS IN ANAEROBIC TREATMENT .............................................................. 15
2.3.2 TWO-STAGE ANAEROBIC WASTEWATER TREATMENT................................................................ 15
2.3.3 FUNDAMENTALS OF ANAEROBIC TREATMENT ............................................................................ 16
2.4 FUNDAMENTALS OF THE FERMENTATIVE PRODUCTION OF HYDROGEN ............. 21
2.4.1 INSTABILITY OF HYDROGEN PRODUCTION AND COMPOSITIONS OF BACTERIAL COMMUNITIES
WITHIN A DARK FERMENTATION ......................................................................................................... 24
2.4.2 PRETREATMENT CONDITIONS TO SCREEN HYDROGEN PRODUCING BACTERIA FROM THE MIX
CULTURE ............................................................................................................................................. 28
2.4.2.1 Heat-treatment ................................................................................................................... 29
2.4.2.2 Acid/Base Treatment .......................................................................................................... 30
2.4.2.3 Lack of Carbon Source....................................................................................................... 31
2.4.2.4 Methanogen Inhibitors ....................................................................................................... 31
2.5 FERMENTATION PROCESS THROUGH GRANULATION .................................................. 31
2.6 GRANULE MICROSTRUCTURE................................................................................................ 32
2.6.1 FORMATION OF LAYERED MICROSTRUCTURE ............................................................................. 32
2.6.1.1 Biogranules with Layered Microstructure ......................................................................... 36
2.6.2 FORMATION OF UNIFORM MICROSTRUCTURE ............................................................................. 36
2.6.2.1 Biogranules with uniform microstructure .......................................................................... 37
2.6.3 HYDROGEN PRODUCTIVE GRANULES ......................................................................................... 37
2.6.3.1 Microstructure of Hydrogen-Producing Granule .............................................................. 38
CHAPTER 3: MATERIAL AND METHODS.................................................................................... 39
3.1 REACTOR CONFIGURATION.................................................................................................... 39
3.2 GAS COLLECTION SYSTEM...................................................................................................... 41
3.3 SUBSTRATE PREPARATION ..................................................................................................... 41
3.4 START-UP AND MONITORING BIOREACTOR OPERATION ............................................ 44
3.4.1 SEED SLUDGE ............................................................................................................................. 44
3.4.2 MONITORING BIOREACTORS PERFORMANCE .............................................................................. 44
3.4.2.1 pH ....................................................................................................................................... 44
3.4.2.2 Solids .................................................................................................................................. 45
3.4.2.3 Gas Composition ................................................................................................................ 46
II
3.4.2.4 Volatile Acids ..................................................................................................................... 46
3.4.2.5 Total Organic Carbon ........................................................................................................ 47
3.4.3 MICROBIAL ANALYSIS ................................................................................................................ 47
3.4.3.1 Scanning Electron Microscopy (SEM) ............................................................................... 47
3.4.4 MOLECULAR MICROBIAL DIVERSITY ANALYSIS ........................................................................ 48
3.4.4.1 DNA Extraction .................................................................................................................. 48
3.4.4.2 Polymerase Chain Reaction. .............................................................................................. 48
3.4.4.3 Terminal-Restriction Fragment Polymorphism analysis. .................................................. 49
CHAPTER FOUR: RESULTS AND DISCUSSION .......................................................................... 50
4. SAMPLING AND RESULTS ........................................................................................................... 50
4.1 OPTIMIZE THE ANAEROBIC H2 PRODUCTION ................................................................... 50
4.2 CULTURE ENRICHMENT (PRE-TREATMENTS AND START UP) .................................... 50
4.2.1 INITIAL HEAT TREATMENT .......................................................................................................... 51
4.2.2 ACID TREATMENT....................................................................................................................... 51
4.2.2.1 Enhancement pH ................................................................................................................ 51
4.2.2.2 Cultivation pH .................................................................................................................... 51
4.3 BIOLOGICAL HYDROGEN PRODUCTION MEASURED IN BATCH ANAEROBIC
RESPIROMETERS AFTER HEAT TREATMENT ......................................................................... 53
4.3.1 BIOLOGICAL HYDROGEN PRODUCTION MEASURED IN ASBR AFTER HEAT TREATMENT ........... 60
4.4 BIOLOGICAL HYDROGEN PRODUCTION MEASURED IN ASBR AFTER LACK OF
CARBON SOURCE TREATMENT.................................................................................................... 61
4.5 MICROBIAL SHIFT ...................................................................................................................... 65
4.6 DYNAMIC CHANGES DURING ONE CYCLE OF ASBR ....................................................... 68
4.7 CHARACTERISTIC OF ENRICHED ACIDOGENIC GRANULES ....................................... 70
4.7.1 SIZE (AVERAGE GRANULE DIAMETER) DISTRIBUTION AND SETTLING VELOCITY ...................... 70
4.7.2 MORPHOLOGY AND MICROSTRUCTURE OF GRANULES ................................................................ 71
4.8 BIOLOGICAL HYDROGEN PRODUCTION MEASURED IN UASB WITH ENRICHED
GRANULE ............................................................................................................................................. 76
CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATIONS .............................................. 78
5.1 APPLICATION AND CONTRIBUTION OF THIS RESEARCH ............................................. 78
5.2 CONCLUSIONS .............................................................................................................................. 78
5.3 RECOMMENDATIONS FOR FURTHER RESEARCH............................................................ 79
BIBLOGRAPHY ................................................................................................................................... 81
III
SUMMARY
Energy supply and environmental protection are two crucial issues for sustainable
development of today's world. Among renewable energies, hydrogen produced by
biomass is a completely carbon-free fuel with a high energy yield (122 kJ/g), and
considered a feasible alternative to fossil fuels. Harvesting hydrogen by fermentation
process has attracted many researchers in recent years.
This study demonstrated acidogenic sludge (pH 5.5) could produce hydrogen and also
granulate in an anaerobic sequencing batch reactor (ASBR) fed with synthetic
wastewater containing glucose with 6.7h hydraulic retention time (HRT) at ambient
temperatures. Optimization, enrichment, and stability of the acidogenic granular sludge
have been investigated at a constant loading rate of 25 g-glucose/ (L.d). Results
showed that hydrogen in the biogas increased from 15% to 48% by subjecting the
biomass to a combination of heat-treatment, acidic pH, and carbon source limitation.
The maximum hydrogen, yield, and production rate was 73%, 2.5 molH2/mol glucose,
and 0.34 molH2/d, respectively. Microbial analysis indicated that enrichment by
granulation was successful and microbial diversity changed significantly after
treatments. The ASBR was operated for 445 days.
The hydrogen producing granules were characterized. A typical matured granule was
1.7mm in diameter with an average of 43 m/h settling velocity. Moreover, as
morphological analysis demonstrated, the inner and outer surface of the granules was
comprised the same types of bacteria and hence had non-layered structure. A hydrogen
producing granule had multiple cracks on the surface. The acidified effluent comprised
volatile fatty acids (VFA) and alcohols. The VFA comprised acetate (73%), butyrate
(23%), propionate (1.5%), caproate (0.69%), valerate (0.58%).
Key words: acidogenic, hydrogen, granule, glucose, synthetic wastewater
IV
LIST OF FIGURES
FIGURE 1.1............................................................................................................................................... 4
FIGURE 1.2............................................................................................................................................... 5
FIGURE 1.3............................................................................................................................................... 9
FIGURE 2.1......................................................................................................................................... 2020
FIGURE 2.2........................................................................................................................................... 333
FIGURE 2.3........................................................................................................................................... 355
FIGURE 3.1......................................................................................................................................... 4040
FIGURE 4.1........................................................................................................................................... 522
FIGURE 4.2........................................................................................................................................... 533
FIGURE 4.3........................................................................................................................................... 555
FIGURE 4.4. .......................................................................................................................................... 577
FIGURE 4.5........................................................................................................................................... 599
FIGURE 4.6......................................................................................................................................... 6161
FIGURE 4.7......................................................................................................................................... 6262
FIGURE 4.8......................................................................................................................................... 6364
FIGURE 4.9......................................................................................................................................... 6768
FIGURE 4.10....................................................................................................................................... 6970
FIGURE 4.11....................................................................................................................................... 7071
FIGURE 4.12....................................................................................................................................... 7172
FIGURE 4.13......................................................................................................................................... 734
FIGURE 4.14......................................................................................................................................... 744
FIGURE 4.15......................................................................................................................................... 745
FIGURE 4.16. ........................................................................................................................................ 755
FIGURE 4.17......................................................................................................................................... 766
FIGURE 4.18......................................................................................................................................... 777
V
LIST OF TABLES
TABLE 2.1. ............................................................................................................................................. 14
TABLE 2.3 .......................................................................................................................................... 2727
TABLE 2.4 .......................................................................................................................................... 3434
TABLE 3.1 .......................................................................................................................................... 4242
TABLE 3.2 .......................................................................................................................................... 4343
VI
Chapter One
Introduction
Chapter 1: Introduction
1.1 Introduction
Decreasing fossil fuel reserves, global warming, and the need for energy efficiency has
attracted many researchers to work on hydrogen production. There has been some 200
publications related to fermentation hydrogen production from wastewater and solid
wastes by mixed cultures over the past three decades [1]. The reason for this attraction
is because wastewater treatment is one of the vital areas in our well being. In addition,
the production of hydrogen gas from wastewater makes wastewater treatment more
economical.
Hydrogen gas shows promise as a non-polluting fuel since it produces water instead of
green house gases when combusted [2]. Furthermore, hydrogen has a high-energy
yield (122 kJ/g) that is about 2.75 times that of hydrocarbon fuel [3]. Despite the
"green" nature of hydrogen as a fuel, it is still primarily used from non-renewable
sources such as fossil fuels via steam reforming [4], which indicates that bio-hydrogen
production from photosynthetic or fermentative routes using renewable substrate has
not become economically and technically feasible yet.
Fermentative hydrogen is usually produced from the break-down of sugars during
anaerobic glycolytic. Pyruvate driven from various substrate catabolismes, such as
glycolysis could be the source of the majority of microbial hydrogen production. This
compound can be broken-down and catalyzed by "pyruvate formate lyase" (first
reaction) and "Pyruvate ferredoxin oxidoreductase" (second reaction) enzymes [5].
1 − ( PFL) Pyruvate + CoA → acetyl − CoA + formate
−
−
CH 3COCOO + CoA → CH 3CO + CoA + HCOO
(1.1)
−
2 − Pyruvate + CoA + 2 Fd (or ) → acetyl − CoA + CO2 + 2 Fd (red )
(1.2)
1
Chapter One
Introduction
As shown in equitation 1.1.1, Acetyl-CoA from pyruvate break-down can be the
source for Adenosine 5'-triphosphate ATP and either formate or reduced ferredoxine
(Fd (red)), can be the source for hydrogen [6]. Generally, from the pyruvate
metabolism one or two hydrogen molecules can be produced which constitutes a
relatively low yield. According to Hallenbeck (2005), there may be several reasons for
this low productivity. The produced formate from pyruvate (equation 1.1) is further
broken down to H2 and CO2 by hydrogen lyase complex under acidic conditions only
when the formate concentration is high. Therefore, under most conditions which the
formate concentration is not enough high in the environment, the degradation of
formate is incomplete which leads to less than stoichiometric hydrogen production. In
addition, fermentation has been optimized by evolution to produce cell biomass,
methane and carbon dioxide. Thus, hydrogen is only a by-product of the anaerobic
fermentation. Besides, in many organisms the actual yields of hydrogen production
decrease by hydrogen recycling due to the presence of uptake hydrogenases1, which
consume a portion of the hydrogen produced.
An additional factor that may reduce hydrogen productivity is the fact that in mixed
acid fermentation, the mixed cultures present in the environment may lead to other
fermentation end products. A typical fermentation might yield lactate [7], ethanol [8],
acetate, formate, H2, CO2, succinate and butanediol. Some of these reactions lead to a
decrease in the reducing power of pyruvate for hydrogen production and some others
are hydrogen consuming reactions. As a result, even though the maximum possible
yield of hydrogen from the enteric-type fermentation of glucose is two mol of H2 per
mole of glucose, actually only about one-half of this amount is observed.
1
Hydrogenase is an complex enzyme carries out chemical reversible reaction of : 2 H
+
+ 2e − ↔ H
2
2
Chapter One
Introduction
Other than enteric-type fermentation, a different pathway of fermentation is typified by
those carried out by clostridia. These species can break down the pyruvate by
pyruvate:ferredoxin oxidoreductase (PFOR), generating reduced ferredoxin and acetylCoA (figure 1.1). Through this reaction several solvents such as acetone, butanol,
butyrate and ethanol could be produced. Little or no hydrogen is produced during
production of these solvents. The maximum theoretical yield for these fermentative
processes is calculated at 4 mol of H2 per mole glucose [9]. The origin of this higher
amount is by the action of NADH:ferredoxin oxidoreductase which recycles the NAD
and produces Fd (red) which can in turn to drive hydrogen evaluation [10]. However,
the reduction of hydrogenase enzyme by NADH is an energetically unfavorable
reaction and it can only be completed at very low partial pressure of hydrogen. In
natural environments the very low partial pressure can be obtained from very active
hydrogen consumption provided by methanogenesis. Nevertheless, in laboratory
experiments researchers have tried to decrease the hydrogen partial pressure by
sparging of gases such as nitrogen.
From the facts presented, the main natural limitations of hydrogen production process
can be highlighted as:
Overall yield for hydrogen productions is one or two molecules of hydrogen
per molecule of pyruvate which is considered low production.
The reduction of hydrogenase by NADH is an energetically unfavorable
reaction and it only proceeds at very low partial pressure of hydrogen, below
10-3 atm, when the free energy change is negative.
Typical fermentation process might yield lactate, ethanol, acetate, formate,
butanediol, succinate and other solvents to receiver NADH. Production of these
solvents decreases the hydrogen production yield.
3
Chapter One
Introduction
2 Ferredoxin (oxd)
Glucose
2 NAD
2ADP
2 NADH
2 Ferredoxin (red)
2 ATP
2 Pyruvate
2 Fd (oxd)
H2 ase
2 Fd (red)
2 Acetyl-CoA
2 CO2
2ADP
2 ATP
4 H2
2 Acetate
Figure 1.1 Hydrogen production by clostridia carrying out acetate fermentation [6]
The presence of methanogenesis in mixed anaerobic cultures which consume
the hydrogen.
According to these natural limitations, it is impossible or difficult to go against the
natural approach to obtain higher yield of hydrogen production. In order to overcome
these limitations, many researchers have applied controlling methods such as:
•
Repeated heat treatments were used to screen hydrogen productive species with
the ability of sporulation during unfavorable conditions and to sustain high
hydrogen production in long-term experiments [11 and12].
•
Vacuum, vigorous stirring, immobilizing cells and nitrogen gas sparging was
utilized to decrease the hydrogen partial pressure inside the reactor [13 and14].
4
Chapter One
•
Introduction
Operating under thermophilic condition to increase the entropy term and as a
result promote the reduction of hydrogenase enzyme by NADH which is
energetically an unfavorable reaction [6].
•
Aeration, addition of toxic chemical, operation at a short HRT to wash out
them, and operation at low pH to inhibit the activity of hydrogen-consuming
methanogenesis [15].
These controlling methods, as shown in figure (1.2), can be both energetically and
economically intensive.
Figure 1.2 Natural limitations of hydrogen production and its control parameters
1.1.1 Research Objectives
The intention of this study was to optimize hydrogen production as well as avoiding
the controlling methods by enriching the biomass by subjecting it to a combination of
acid treatment, heat treatment and lack of carbon source treatment. These biomass
treatments make use of special property of hydrogen productive species, which is
sporulation during unfavorable conditions, to screen them from other species. The
enriched cultures resulted from these treatments were examined in a batch and
continuous approach by an anaerobic sequencing batch reactor (ASBR) and up-flow
anaerobic sludge blanket (UASB) to study the stability of hydrogen production.
5
Chapter One
Introduction
The procedures discussed as controlling methods, to overcome the natural limitation of
hydrogen production are both energy consuming and costly. Therefore, the aim of this
study was to avoid these controlling methods and as result obtain sustainable and
economical hydrogen production. The proposed idea in this research was supported by
four theoretical facts as follows:
1. Sludge can be treated to form hydrogen-productive granules [16].
2. Granules can be retained because of superior settling characteristics [17 and18]
and since, low HRT favors hydrogen productivity; therefore this characteristic
of granules can be beneficial for hydrogen production.
3. Hydrogen producing Clostridium and bacillus are able to produce endospores
in harsh conditions such as heat, chemical toxicity, lack of carbon source,
ultraviolet, ionizing radiation, Acid/base conditions, and Desiccation [19].
4. Granules treating carbohydrates have layered structure [20].
The overall objective of this study is to optimize and stabilize the hydrogen production
by granule enrichment. The following specific studies were conducted to achieve the
overall objective:
1. Optimize and stabilize the anaerobic H2 production through enrichment of granular
culture by acid treatment, heat-treatment, and lack of carbon source.
2. Characterize the SBR hydrogen producing granules.
3. Comparison of the hydrogen production stability of the enriched granule between
the batch and continuous approach.
1.1.2 Hypotheses
1. Make use of special property of Clostridium and Bacillus species inside the
granules (sporulation in harsh conditions), to screen them from other species inside
6
Chapter One
Introduction
the mixed culture through acid treatment, heat treatment and lack of carbon source
treatment .
2. Afterwards, retain only the granules inside the sequencing batch reactor by special
properties of granules which possess higher settling velocity.
1.1.3 Chapters in Brief
The following flowchart (Figure 1.3) summarized the complete research plan. As
shown in the flow chart, first of all, the sludge collected from a local anaerobic
digestion was subjected to heat and acidic treatment to enrich the culture towards
hydrogen production and to induce sludge granulation. The enriched culture obtained
from the treatments was activated in an ASBR using synthetic wastewater. After the
lag time, the 10 liter reactor was operated at pH 5.5, and HRT of 6.66 hours.
The reactor performance was monitored through several parameters such as suspended
solid, gas production, gas composition and volatile fatty acids concentrations. These
parameters were analyzed by equipments such as GC-FID, GC-TCD and
spectrophotometer. Microbial analyses such as T-RFLP and SEM were conducted after
the ASBR reached the steady state. After reaching the steady state, data was analyzed
to determine hydrogen production yield and rate.
In order to optimize the hydrogen productivity of granules, heat and lack of carbon
source treatment is applied to the culture after reaching the steady state. After each
treatment, the performance of the reactor is determined and the data collected from the
reactor running, is analyzed for hydrogen productivity. Finally, the hydrogen
production stability of the enriched culture is compared in a continuous approach by
UASB and a batch approach by an ASBR for stable hydrogen production.
.
7
Chapter One
Introduction
Hydrogen Production by Enrichment Granules in an Acidogenic Fermentation Process
Seed from anaerobic digestion treatment
Heat treatment
Acidic treatment
Enriched culture
Synthetic
wastewater
Lag period and start up
Constants:
pH: 5.5
Ambient temperature
V: 10L
HRT: 6.7 h
Operating the reactor
Equipments:
• GC-FID
• GC-TCD
• pH controller
• Water displacement
method
• Phenol-sulfuric acid
method with UV
• Programmable Logic
Controller (PLC)
Monitoring the performance of anaerobic
sequencing batch reactor (ASBR) based on:
• H2 , CO2, CH4 and N2
composition
• Effluent analysis, VFA
• Mass balance
• Suspended Solids
• Gas production rate
• Glucose concentration
Continued in next page
8
Chapter One
Introduction
Reach the quasi steady-state
Equipments:
SEM
Light microscope
Microbial community studies:
Morphological analysis
T-RFLP
Data analysis
Determine:
H2 production yields
H2 production rate
Apply enrichment treatments
2- Lack of carbon
source treatment
1- Heat Treatment
Data Analysis
Determine:
H2 production yields
H2 production rate
Determine the stability of the resulted culture in continues and
batch reactors
SBR
UASB
Monitoring the performance
Study the stability
Figure 1.3 Experimental Protocol
9
Chapter Two
Literature Review
Chapter 2: Literature Review
2.1 Energy Supply and Environmental Protection
Energy supply and environmental protection are two crucial issues for the sustainable
development of today's world. At present, fossil fuels provide over 80% of the energy
consumption of the world [21]. There are different predictions regarding fossil fuel
depletion. Nevertheless, it is definite that fossil fuels will eventually become depleted
in the near future. Furthermore, burning these fuels contributes to climate change and
environmental pollution [22]. Hence, many studies have been conducted on alternative
energy sources.
Having taken into account the ever-growing demand for energy, it seems inevitable to
seek a sustainable energy source. Contrary to fossil fuels, renewable energy is energy
derived from resources that are regenerative and cannot be depleted [23]. Therefore,
renewable energy sources are fundamentally different from fossil fuels, and do not
produce as many greenhouses gases and other pollutant as does fossil fuel combustion.
Various kind of renewable energies such as wind, water, and solar energy had used
traditionally. However, because of the threats of climate change due to pollution and
the exhaustion of fossil fuels there are many other suggestions as renewable energies.
These include biofuel (liquid biofuel, biodiesel [24], ethanol, solid biomass [25] and
biogas), geothermal energy [26], geo-energy, nuclear energy, and hydrogen fuel are
some examples from on-going natural processes. Energy sources that could maintain
our standard living without causing adverse affects to the environment includes wind,
solar, and biomass, among others, these three will arguably be the best sources in the
future [27].
10
Chapter Two
Literature Review
2.2 The Transition to the Renewable Energy Economy
From the facts presented previously, it is easy to see that there is enough renewable
energy in the world to satisfy our needs. Many technologies are available and some of
these technologies cost more than others. It seems the only question that remains is
when we will make the transition to a new renewable energy economy.
The current political powers in most countries seem to prefer to continue with the
fossil fuel energy infrastructure. This results in high demand for oil and gas and violent
circumstances in Middle East where most oil is.
The concern is how much time must pass before we arrive with our new renewable
energy infrastructure? Do we have to wait until all the fossil fuel resources depleted
and then face the problem? How many more years of increasing greenhouse gas
emissions and rising energy prices can the world sustain?
2.2.1 Hydrogen Production from Renewable Energy
Amongst renewable energies presented in section 2.1, hydrogen obtain from biomass
is a completely carbon-free fuel with a high combustion enthalpy (185 kJ L-1) and is
considerable a feasible alternative to fossil fuels. With technology for hydrogen as fuel
for transportation already well established [14], a solution for energy supply and
mitigating global warming could be achievement of a hydrogen economy where the
hydrogen gas is produced by renewable energy.
For more than three decades, researchers have been trying to produce hydrogen from
various sources. Throughout these studies, different amounts of hydrogen have been
obtained. However, there has not been an efficient, simple, robust and most
importantly affordable method found, both from an investment capacity and operating
costs of point of view.
11
Chapter Two
Literature Review
Hydrogen has a great potential for use as primary or secondary energy source for
chemical synthesis or for electrical storage and generation with fuel cells [28].
2.2.2 Renewable Energy through Anaerobic Treatment Technology
Wastewater treatment can be a possible source of energy, since; anaerobic treatment of
wastewater can produce methane as well as hydrogen. These gases can generate energy
by combustion or via fuel cells.
Biogas is considered a traditional energy. Unreliable evidence indicates that biogas
was used for heating bath water in Assyria during the 10th century BC and in Persia
during the 16th century AD. However, methane was first known as having useful and
profitable value in England, where a particularly designed septic was used to generate
gas for the purpose of lighting in the 1890s [29]. There are also reports of successful
methane production units around the world, and many farmers wonder if small scale
methane production units can be set up at their farms to convert manure and waste into
energy [30].
Recent interest in the use of anaerobic treatment at sewage treatment facilities is
increasing, as anaerobic treatment reduces the ultimate volume of bio-solids needing
disposal by 50-80 percent. In addition, during anaerobic treatment, methane or
hydrogen is produced which has energy value, and the residual bio-solids can be safely
used as a humus-rich compost if it is low in heavy metal content.
2.2.3 Hydrogen Production Feasibility
Biomass obtained from different sources can have various hydrogen productions
depending on the kind and concentration of carbohydrates present in the biomass.
Generally, the most common products in the fermentation of carbohydrates are acetate
and butyrate. This acidification process may be expressed by following reactions:
12
Chapter Two
Literature Review
C 6 H 12 O6 + 2 H 2 O → CH 3COOH + 2CO2 + 4 H 2
(2.1)
C 6 H 12 O6 → CH 3 CH 2 CH 2 COOH + 2CO2 + 2 H 2
(2.2)
As shown in the equations, the stoichiometric yields are 4 moles of hydrogen per mole
of glucose in the production of acetic acid (maximum theoretical hydrogen), and 2
mole of hydrogen in the production of butyric acid. However, several volatile fatty
acids and alcohols such as propionate, hexanoate, ethanol, and butanol can be as
fermentation products during hydrogen production process. H2 can readily be produced
from a range of biomass materials. However, without substantial improvement, most
yields are probably too low to be practically useful. As an example, Antonopouluo et
al. (2007) had produced 10.4l hydrogen per kg of sorghum biomass [31]
The demand of hydrogen as a new clean energy source is rapidly increasing.
Therefore, low-cost technology for bio-production of hydrogen is being developed in
many countries. Improving bio-hydrogen-producing capacity and reducing cost is the
key to achieve industrialization. The microbiological conversion of organics into
hydrogen is comparatively inefficient (15-30% efficiency). However, hydrogen gas
has an advantage of high conversion rate (90%) of gas to electricity. Therefore,
hydrogen production from wastewater is a relatively feasible option since the market
value of hydrogen gas is nearly 20 times higher than methane gas. In all cases, the
overall efficiency from waste to electricity via hydrogen gas remains relatively low (is
1 kW-h every kilogram of biodegradable waste) mainly due to the low efficiency in the
fermentation step [32]. Table (2.1) has shown hydrogen production cost and amount of
energy used from various kinds of biomass sources.
13
Chapter Two
Hydrogen from
Biomass
Dairy Manure
Ag Residues
Wood Waste
Energy Crops
MSW
Landfill Gas
Literature Review
H2
Number of H2
H2 Production Potential FCVs
Cost
(Q-Btu) Supported ($/lb)
Technology
Anaerobic
Digestion
0.0068
278,208
1.04
Gasification
0.0007
28,639
1.20
Gasification
0.0164
670,971
1.20
Gasification
0.0040
163,651
1.23
SMR
0.0100
409,129
1.10
SMR
0.0071
290,481
1.17
H2
Cost
wo/Taxes
($/gal gas
equivalent)
H2
Cost
w/Taxes
($/gal gas
equivalent)
1.06
1.22
1.22
1.26
1.12
1.20
1.56
1.72
1.72
1.76
1.62
1.70
Table 2.1 Cost of the Hydrogen Technology from various Biomass Sources.
Notes:
* Biomass data from Directed Technologies, 2003, DOE Grant No. DE-FG0199EE35099.
* Fuel Cell Vehicle (FCV)
* SMR = Steam Reformation; Ag = Agriculture; MSW = Municipal Solid Wastes.
* The average fuel economy of FCVs is assumed to be 25 mi/lb H2.
* A pound of H2 = 51,500 Btu; a gallon of gasoline = 115,500 Btu; (both at net heating value).
2.3 Anaerobic Wastewater Treatment
Louis Pasteur was the first scientist to discover anaerobic organisms during his
research on fermentation microorganisms in 1861 [33]. In 1881, Mouras' Automatic
Scavenger used anaerobic microorganisms to treat waste for the first time [34 and35].
Currently, anaerobic digestion is an established technology for the treatment of wastes
and wastewater. Anaerobic treatment is applicable for a wide range of users, from
industry to farming, waste-treating companies, water boards and individual farms or
households. The technology is widely applied in industry, especially in the food and
beverage and pulp and paper industry, particularly for wastewater treatment.
Throughout time, studies and experiments has shown various advantages and
disadvantages of anaerobic treatment. The rationale and interest in the use of an
anaerobic treatment process can be explained by considering the advantages and
disadvantages of this system [36]. One of the important advantages of anaerobic
process is that, this process can be net energy producer instead of energy user. In
addition, some of the advantages of these systems are as follows; they have lower
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biomass production, fewer nutrients requirement, rapid response to substrate addition
after long period without feeding and finally they can use high volumetric loading rate
and therefore, smaller reactor volume and less space is required for treatment [37].
Potential disadvantages also exist for anaerobic process such as longer start-up time to
develop necessary biomass inventory, may require alkalinity addition to adjust the pH,
biological nitrogen and phosphate removal is not effective, the process is sensitive to
the adverse effect of lower temperatures on reaction rates, potential for production of
odors and corrosive gases and may require treatment with an aerobic treatment process
to meet discharge requirements. According to these advantages and disadvantages, this
process is beneficial for treatments of specific wastewaters.
2.3.1 Important Parameters in Anaerobic Treatment
The complexity of anaerobic wastewater treatment, relative to the microbial consortia
and reactions involved, specify that certain parameters have particular significance for
process control and system stability. These parameters include the reactor environment
and operational parameters. The environmental parameters include: temperature, pH,
alkalinity, volatile acids, ammonia, sulfate, toxic metals, salts and inhibitory
intermediate products [38]. The operational parameters included solids concentration
(MLSS), solids retention time (SRT), food to microorganisms ratio; (F/M) ratio,
organic concentration and loading rate, and hydraulic retention time (HRT) [39]. The
current study has attempted to pay attention to environmental and operational
parameters for the particular anaerobic reactors used.
2.3.2 Two-Stage Anaerobic Wastewater Treatment
Hydrogen and methane production can be easily linked, using a two-stage process. The
first stage is designed for the initial fermentative/acidogenic degradation. During these
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processes hydrogen could be recovered. The second stage is for the subsequent
acetogenic/methanogenic degradation of the intermediate fatty acids. This process
would require longer hydraulic detention times and would produce methane gas [40].
The two processes could theoretically be separated by controlling pH and hydraulic
detention times [41].
Although two-stage anaerobic treatment systems have been used, none have yet been
designed or operated at full scale for hydrogen production and recovery [42].
The present study has focused on the acidogenic process which is the initial stage of
the complete process of fermentation. The degradation process is not accomplished by
only acidogenic process; therefore, chemical oxygen demand (COD) removal is not
completed by acidogenic
process.
The
process should be
continued
by
acetogenic/methanogenic degradation in a separate reactor to achieve high COD
removal.
2.3.3 Fundamentals of Anaerobic Treatment
Organic molecules can be degraded by aerobic or anaerobic reaction. In aerobic
degradation free molecular oxygen is used while, in anaerobic degradation there is no
free molecular oxygen involved. If the degradation of glucose (C6H12O6) occurs with
free molecular oxygen, the degradation is referred to as aerobic respiration. Aerobic
respiration occurs in the aerobic tank of an activated sludge process and it results in the
production of bacterial cells (sludge), carbon dioxide, and water.
C 6 H 12 O6 + 6O2 → 6CO2 + 6 H 2 O
(2.3)
There are four important forms of anaerobic degradation of organic molecules that
occur at wastewater treatment plants. These four are nitrate reduction (denitrification),
sulfate reduction, methanogenesis (methane production), and fermentation [43].
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Nitrate reduction commonly take places in an anoxic selector, denitrification tank, and
a secondary clarifier and it can be completed by facultative anaerobic bacteria. Nitrate
reduction results in the production of bacterial cells (sludge), carbon dioxide, water
and molecular nitrogen.
−
C 6 H 12 0 6 + 4 NO3 → 6CO2 + 6 H 2 O + 2 N 2
(2.4)
Sulfate reduction results from obligatory bacteria. Sulfate reduction typically occurs in
an anaerobic digester. However, this reaction also occurs in sewer systems, secondary
clarifier and a thickener if the settled solids remain too long in theses treatment units
without the presence of oxygen and nitrate. Bacterial cells, carbon dioxide, water,
sulfide, and different kind of organic compound, mostly acids and alcohols can be
produced by sulfate reduction.
2CH 3 CHOHCOOH + SO4
2−
+ H + → 2CH 3COO − + 2CO2 + 2 H 2 O + HS −
(2.5)
Methanogenic bacteria can produce methane through two pathways. The major route is
from acetate by aceticlastic, methane-forming bacteria. Another way of methane
production is the reduction of carbon dioxide by hydrogen-oxidizing, methane forming
bacteria. This route results in the production of bacterial cells, methane, and water.
CH 3COO − + H 2 → CH 4 + H 2 O + OH −
(2.6)
4 H 2 + CO2 → CH 4 + 2 H 2 O
( 2. 7 )
The fermentation reaction differs according to the sugar being used and the product
produced. If the sugar is glucose (C6H12O6), the simplest sugar, the product can be
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ethanol (C2H5OH). This is one of the fermentation reactions carried out by yeast, and it
can be applied in food production.
C6H12O6 → 2C2H5OH + 2CO2 + 2 ATP (Energy Released:118 kJ/mol)
(2.8)
Glucose undergoes glycolysis as it is degraded. At the end of gylcolysis two molecules
of pyruvate (CH2COCOOH) are produced. One pathway to degrade pyruvate is
through fermentation to volatile fatty acids such as acetate, alcohols such as ethanol
and gases such as hydrogen and carbon dioxide [44]. One more pathway is through
methanogenesis.
Fermentation is a process of energy production in a cell compounds with no free
molecular oxygen, carbon dioxide or sulfate. Typical examples of fermentation
products are ethanol, lactic acid, and hydrogen. However, more compounds can be
produced by fermentation, such as butyric acid and acetone. The ratio of these VFA
changes with different feedstock, although typically acetate is the major product [45].
Fermentation requires the use of an organic molecule to remove the electrons from the
degrading compound; furthermore fermentation is an inefficient process, and it
releases little energy to the bacteria [46]. The reason is most of the energy released by
the degraded compound remains in the fermented products. This process normally
occurs in an anaerobic digester, but it may also take place in sewer systems, a
secondary clarifier, or a thickener. Fermentation can be completed by both facultative
anaerobic bacteria and strict anaerobic bacteria and can occur by many different
pathways with many different products. The types of organic compounds produced
during this process are dependent on the type of bacteria and the existing operational
conditions.
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In many of the fermentative pathways, hydrogen is produced. The production of
hydrogen gas is important in anaerobic digesters because, hydrogen is one the main
substrates for the production of methane and also hydrogen pressure may inhibit the
acetogenic bacteria.
Strict anaerobic bacteria are more important than facultative bacteria in fermentation
process. Bacteroides, Bifidobacteria, and Clostridium are some examples of these
strict anaerobic bacteria.
There are two important groups (which are the concern of this study) of fermentative
bacteria: the acidogenic bacteria and the acetogenic bacteria. The acidogenic bacteria
or acid-formers such as Clostridium convert simple sugars, amino acids, and fatty
acids to organic acids, alcohols, acetone, carbon dioxide, hydrogen, and water. Several
of these compounds are volatile and malodorous [47]. In addition, some of these
compounds can be used directly by methane-forming bacteria, while other compounds
can be converted to compounds that can be used by methane-forming bacteria.
Acetogenic bacteria produce acetate and hydrogen that can be used straightforwardly
by methanogenesis bacteria. Acetogenic bacteria convert several of the fatty acids that
are produced by acidogenic bacteria to acetate, hydrogen and carbon dioxide.
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Insoluble, complex organic substrates
HYDROLYSIS
PHASE 1
Soluble, simple organic substrates
ACID AND ALCOHOL
PRODUCTION
Organic acids and alcohols
PHASE 2
ACETOGENESIS
Acetate, carbon dioxide, hydrogen
METHANOGENESIS
PHASE 3
Methane
Figure 2.1 Biological phases of anaerobic degredation
As shown in figure (2.1) there are three basic biological phases that occur in municipal
anaerobic digesters with respect to methane production. According to Thiele (1991),
[48], and Henze (1983), [49] three major biological reaction steps are involved in
anaerobic treatment. These are hydrolysis, fermentation/acetate production, and
methanogenesis. During hydrolysis fermentative organisms break down more
complicated and large organics to simpler compounds. Afterward, during fermentation
products of hydrolysis are converted to organic acids, alcohols, carbon dioxide, and
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hydrogen by syntrophic acetogenic bacteria. During the next stage of fermentation,
most of the acids and alcohols are converted to acetate. Finally, during
methanogenesis, methane-forming bacteria covert carbon dioxide, hydrogen, acetate
and several others limited substrate to methane gas.
The anaerobic treatment process is therefore a complex reaction and a given anaerobic
reactor requires the presence of the right microbial consortium which must live in a
dynamic state for a successful system operation. It is obvious that species diversity is a
source of difficulty on process control and failure of anaerobic reactors. These
problems are only overcome if there is a balance in the species population [50].
2.4 Fundamentals of the Fermentative Production of Hydrogen
From the facts presented in previous section, hydrogen can be produced during
acidogenic and acetogenic phases of the process of anaerobic treatment. Therefore, the
possible reactions, which are supposed to occur in hydrogen productive reactors are
hydrolysis, acidogenesis and acetogenesis.
Hydrolytic bacteria and fermentation: According to Novaes (1986), [51], most
organic wastes contain carbohydrates, lipids and protein. The function of the
hydrolytic bacteria is to reduce the high molecular weight organics to low molecular
weight substances. Specifically, they produce enzymes which hydrolyze organic
compounds such as cellulose, hemicellulose, pectin, starch (polysaccharides) and
others into smaller molecular weight materials such as monosaccharide.
As Daniels (1984), [52] stated the hydrolytic bacteria include obligate anaerobes like
clostridium, bacteroides, ruminococcus, and butyrivibrio species and facultative
anaerobes such as escheichia coli and bacillus species.
Acidogenic/Acetogenic (syntrophic) bacteria: Several studies have reported on the
acid forming phase of anaerobic treatment. This attraction is because of the importance
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of the acid forming phase is important in overall anaerobic treatment as well as its role
in the formation of granules. Subsequently, the degradation of organic acids is
performed by the acetogenic bacteria. That is, the acetogenic bacteria oxidized
hydrolytic fermentation products to acetate and other fatty acids. This group of
bacteria, which have also been studied by Bryant (1979), [4], includes both facultative
and obligate microbes that can ferment organic molecules larger than acetic, such as
butyrate and propionate, in addition to compounds larger than methanol to hydrogen
and acetate. The acetogenic bacteria can be grouped into two categories depending on
whether H2 is produced (H2-producing acetogens) or hydrogen is consumed
(homoacetogens).
The H2-producing acetogens exist in a syntrophic association with H2-utilizing
bacteria. H2-producing can grow only in an environment with extremely low partial
pressure of hydrogen in the reactor (as explained in chapter one). This low partial
pressure can be naturally provided by methanogenesis. One explanation for the
dependency of the H2-producing bacteria on the methanogens has been presented by
Bryant (1979), [4] which is summarized in Table (2.2).
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A. Propionate-catabolilzing acetogenic bacterium
−
CH 3 CH 2 COO − + 3H 2 O → CH 3 COO − + HCO3 + H + + 3H 2
= +18.2 Kcal/reaction
B. Butyrate-catabolizing acetogenic bacterium
CH 3 CH 2 CH 2 COO − + 2 H 2 O → 2CH 3 COO − + H + + 2 H 2
= +11.5 Kcal/reaction
C.H2-utiliziing methanogenic bacterium
−
HCO3 + 4 H 2 + H + → CH 4 + 3H 2 O
= - 32.4 Kcal/reaction
D. SUM A+C Syntrophic association
−
4CH 3CH 2COO − + 3H 2O → 4CH 3COO − + HCO3 + H + + 3CH 4
= - 24.4 Kcal/reaction
E.SUM B+C syntrophic association
−
2CH 3CH 2CH 2COO − + HCO3 + H 2O → 4CH 3COO − + CH 4 + H +
= - 9.4Kcal/reaction
Table 2.2 Stoichiometry and the change of free-energy for catabolism of propionate and butyrate
by H2-producing acetogens and H2-utilizing methanogens [4].
Table (2.2), has showed that the catabolism of propionate to acetate, CO2 and H2
(reaction A) and the catabolism of butyrate to acetate and H2 (reaction B) would not
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proceed alone because of the highly positive free-energy for both reactions (4).
However, the H2-prutilization reaction by methanogens (reaction C), would proceed
because of its highly negative free-energy. Therefore, when the H2-producing
acetogenic bacteria are placed in syntrophic association with the H2-utilizing bacteria
(sum of A+C, and sum of B+C), then the combined reactions (reactions D and E)
become energetically favorable [4].
According to Daniels (1984), for the reactions to favor H2- producing bacteria, which is
with a negative free energy, the H2 partial pressure must be less than 10-3 atm for the
use of butyrate and 10-4 atm for the use of propionate. That is, propionate acetogenesis
is more sensitive to H2 partial pressure than butyrate acetogenesis.
2.4.1 Instability of Hydrogen Production and Compositions of Bacterial
Communities within a Dark Fermentation
One of the key problems researchers have faced on the subject of hydrogen production
is to have stable hydrogen production over a long term period. The average biogasproduction rate, hydrogen percentage, bacterial density, volatile fatty acids and
alcohols productions are registered as steady-state values. Normally when the variation
is less than 10% it is considered to be steady-state [51].
As studies have shown, under non-sterile conditions, the culture will shift from "H2
producing bacteria" to "hydrogen consuming bacteria" such as methanogenesis or
"hydrogen producer's competitors" such as acetogens, propionate, and lactate
producers. However, by using controlling methods to inhibit hydrogen consuming
bacteria and hydrogen producer's competitors such as heat treatment and acid/base
treatment it is possible to achieve stable hydrogen production. However, there has not
yet been a promising solution to maintain this stable state, for long term. As an
example, the heat-shocking process produced a stable inoculum for biogas production.
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Van Ginkel (2005) used the same heat shocked repeated five times over a 30-days
period produced an average of 262 ± 24 mL of biogas with no apparent trend in gas
production [53].
There has been a range of periods reported before reaching the steady-state operation.
As Liu and Fang (2002) have stated, pseudo-steady state operation has been reached in
14-21 days in various HRT (between 4.6 to 28.8 days) [54]. Moreover, according to
Lin and Chaia-hong (2003), it took 44days for the reactor to reach stable performance
with HRT of 12h, and the stable reactor performance lasted for 13 days [55].
The reason for this short period of steady-state is that there are several reactions take
place inside the mix culture. A number of these reactions lead to hydrogen production
and others may result in hydrogen consumption. Optimum H2 yield should be achieved
with acetate as the fermentation end product. However, in practice, high H2 yields are
usually related with butyrate production, and low yields with the production of
propionate, and reduced end products such as alcohols and lactic acid. Some Clostridia
species such as C. butyricum produce predominantly butyrate. On the other hand, C.
propionicum types produce mainly propionate. Vavilin et al. (1995) [56] gave the
overall equation for the production of propionate from hexose, showing that the
reaction involves the consumption of H2:
C 6 H 12 O6 + 2 H 2 → 2CH 3CH 2 COOH + 2 H 2 O
(2.8)
To obtain higher amount of hydrogen production, propionate production should be
avoided. According to Vavilin et al. (1995) the limiting substrate for butyrate
production is glucose, while the limiting substrate for propionate production is
hydrogen, and the two groups of microorganisms producing these end products are in
balance in the microbial consortium. Since, C. propionicum is a non-spore former
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bacteria; therefore, it can be inhibited by heat treatment of the inoculum and as a result
may assist in biasing the community towards hydrogen production. In addition, Cohen
et al. (1985), [57] demonstrated that irregular feeding rate (a 2 h daily interruption of
supply) to a reactor inoculated with un-pasteurized activated sludge strongly selected
for non-spore forming propionate-formers. The culture which had previously produced
butyrate and H2 after a one-off cessation of the feed supply for 6h, or regular feed
interruption for 1h per day gave similar shifts in product formation, thought to be
related to a population shift away from butyrate/H2 producing spore formers and
towards propionate producing non-spore formers. The semi-continuous feeding mode
used in some studies at laboratory scale, thus gave poor performance comparing to
feeding in continues mode. Also, under continuous operation, it is possible that a short
process disturbance commit the spore-formers irretrievably to sporulation, and the
spore-forming population may be then depleted by wash-out if the HRT is short, even
though normal feeding is quickly resumed [58]. All these factors affected hydrogen
production stability by a mix culture.
Chang (2004) has reported that the steady-state operation can be maintained for 20
days at the HRT of 0.5h with the average hydrogen production rate and yield boosted
to 9.72 L/h/L, 3.89 mol-H2/mol-sucrose, respectively [59]. Another study by Nanqi
Ren et al. (2003) has revealed that after 33 days stable conditions could be obtained.
Gas production rate was 24.96 L/d during stable condition. However, after day 21, gas
production rate and fermentation products decreased and there were significant
changes in the composition of the products. The ratio of butyric acid within total
products drastically decreased from 974.5 mg/L to about 500 mg/L, whereas the ratio
of propionic acid gradually increased from 179.6 mg/L to 547.1 mg/L. Such changes
suggested the conversion of the metabolic pathway of the microbes within the reactor.
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When the system stabilized again after day 33, the average concentration of ethanol,
acetic acid, propionic acid, butyric acid and valertic acid were 847.5 mg/L, 868.3
mg/L, 553.7 mg/L, 402.6 mg/L and 214.2 mg/L respectively, which presented a mixed
acid fermentation [60]. Table (2.3) has summarized results regarding the steady-state
conditions.
Time taken to reach Duration
that
Author
Year
the steady state (d)
steady state lasted
HRT
Jiunn-Jyi Lay, [61]
2000
60
-
-
Herbert H.P. Fang et al.
2001
60
-
6h
H. Liu and Herbert Fang
2002
14-21
-
4.6-28.8 days
44
13 days
12h
Chiu-Yue
Lin
and 2003
Chaia-hong Jo
Ea-San Chang
2004
33
-
-
Nanqi Ren
2003
33
-
-
Table 2.3 Different studies' comparison on steady state condition for hydrogen production
The complex nature of consortia in H2 producing microf1ora and the existence of
shifts in population are now being demonstrated using genetic techniques. Pertu E. P.
Koshkinen et al. (2006), [62] has studied microbial community composition dynamics
during glucose fermentation in a fluidized bioreactor (FBR). According to his work the
prompt onset of H2 production was due to the rapid growth of Clostridium butyricum
(99-100%) affiliated strains after starting continuous feed. The proportion trend of C.
butyricum in FBR attached and suspended-growth phase communities coincided with
H2 and butyrate production.
To summarize, after screening the H2 productive bacteria by using their special
property of sporulation during harsh conditions the hydrogen productivity increased.
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However, after this stage the new bacterial community will be activated and bacterial
community diversity increased and as a result the composition of the microbial
pathways will also change to different VFA and alcohols. Consequently, hydrogen
productivity decreased inside the reactor and cause instability. Several solutions have
been suggested to disclose this undesired phenomenon as mentioned.
2.4.2 Pretreatment Conditions to Screen Hydrogen Producing Bacteria
from the Mix Culture
There is a need to use mixed cultures that are present within the natural environment in
order to have more practical wastewater treatment. Additionally, processes using
mixed cultures are easier to operate and also they are able to have a wider choice of
feedstock [63]. However, these mixed cultures can be used as long as hydrogen
consumption by methanogens and other bacteria are inhibited through seed sludge
pretreatment such as heat-treatment, acid/base inhibitors, and lack of carbon source
treatment [64]. These pretreatments could be applied before start-up of the reactor
operation while there are other inhibitors such as, hydraulic retention time (HRT),
temperature and pH which are applied during reactor's operation [65 and 66].
Seed sludge pretreatments are achieved by relying on the spore-forming
characteristics of the hydrogen-producing Clostridium [19]. These kinds of bacteria
have an ability to produce spores in harsh environments such as high temperature,
desiccation, lack of nitrogen and carbon source, chemical toxicity, acidic or basic
conditions, aeration, ultraviolet and ionizing radiation. When favorable conditions
return, the spores become vegetative cells [67 and 68]. The purposes of all these
pretreatments are to suppress as much hydrogen-consuming bacterial activity as
possible while still preserving the activity of the hydrogen-producing bacteria [19].
Each of these inhibiters has their own advantages and disadvantages. Present study has
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tried to apply the most economical and common pretreatments on the sludge which are
heat-treatment, acid treatment and lack of carbon source treatment.
2.4.2.1 Heat-treatment
In 1977, Alexander has used the characteristic of Clostridium species, which is
sporulation in harsh conditions such to screen them from non-spore forming species
[69].
Heat treatment has been the most common treatment to screen of hydrogen producing
bacteria [1]; the reason could be because of, the quick and inexpensive practice.
However, heat treatment practice has a number of disadvantages, the process requires
energy for heating and because of anaerobic condition it is difficult and energy
consuming to heat the anaerobic biomass inside the reactor. In addition, Oh, et al.
(2003), has reported that heat treatment could not inhibit the activity of all hydrogenconsuming bacteria such as homoacetogenic bacteria [62]. This kind of bacteria may
survive the heat treatment, and consume hydrogen for the production of acetate, and
therefore, the overall hydrogen production decreases. Lastly, repeated heat treatment is
required to sustain hydrogen production over long term experiments.
To be more precise, germination of a spore involves three steps: activation,
germination and outgrowth. Heat treatment is one way to start spore germination.
Germination of spores is considered a rapid process, which generally takes 60 to 90
minutes [70]. A number of parameters affect spore germination such as incubation,
pH, prior heat treatment, and reducing conditions [66]. Moreover, sporulation takes 6
to 8 hours in most spore-forming species [70]. Naturally the duration depends on
environmental conditions and the kind of spore-forming bacteria.
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In this study, heat treatment of the inculumn was employed as one of the methods to
increase hydrogen production by inactivation of non-spore forming hydrogen
consuming microorganisms.
2.4.2.2 Acid/Base Treatment
There are two kinds of selection by pH, enhancement pH and cultivation pH. The first
one is applied for short term period and it selects the species which can survive after
such high or low pH. Second enrichment however, is a long term practice which
chooses the kind of bacteria with the ability to tolerate this pH during reactor operation
[71]. The current study has applied both enrichment techniques.
Chang et al. (2002), [72] have reported a great increase in hydrogen yield after treating
the sludge with acid and base treatment for about 24h. This increase of hydrogen is
because; the hydrogen utilizing methanogenesis were killed or inhibited during the
enrichment. However, clostridia remain in the culture by producing spores during the
enrichment [73]. Endospores are very resistance to acid and base because of their
complex, multilayered structure which is structurally different from vegetative cells
[19].
According to Zhen-Peng Zhang et al. (2006), [74] there is a rapid formation of
granules in an anaerobic reactor by acid incubation for 24h by shifting the culture pH
from 5.5 to 2.0. The same concept has been used for this study to form granules inside
the ASBR.
Based on numerous experiment results, the optimum cultivation pH value for
acidogenic hydrogen production is around 5.5 [12 and 75]. Both enhancement pH and
cultivation pH could be very effective in increasing hydrogen production.
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2.4.2.3 Lack of Carbon Source
As mentioned in section (2.4.2) lack of nitrogen and carbon source is considered as
unfavorable conditions which activate spore germination [65 and 66]. However, sporeforming bacteria enrichment is not commonly achieved through lack of carbon source.
However, one of the advantages of this practice is that it is economically favorable
since no energy input is required [54].
2.4.2.4 Methanogen Inhibitors
Since, methanogens are very sensitive to sudden pH variations, chemical toxicity,
oxygen and heat, therefore, inhibition of these species is achievable. Forced aeration
has been used by Ueno et al, (1995) and results showed that 330-340 ml H2/g hexose
could be produced without production of methane gas [76].
Moreover, 2-Bromoethanesulfonate (BES) is considered as a methanogen inhibiter.
Use of BES at concentrations up to 25 mM reported to be effective to inhibit the
methanogenesis and thus increasing hydrogen production [77]. Although, BES is an
analog of the coenzyme in methanogen and thus is very specific against methanogens
[78], however, Koskinen et al. (2006), [60] has reported BES in batch-culturing
resulted in low diversity of Clostridia and did not eliminate all H2 consumers.
2.5 Fermentation Process through Granulation
The advantages of granulation process through reactor are biomass enhancement,
increase of reactor efficiency in organic removal, and increase of methane production
has discovered by numerous studies. However, studies on the application of granular
sludge processes for anaerobic hydrogen production only began in recent years. These
studies have shown a superior potential of hydrogen productivity by granules as
compared to suspended sludge system [79]. Understanding the mechanisms of
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granulation process is necessary to for improve reactor design and performance [80
and 81]. Due to the absence of initial cores for cell attachment, microbial granulation is
preceded with a selfadhesion or aggregation of microbial cells [82]. This process thus
can be defined in terms of energy involved in the interaction of cell to cell and is
governed by the surface physicochemical characteristics of microbial cells. In a
thermodynamic sense, microbial sludge stability is governed by a charge balance
among several repulsive forces which include electrostatic, salvation (hydration) and
steric forces, and attractive forces including van der Walls, short range hydrogen
bonds, and electrostatic forces [83]. Some physicochemical models therefore, have
been proposed, as an example, secondary minimum adhesion model [84], extracellular
polymers (ECPs) bonding model [85], and inert nuclei model [18]. The granulation of
microbial cells is a complicated process, in which biological, microbiological and
hydrodynamic factors are also involved other than physicochemical forces. For
example, different models and hypotheses based on aforementioned factors have been
proposed for anaerobic granulation, including structural models, proton translocationdehydration theory, cellular automaton model and cell-to-cell communication model
The formation and mechanisms of conventional granulation of anaerobic sludge in
UASB reactor have been well documented [86].
2.6 Granule Microstructure
2.6.1 Formation of Layered Microstructure
During
anaerobic
degradation,
complex
substrates
are
converted
by
fermentative/acidogenic bacteria into volatile fatty acids (VFA), which are further
converted by acetogens forming bacteria to acetate, prior to formation of methane by
methanogens. The rate of each step depends on the concentration of the reactants,
bacterial species, and a number of environmental parameters, such as pH and
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temperature. In the cases where the initial step of the substrate degradation is
significantly faster than the subsequent degradation of the intermediates, most of the
substrates are consumed by bacteria near the bio-granule surface. The concentration of
intermediates would build up, causing them to diffuse toward bio-granule interior, due
to concentration gradients, leading to further degradation. As a consequence, the biogranule develops a layered structure, where the outer layer is mainly responsible for
the rapid initial step of substrate degradation and the inner layer(s) for the subsequent
degradation of the intermediate (figure 2.2).
Figure 2.2 Effect of degredation kenetics on biogranule microstructure [85]
Table (2.4) summarizes the maximum specific substrate rate of enrichment culture at
35 °C for a number of substrates. Propionate was converted to acetate at the rate
similar to the subsequent conversion of acetate to methane. Acetogenesis of butyrate
was however twice as fast. On the other hand, carbohydrates, including glucose,
sucrose and starch, were converted to VFA at rates about 5-10 times faster than
acetotrophic methanogenesis.
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substrate
Max
Reference
SSUR
Acetotrophic methanogenesis
Acetate
4.8
Zenhder, et al (1980)
Acetotrophic methanogenesis
Acetate
6.8-13.1
Lawrence and McCarty (1969)
Acetotrophic methanogenesis
Acetate
4.4-11.6
Noike, et al (1985)
Acetogenesis
Propionate
6.2
Gujer and Zehner (1983)
Acetogenesis
Propionate
8.2-12.2
Lawrence and MaCarty(1969)
Acetogenesis
Butyrate
16.6
Lawrence and MaCarty(1969)
Fermentation of carbohydrate
Glucose
72
Zeotemeyer, et al. (1982)
Fermentation of carbohydrate
Sucrose
71
Noike, et al (1985)
Fermentation of carbohydrate
starch
40
Noike, et al (1985)
Table 2.4 Maximum substrate utilization rate at 35°C (g-COD/g.VSS.d), [87]
Fang et al (1995) studied the degradation of butyrate in a UASB reactor and confirmed
that acetotrophic methanogenesis was the rate-limiting step [88]. This was also
reflected on the two-layered microstructure of butyrate-degrading biogranules. Most of
the butyrate was readily converted to acetate in the outer layer, while the acetate
produced was allowed to diffuse to the interior before methanogenesis could take
place. Li et al. (1995), based on some interesting observations, suggested that benzoate
degradation is a two-step process [89]. Bacteria such as Syntrophus buswelli, rapidly
converted benzoate inside their cells directly into acetate, which is subsequently
converted to methane by methanogens at a slower rate. Like those degrading butyrate,
benzoate-degrading granules also exhibited a two-layered microstructure.
34
Chapter Two
Literature Review
Figure 2.3 Structure of carbohydrate treating granules [85]
In the degradation of carbohydrates, the rate-limiting step is the acetotrophic
methanogenesis [46], as shown in Table (2.4) the initial acidogenesis of carbohydrate
took place rapidly at the outer layer; the intermediate VFA, including butyrate and
propionate, degraded at lower rate, and diffused to the interior of the biogranule. These
intermediates are then degraded by acetogens in the middle layer forming acetate.
Since the methanogenesis step is rate-limiting, the acetate diffused toward the
biogranule centre where methanogenesis took place. As a result, carbohydratedegrading biogranules develop a three-layered microstructure.
35
Chapter Two
Literature Review
The thickness of the outer layer, where fermentation and acidogenesis took place, is
dependent on the complexity of the carbohydrate. The trend seems to be the more
complex the substrate, the thicker the outer layer.
2.6.1.1 Biogranules with Layered Microstructure
Biogranules having layered microstructure included utilizing butyrate, sucrose, starch
and carbohydrates in brewery wastewater as organic substrates. Among them, butyrate
and benzoate degrading biogranules exhibited a two-layered microstructure, while the
three carbohydrate degrading ones exhibited a three layered microstructure. As Mu et
al. (2006), the outer layer of the butyrate degrading biogranules [79] have a thickness
of 20-40 µm . Under epi-flurescence at 350nm and 420nm [90], this layer emitted
intense fluorescence due to the presence of hydrogenotrophic methanogens. SEM
micrographs showed that this layer packed with micro-colonies composed of two
species of bacteria which appeared to be juxtaposed for syntrophic association. The
interior composed of uniformly distributes Methanothrix-like bacteria, which emitted
much dimmer flurescence [91]. Similar to these degraded butyrate, benzoate-degrading
biogranules also exihibited a two-layered microstructure, in which the outer layer was
composed of an abundance of Syntrophus buswellii-like [92], which converted
benzoate into acetate, whilst the interior was mainly composed of Methanithrix-like
filaments.
2.6.2 Formation of Uniform Microstructure
Within the process of decomposition of organic compounds the, granules degrading
simple substrates or the final stage of the process have simpler structure. As an
example, in the degradation of formate and acetate, the substrate was converted into
methane in a one-step process, each by a predominant species of methanogen, the bio-
36
Chapter Two
Literature Review
granules resulting in a simple, uniform microstructure. In addition, these two biogranules were smaller in size comparing to other kind of biogranules.
For biogranules degrading substrates of which the initial degradation was slow relative
to the subsequent degradation of intermediates, a considerable fraction of substrate
would diffuse toward the interior before being degraded. Substrate concentration
becomes quite uniform over the biogranule cross-section; as a result biogranules
developed a uniform microstructure with even distribution of all sorts of bacteria
involved at various stages of degradation. This was the case for the propionate- and
peptone- biogranules. For these substrates, the initial steps of degradation, including
acetogenesis of propionate [93], hydrolysis of protein [94] and acidogenesis of
glutamate, are rate limiting.
2.6.2.1 Biogranules with uniform microstructure
Biogranules degrading formate, acetate, propionate and peptone wastewater had a
uniform microstructure. Conversions of formate and acetate into methane are one-step
process. Biogranules degrading formate and acetate were small, and each had a simple
uniform microstructure composing of a predominant species of methanogen. Formatedegrading biogranules were of irregular shape with sizes less than 0.5 mm, were
mainly composed of rod-shaped filamentous Methanobacterium formicicum-like
bacteria [95]. Acetate-degrading biogranules were also small (less than 1 mm in size):
they were predominantly composed of Methanothrix-like filaments and scattered
clusters of Methanosarcina-like cysts [91].
2.6.3 Hydrogen Productive Granules
It was found in recent studies that the hydrogen-producing sludge could form granules
with high bioactivity [16].
37
Chapter Two
Literature Review
The physical characteristic of these kinds of granules has been investigated by
measuring the average diameter of the granules and settling velocity. The
microstructural characteristics have been analyzed by scanning electron microscopic
(SEM), Transmission Electron Microscopy (TEM), and extraction of extracellular
polymeric substances.
EPS are products of bacteria that accumulate on the bacterial cell surface [96]. They
form protective layer for the cells against the harsh external environment, and also
provide carbon and energy during starvation. EPS were found to be essential to the
flocculation of activated sludge [97 and 98] and to the microstructure of methanogenic
granular sludge [99]. EPS are composed of variety of substances, including
carbohydrate, protein, humic substances, uronic acid, and DNA.
Based on Fang et al. (2002) results, the characteristic of a hydrogen producing granul
(HPG) was 1.6+0.2 mm in size, 1.038 g/ml in density, >50 m/h in settling velocity, and
11+1% in ash content. These characters are comparable to methanogenic granular
sludge except that the ash content which is lower for HPG [17].
2.6.3.1 Microstructure of Hydrogen-Producing Granule
The structural surface of the HPG as determined by SEM photos is porous with
multiple cracks. This kind of structure is likely to facilitate the passage of nutrients and
substrate as well as the release of hydrogen, which has a very limited solubility of 1.58
mg/L in water. In addition, as shown in several studies, HPG granule, unlike those of
methanogenic UASB granules did not exhibit a layered structure because of the
simplicity of the acidogenic process. The microbial species responsible for the
formation
of
hydrogen
producing
granules
is
still
unclear.
38
Chapter Three
Material and Methods
Chapter 3: Material and Methods
3.1 Reactor Configuration
ASBR and UASB reactors were used for conducting the research. The reactors were
identical in all aspects. Photographs of the reactors are shown in plates (3.1.1a) and
(3.1.1b). The total reactor volume was 14 L of which 10 L were used as the working
(fluid) volume while the remaining 4 liters was head-space.
Six ports were installed in the ASBR, and five ports in the UASB for sampling,
injecting NaOH, feeding, de-sludge, and decanting. The ports were 9cm long and
0.8cm inside diameter and were made of stainless steel tubes.
The top of the reactor was fitted with a plate having the same outside diameter
(19.5cm) as the flange of the reactor. The plate and flange were 1cm thick. The plate
and flange had eight holes in which threaded rods were used to fasten them.
The top plate of each reactor had two more holes. One of the holes was used for biogas
removal. The second hole was fitted with 2.5cm inside diameter stainless steel tubing
and 37cm long, to secure the pH controller probe.
The reactor volumes were calibrated using measured amounts of tap water. Peristaltic
pumps were used for the feeding the substrate concentrate, dilution water, and for
effluent withdrawal. Three pumps were used for the ASBR and three for the UASB.
Dilution water and decant pumps were fitted with size 15 pump heads, while the
feeding pumps were fitted with size 13 pump heads.
A programmable logic controller (PLC) was used for the ASBR. The substrate
concentrate was kept at 4°C before dilution with tap water. Plate (3.1.1c) shows the
experimental set-up of the ASBR and UASB system. Mixing was done with USP5402E impellers operated at 100rpm.
39
Chapter Three
Material and Methods
a
UASB
pH Controler
Decant pump
Feed pump
b
PLC
Impeller
motor
ASBR
Feed pump
Figure 3.1 a) Up flow Anaerobic Sludge Blanket, b) Anaerobic Sequencing Batch Reactor
40
Chapter Three
Material and Methods
3.2 Gas Collection System
The gas exits the reactor through one of the holes on the top plate and was conveyed to
the gas collector by way of tygon tubing. The top of the water-displacement gas
collector had two holes one for connecting to the reactor and the other for venting the
gas. A sampling part fitted with a septum allowed for sampling with a syringe. A ruler
was attached to outer surface of the gas collector to measure the amount of gas
production. The water's pH inside the water reservoir was kept at less than 3 with HCl
to reduce carbon dioxide dissolution.
3.3 Substrate Preparation
The synthetic feed used for fermentation contained 6.94g glucose/L as the sole carbon
source as well as a sufficient amount of inorganic supplements. Properties of the
substrate are shown in Table (3.1).
Since glucose in the synthetic substrate can be easily degraded; therefore, the substrate
concentrate was refrigerated at 4°C. In addition, to obtain a well dissolved and
homogenous substrate without any degradation during feeding, the concentrated
substrate was autoclaved at 120°C for 20 minutes before refrigeration. As mentioned
before (3.1) prior to feeding this concentrated substrate to the reactor it was diluted 74
times with water.
Since spore germination, and the granulation process require specific nutrients [100]
substrate properties should be selected with care. Magnesium chloride (MgCl2) and
calcium chloride are two inorganic supplements required in the process of granulation
[77]. Moreover, the hydrogenase enzyme contains a unique, complex nickel-iron
center (Ni-Fe); therefore, ferrous chloride (FeCl2) and nickel sulfide (NiSO4) are also
41
Chapter Three
Material and Methods
considered key trace minerals during hydrogen production [6]. The other minerals are
necessary for typical bacteria activities (Table 3.1).
Parameter
Value (mg/L)
Carbon Source
Diluted Glucose Concentration
6944
NH4Cl
200
CaCl2.2H2O
50
Trace Minerals
KH2PO4
30.47
MgCl2.6H2O
100
MnCl2.2H2O
12.5
Na2MoO4.2H2O
10
FeCl2.4H2O
30
NiSO4
32
Table 3.1 Substrate composition
The synthetic substrate concentrate was made up every five days for both the ASBR
and UASB reactors. A substrate volume of 5 L was prepared for each reactor and after
autoclaving was kept refrigerated over five days. Before reactor feeding the substrate
was diluted for SBR and UASB with 74 and 12.48 times with water respectively.
Since the organic loading rate (OLR) was high (25g/L.d) for both ASBR and UASB,
also the reaction occurring was an acidic reaction, pH can drop. Therefore, sodium
hydroxide (NaOH) 1 N was used to adjust the pH.
42
Chapter Three
Material and Methods
The organic loading based, on the substrate strength and reactors HRT was kept
constant at 25 g/d.L for both the ASBR and UASB (Table 3.2).
Operational parameters
Reactors
ASBR
UASB
HRT (h)
6.7
48
Concentration (g/L)
6.94
12.5
25
25
Organic Loading Rate (g/L.d)
Table 3.2 ASBR and UASB operational parameters
The volume of substrate fed and decant during 4 hour cycle operation, along with other
operational variables are shown in Table (3.3).
Operational variables
ASBR
Number of cycle per day
6
Length of cycle, hours
4
Volume of feed per sequence, liters
6
Volume of feed per day, liters
36
Volume of decanted per sequence, liters
6
Volume of decanted per day, liters
36
Length of feeding time, min
10
Length of react time , min
190
Length of settling time, min
30
Length of decanting time, min
10
Reaction/settling ratio
6.3
Table 3.3 ASBR operational variables
43
Chapter Three
Material and Methods
6.7h HRT and 48h HRT were used for the ASBR and UASB operation. The
sequencing batch reactor was operated for 519 days, from 13th March 2005 to 15th July
2007, and the UASB from 10th of May 2007, to 10th of July 2007 for 66 days.
3.4 Start-up and Monitoring Bioreactor Operation
3.4.1 Seed Sludge
The seed sludge was obtained from a local wastewater treatment plant. The H2producing ability of the sludge was improved via heat treatment of the seed sludge at
80˚C, followed by acid pretreatment, in which the pH of the sludge was adjusted to 3.0
with 0.1 N HCl and was restored to 5 with NaOH afterwards.
3.4.2 Monitoring Bioreactors Performance
To determine reactors performance, several parameters were regularly analyzed such
as total organic carbon (TOC), gas composition, VFA composition, VFA
concentration, and gas production rate. Other analytical parameters were determined
less frequently such as morphological analysis of the granules, chemical oxygen
demand (COD), alkalinity, and glucose concentration. Granules were morphologically
analyzed at the end of the study to determine the hydrogen producing granule
characteristics.
Optimum temperature for mesophilic hydrogen production was found to be 30°C
[101]. The temperature of Singapore, where the experiment was conducted, varied
from 24°C to 32°C [109]. In addition, according to Chiu-Yue Lin, anaerobic sewage
sludge could be acclimated to produce hydrogen at ambient temperature [102].
Considering the reactor volume of 10 L, low HRT of 6.66 and the reactor
configuration, a significant amount of energy is required to bring the reactor
44
Chapter Three
Material and Methods
temperature up to the optimal mesophilic temperature. Therefore, along with
objectives of this study which is to reduce controlling method to increase hydrogen
production feasibility, AnSBR was operated at ambient temperature.
3.4.2.1 pH
Throughout operation pH was controlled with pH controllers dosing NaOH for both
the SBR and UASB. pH measurements were also performed on grab samples using an
Alex pH meter. The pH meter was calibrated each time it was used according to
standard procedures with pH 4.00, 7.00 and 9.00 buffer solutions. The pH
measurements were made immediately after taking the samples from the reactor to
minimize the loss of dissolved carbon dioxide. Measurements were made by inserting
the pH probe into the sample after rinsing with distilled water and samples. The pH
probe was a standard glass membrane-type probe, which was gel-encased.
Frequent pH measurements were made to monitor the reactor system, because changes
in the pH value, especially towards acidic conditions, indicate a potential imbalance in
the reactors. A drop in pH would indicate an accumulation of volatile fatty acids. A
drop to pH 3 could inhibit bacterial activities
3.4.2.2 Solids
On accordance with Standard Methods [112] the samples were filtered through 9 cm
GF/C filter paper. The filter paper was pre-dried and weighed. Pre-drying the filter
paper involved ignition at 550˚C for 20 minutes and cooling in a desiccator. The initial
weight of the filter paper was recorded after ignition by weighing with a laboratory
balance. The samples were then filtered through the filter paper using vacuum
filtration. The filter paper with the captured solids was then placed in the oven at
103°C for one hour to dry to constant weight and then cooled in a desiccator to be
45
Chapter Three
Material and Methods
weighted for the second time. The total suspended solids and volatile suspended solids
were calculated using the formula:
Total suspended solis, mg / L =
B− A
Volume of samples, ml
Volatile suspended solids, mg / L =
B−C
Volume of sample, ml
(3.4.2.2.1)
(3.4.2.2.2)
Where:
A= Initial weight of filter, mg
B= weight of filter after drying in a 103°C oven
C= weight of filter after ignition in a 550°C oven
3.4.2.3 Gas Composition
The biogas composition was determined by gas chromatography (Shimadzu GC-17A
equipped with Thermal Conductivity Detector) on a Porapak N 80/100 mesh column.
The column temperature was maintained at 60oC. The temperature for the detector and
injection port was maintained at 120oC. Argon was used as the carrier gas. A standard
gas having 25% hydrogen, 25% nitrogen, 40% methane and 10% carbon dioxide was
used to calibrate the GC for biogas composition.
3.4.2.4 Volatile Acids
Analysis of VFA was made by gas chromatography (Shimadzu GC-14B equipped with
Flame Ionization Detector) on a 25m x 0.32 mm HP-FFAP fused silica capillary
column in accordance with the method described by Yu and Fang (2003). Prior to
analysis the samples were acidified to a pH < 2 with methanoic acid [103].
46
Chapter Three
Material and Methods
3.4.2.5 Total Organic Carbon
Analysis of total organic carbon was made by Shimadzu TOC-Vcsh, Japan. Hightemperature combustion method was used to measure the total organic carbon (TOC).
3.4.3 Microbial Analysis
3.4.3.1 Scanning Electron Microscopy (SEM)
Fang and Chui's (1993) protocol was used for granule sectioning and sample
preparation prior to SEM observation [104].
SEM sample preparation protocol:
1. Granules were fixed by soaking in a 0.1 M phosphate buffer solution (pH 7.2)
with 4% glutaraldehyde for 2 hours
2. Sliced in half after being frozen with liquid nitrogen
3. Dehydrated with a series of water/ethanol solutions followed by another series
of ethanol/carbon dioxide solutions:
30% alcohol-10-15min
50% alcohol-10-15min
70% alcohol-10-15min
80% alcohol-10-15min
90% alcohol-10-15min
95% (2 changes)-10-15min
100% (2 changes)-10-15min
4. Critical point dried with carbon dioxide using a Blazers CPD 030 Critical Point
Dryer
5. Mounted on stubs with silver paint
6. Coated with gold-palladium
47
Chapter Three
Material and Methods
After coat with gold-palladium the sectioned granules were viewed and photographed
under SEM. Different magnifications of 50, 75, 100, 350, 1000, 2000, 3500, 5000, and
10000 were used for morphological analysis. This rang of magnification from 50 to
10000 were used because granule's structure, granule's size, bacteria's distribution, and
bacteria's morphology were analyzed under SEM.
3.4.4 Molecular Microbial Diversity Analysis
The bacterial community of the ASBR and UASB was analyzed by T-RFLP. DNA
extraction and PCR are processes prior to T-RFLP as sample preparation.
3.4.4.1 DNA Extraction
Cells (1 ml) used for DNA extraction were withdrawn after vigorous shaking from the
reactors by 1-ml disposable syringe and needles following the protocol [105] and
centrifuged immediately at 10,000 for 10 minutes at 4°C. The cell pellets were stored
at -20oC until processing. Genomic DNA was extracted from frozen cell pellets using
the DNeasy Tissue Kit (QIAGEN GmbH, Germany) according to the manufacturer’s
instructions. Molecular techniques such as polymerase chain reaction (PCR), terminalrestriction fragment length polymorphism (T-RFLP) were used to characterize and
identify the genera of current active culture subsequently [106].
3.4.4.2 Polymerase Chain Reaction.
PCR was performed in a total volume of 20 µl by Bio-Rad thermal cycler (Bio-Rad
Laboratories, Inc., California) and the products were analyzed on an Agilent
Bioanalyzer using DNA7500 Labchip Kit. The initial amplification was performed
with a pair of universal Eubacterial primers 8F [5’AGAGTTTGATCCTGGCTCAG3’]
and 1541R [5’AAGGAGGTGATCCAGCCGCA3’]) [107]. The following PCR
48
Chapter Three
Material and Methods
parameters were used for the amplification: 130s at 94º; 30 cycles of 30s at 94º, 45 s at
55º, and 130s at 72º; and a final extension of 6 min at 72º.
3.4.4.3 Terminal-Restriction Fragment Polymorphism analysis.
T-RFLP with the restriction enzyme provides an overview of the microbial community
composition over time during the enrichment process to estimate the microbial
diversity. By using the protocol [108] the amplified fragments by universal primer (8F
and 1541R, 8F labeled with Cy5) were digested with the restriction endonucleases Hha
I (NEB, USA). The enzyme was deactivated by heating at 65 ºC for 10 min.
49
Chapter Four
Results and Discussion
Chapter Four: Results and Discussion
4. Sampling and Results
The steady state performance of the ASBR could be regarded to quasi steady state.
Because the ASBR process is operated in cyclic mode, its steady state condition is
quasi. The quasi steady-state are considered to be established when gas quality and
total organic carbon concentration of the effluent are stable (less than 10% variation),
[55] during 20 days of ASBR operation. The results for the ASBR parameters
monitored were obtained under quasi steady state conditions during each operational
treatment. However, for UASB reactor steady state conditions were obtained as
indicated by constant hydrogen production [53].
4.1 Optimize the anaerobic H2 production
In the beginning of the experiments the performance of the ASBR was optimized by
selecting the optimum parameters from other studies on hydrogen production such as
pH 5.5, HRT 6.7h, and reaction/settling ratio 6.33 [12, 16, 49, 57 and 74].
The initial biomass concentration was set at 8.8 g/L which increased afterwards
because of the granulation. Organic loading rate was selected according to glucose
concentrations typically found in the sugary factory processing industry. Since, the
reactor is operated in Singapore; therefore, the ambient temperature varies from 22 to
34 °C [109].
4.2 Culture Enrichment (Pre-treatments and Start up)
Prior to reactor start up, two kinds of treatments (heat and acid treatments) were
applied to the seed from the anaerobic digestion of a local wastewater treatment plant.
50
Chapter Four
Results and Discussion
4.2.1 Initial Heat treatment
The wet sludge was heat treated for 20 minutes at 80°C before start up. The reasons for
this treatment was to select Clostridium spore-formers from natural environment and
pasteurized the activated sludge [58 and110].
4.2.2 Acid Treatment
There are potentially two kinds of pH enrichments, enhancement pH and cultivation
pH.
4.2.2.1 Enhancement pH
According to Zhang 2006, to induce microbial granulation, the acclimated culture
could be subjected to an acid incubation [74]. Microbial aggregation could take place
immediately with initiation of acid incubation and granule can be developed rapidly.
The same concept had used in this study for granulation. Therefore, the culture after
heat treatment was subject to an acid incubation for 24 h shifting the culture pH to 3.0
by HCl. The culture was resumed to pH 5.5 after the incubation and the reactor start
operation at hydraulic retention time (HRT) of 6.7 h.
Enhancement pH was applied only for short period and it selects the species which can
survive in such low pH.
4.2.2.2 Cultivation pH
Second enrichment, is a long-term practice which selects the species with the ability to
tolerate the specific pH during reactor operation. In the present study, a constant pH of
5.3 was selected to screen acidogenesis culture and suppresses the methanogenesis
activity.
After 14 days of ASBR operation with stirring rate of 100rpm, granules appeared as
shown in figure (4.1).
51
Chapter Four
a
c
Results and Discussion
600µm
300µm
b
300µm
d
300µm
Figure 4.1) Microscopic images of granules after 14 days of ASBR operation.
As can be seen in figure (4.1) the granules have a hirsute and squashy structure. The
average size of these immature granules was 1.5mm.
Before the heat treatment practice the reactor was held at quasi steady state for 42
days. During this period biogas composition was mostly carbon dioxide (83%), with
the remaining comprising methane (15%) and a small amount of hydrogen (1.92%).
According to Fang 1995, a sludge bed of bio-granules may contain 50 g/L of
suspended solids (SS), considerably higher than a suspended sludge [94]. In this
study, prior to heat treatment the average suspended solids (SS) concentration was 29
g/L and the maximum concentration was 56 g/L.
52
Gas percentage (%)
Chapter Four
Results and Discussion
100
90
80
70
60
50
40
30
20
10
0
0
10
20
30
40
50
Time (d)
Hydrogen
Methane
Carbon Dioxide
Figure 4.2 Gas composition before heat treatment
4.3 Biological Hydrogen Production Measured in Batch Anaerobic
Respirometers after Heat Treatment
As mentioned in section 2.4.2, heat treatment could be conducted for granule
enrichment. However, resistance of cultures varies due to, the kind of species, and
different conditions of age or growth of the culture. Therefore, to find out the optimum
temperature and duration of heat treatment for specific culture used in this study, series
of serum bottle experiments were conducted. The parameters of these serum bottles
such as loading rate, temperature, pH were the same as the reactor which indicated
25g/L.d, ambient temperature and 5.3 respectively. All batch experiments were carried
out in duplicates and the experiment was done twice. According to Wesley et al.
(1997), it is through the inactivation of one or more essential proteins, such as
enzymes, that heat treatment kills a specific microorganism [111]. The amount of heat
required to kill varies from one organism to another, in fact for any organism both the
amount of heat (i.e., the temperature) to be used and the length of time the material to
be sterilized is maintained at a given temperature must be considered. The bacterial
53
Chapter Four
Results and Discussion
endospores are probably the most resistant form of life known; some will survive
100°C for several hours. There is no standard pattern of heat resistance for endospores,
because their resistance varies not only from species to species but under different
conditions of age or growth. A number of factors have been regarded as contributing to
the heat resistance of spores. Their low water content and thick spore coat are probably
the most important factors. Another important factor to be considered with heat
sterilization is the environmental of the microorganisms being destroyed. Environment
is important for two reasons: (1) in order to kill, the heat must reach the organism, and
(2) more heat than normal is required to kill organisms embedded in protein material.
Vegetative forms of pathogenic organisms are readily destroyed at the temperature of
boiling water. Actually, they are usually killed within a few minutes at 80°C.
However; some bacterial endospores show unusual heat resistance and may survive
boiling temperature for up to 20h. The killing effect of boiling water is greatly
increased by the addition of 2 percent sodium carbonate or detergents. In most cases,
heat activation at 75 to 80°C for 15-20 min is used to inactivate vegetative cells and
activate germination of spores. These factors were considered to choose various
temperatures for serum battle experiment. Five sets of serum bottles with temperatures
of 80°C, 100°C, 120°C, 140°C, and control were examined.
Serum bottle experimental parameters:
•
Volume 150 ml
•
SS the same as the reactor (15g/L)
•
Loading Rate 25 g/L.d glucose
•
Experiment duration: 200 h
54
Chapter Four
Results and Discussion
120
120
100
G a s p e r c e n t a g e (% )
G a s p erc e n ta g e (% )
100
80
60
40
20
80
60
40
20
0
0
0
50
100
150
200
-20
250
0
50
100
-20
Hydrogen
Methane
b
Carbon Dioxide
250
Hydrogen
Methane
Carbon Dioxide
120
120
G a s p e r c e n ta g e (% )
200
Time (h)
Time (h)
a
150
100
G a s pe r c e nta ge (% )
100
80
60
40
20
80
60
40
20
0
0
50
100
150
200
0
250
0
Time (h)
c
Hydrogen
50
100
150
200
250
Time (h)
Methane
d
Carbon Dioxide
Methane
Carbon Dioxide
G a s C o m p o s it io n ( % )
100
90
80
70
60
50
40
30
20
10
0
-10 0
Hydrogen
50
100
150
200
250
Time (h)
e
Hydrogen
Methane
Carbon Dioxide
Figure 4.3 Gas concentration (%) in the vessels head space after heat treatment over time, at a)
80°C, b) 100°C, c) 120°C, and d) 140°C for 25 minutes, e) Control batch experiment without heat
treatment
As shown in figure (4.3) following by a lag phase of 20.3h for 80°C, 23 h for 100°C,
and 25h for 120°C there was gas production for 200h. However, after 200h of
55
Chapter Four
Results and Discussion
experiment there was no activity in terms of gas production for 140°C. Based on these
results, we concluded that biogas production experiments conducted at different
temperatures followed the same trend of gas production quality. After the lag time,
there was an increase in hydrogen production for a period of 99.7h for 80°C, 49h for
100°C and 47h for 120°C; and afterwards the hydrogen was consumed in 27h, 48h and
75h, respectively (figure 4.3a, figure 4.3b and figure 4.3c). To compare the three
batches of 80°C, 100°C, and 120°C, increase of hydrogen per hour were 0.44%,
0.76%, and 0.79% and decrease of hydrogen per hour were also 1.62%, 0.77%, and
0.49% respectively.
The same pattern of H2 production followed by hydrogen consumption was reported
by other researchers [59]. Beeftinkt and van den Heuvel (1987), [112] reported a
similar shift from hydrogen producing process of acetate-butyrate to hydrogen
consuming of without performing any community analysis the metabolic shift was
suggested to have resulted from changes in the microbial community structure rather
than metabolic adaptation. They also showed that differences in bacterial community
structures during acetate–butyrate and acetate–propionate dominated metabolisms.
These results demonstrate that the instability of H2 production in these batch tests
might be due to changes in microbial populations. Oh et al. (2003) found high
hydrogen gas concentrations (57-72%) were produced in all batch tests that
encountered heat treatment at pH (6.2 or 7.5). However, hydrogen gas phase
concentrations in all batch cultures reached a maximum of 57-72% after 30h but
thereafter rapidly declined to non-detectable levels within 80h [62].
As can be understood from these results 80°C showed the shortest lag time, which is
expected because of the lower temperature applied to the culture; and as a result, more
species of bacteria can tolerate 80°C than higher temperatures. In addition, according
56
Chapter Four
Results and Discussion
to Doyle 2002, in most cases, heat activation at 75 to 80°C for 15-20 min is used to
inactivate vegetative cells and activate germination of spores. It means that the
diversity of microbial culture remain in the 80°C batch, after heat treatment, was more
than other serum bottles encountered to higher temperatures, and thus more species of
bacteria are able to consume the hydrogen or produce carbon dioxide. This can be the
reason for rapid decrease of hydrogen at 80°C.
There was no biogas production recorded for 140°C after 200h. As a result, the
experiment was continued for the second batch. The results showed that even though
the lag time is long, however, hydrogen productivity could be maintained for a longer
duration comparing to previous heat treatments (figure 4.4). Nevertheless, because of
long lag time having heat treatment for 140°C does not appear to be a practical option.
Methanogenesis was completely inhibited as shown in Figures (4.3a to 4.3d).
Gas Composition (%)
100
80
60
40
20
0
0
20
40
60
80
100
120
140
-20
Time (h)
Hydrogen
Methane
Carbpn Dioxide
Figure 4.4 Gas concentration (%) after heat treatment over time at 140C for 25 minutes (second
batch of feeding).
Facts presented can conclude that, although heat treatment affects hydrogen
production; however it cannot be the ultimate solution. Although researchers have
applied heat treatment to have stable inoculum for biogas in terms of hydrogen
production, that is only because they have applied it continuously [51].
57
Chapter Four
Results and Discussion
Considering parameters of shorter lag time, longer duration of hydrogen productivity
and maximum hydrogen percentage, 80°C had shown optimum results. Consequently,
this temperature had also been chosen for one more experimental batch test.
These sets of batch tests were conducted to identify the microbial culture responsible
for hydrogen production. The batch set included of four duplicate bottles contained
biomass of the ASBR. The reason for this experiment was to identify the responsible
culture producing hydrogen inside the reactor before further operation of the reactor.
Therefore, these four batch tests with same conditions explained before (4.3) were
selected, first with separated granules; second with only suspended biomass, third with
both granules and separated suspended biomass, and forth with granules and
suspended biomass as control test. Afterwards, heat treatment at 80°C for 25 min was
applied to all batch tests except the control test, and gas concentration (%) was
measured for the duration of 190h. All batch tests were conducted in duplicates. The
results of these batch experiments are shown in figure (4.5).
58
Chapter Four
Results and Discussion
1.2
a
1
gaspercentage
0.8
0.6
0.4
0.2
0
-0.2
0
50
100
150
200
time (hr)
H2
CH4
CO2
1
b
gaspercentage
0.8
0.6
0.4
0.2
0
0
50
100
150
200
150
200
-0.2
time (hr)
c
1.2
gaspercentage
H2
0.8
CH4
CO2
1
0.6
0.4
0.2
0
-0.2
0
50
100
time (hr)
H2
CH4
CO2
d
100
80
60
40
20
0
0
50
100
150
200
-20
Hydrogen
Methane
Carbon Dioxide
Figure 4.5 a) Separated granules, b) Separated suspended biomass, c) Granules with suspended
biomass after heat treatment at 80°C for 25 min, d) Control batch experiment without heat
treatment
59
Chapter Four
Results and Discussion
As can be seen in figure (4.5), separated granules had shown shorter lag time (42.5h)
comparing to suspended biomass (112.5h). However, the lag time for the batch test
containing both suspended biomass and granules was the same as separated granules
(42.5h). Maximum hydrogen proportion was almost the same among three batch tests
which was 45%, and similar to previous batch experiments there was no methane
production. Results of these batch tests concluded that both granules and suspended
biomass are responsible for hydrogen production. This might indicate that the same
species are present in both media.
4.3.1 Biological Hydrogen Production Measured in ASBR after Heat
Treatment
Subsequent to selecting the optimum heat treatment temperature (80°C for 25 min), the
same temperature was applied to ASBR after reaching the quasi steady state. As shown
in figure (4.5a), a similar pattern of hydrogen production was also observed in ASBR.
Although there was an increase from 2% to 16% in average hydrogen production after
heat treatment; however, results confirmed that heat treatment alone, is not the ideal
solution to reach the stable hydrogen production. Figure (4.6 b) shows 30 days of quasi
steady state by the enriched culture after heat treatment.
After applying the heat treatment to ASBR it was followed by a lag time of 3 days the
hydrogen proportion in the biogas increased continuously for 13 days, and afterwards
during 17 days all hydrogen was consumed. A maximum of 37% hydrogen could be
reached after heat treatment.
60
Chapter Four
Results and Discussion
a 100
Gas percentage (%)
90
80
70
60
50
40
30
20
10
0
0
5
10
15
20
25
30
35
Time (d)
Hydrogen
Gas Composition (%)
b
Methane
Carbon Dioxide
100
90
80
70
60
50
40
30
20
10
0
0
5
10
15
20
25
30
35
Time (d)
Hydrogen
Methane
Carbon Dioxide
Figure 4.6 a) ASBR gas concentration (%) after heat treatment over time, b) ASBR quasi steady
state conditions reached after heat treatment
4.4 Biological Hydrogen Production Measured in ASBR after Lack of
Carbon Source Treatment
Enrichment of the culture was continued towards hydrogen production after reaching
the quasi steady state; the culture had encountered lack of carbon source for 20 days.
During these 20 days the sludge was starved and then gas samples were taken.
Since starvation is economical (no additional energy is required), it was chosen as a
novel enrichment method. In addition, after previous treatments the culture had
become more enriched, sudden shock may destroy the species which were beneficial
for hydrogen production. The lack of carbon source is a smooth and long practice
61
Chapter Four
Results and Discussion
which gradually maintains the kinds of bacteria with the ability of sporulation during
of lack carbon and nutrients.
Gas Composition (%)
80
70
60
50
40
30
20
10
0
0
5
10
15
20
25
Time (d)
Hydrogen
Methane
Carbon Dioxide
Figure 4.7 Gas concentration (%) after lack of carbon source treatment during start up
The hydrogen proportion had significantly changed after the treatment. The average
hydrogen concentration (%) increased from an average of 16% to an average of 48%
during quasi steady state and the maximum hydrogen concentration (%) was 73%. In
addition, high hydrogen productivity could be maintained and methanogenesis was
completely inhibited. Comparable hydrogen concentration (%) has reported in other
studies. Liu and Fang (2002) reported that, hydrogen accounted for 57% to 68% of
biogas at HRT ranging 4.6-28.6h and sucrose concentration ranging 4.8-29.8 g/L
during hydrogen production from wastewater by acidogenic granular sludge [52]. Fang
et al. (2002) also reported that methane-free biogas comprised 63% hydrogen, 35%
carbon dioxide, and 2% nitrogen by acidogenic granular sludge degrading sucrose with
HRT of 6h and pH 5.5 [16].
The dominant metabolic products of volatile fatty acids, gas composition, and biomass
production were examined during the first 18 days of start up after encountering to
lack of carbon source for 20 days.
62
a
30
Suspended solid (g/L)
Chapter Four
25
Results and Discussion
20
15
10
5
0
0
5
10
15
20
25
30
35
40
Time (d)
b
1.8
Suspended solid (g/L)
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
5
10
15
20
25
30
20
25
30
Time (d)
Concentration (mM)
c
16
14
12
10
8
6
4
2
0
0
5
10
15
Time (d)
Acetate
Butyrate
Propionate
Figure 4.8 a) Suspended solids concentrations in the ASBR, b) Suspended solid concentrations in
the effluent, c) Dominant volatile fatty acids concentration in the effluent during star up under the
lack of carbon source treatment
63
Chapter Four
Results and Discussion
According to Nandi and Sengupta 1998, the most common products in the
fermentation of carbohydrates are acetate and butyrate. This acidification process may
be expressed by following reactions [113]:
C 6 H 12 O6 + 2 H 2 O → CH 3COOH + 2CO2 + 4 H 2
(4.1)
C 6 H 12 O6 → CH 3 CH 2 CH 2 COOH + 2CO2 + 2 H 2
(4.1)
As shown in the equations 4.1 and 4.1, the stoichiometric yields are 4 moles of
hydrogen per mole of glucose in the production of acetic acid, and 2 mole of hydrogen
in the production of butyric acid. The maximum yield of 2.49 (mol H2/mol glucose)
found during start up of this study was resulted both from acetate and butyrate process.
During this period VFA content was mostly acetate (54%), butyrate (23%), propionate
(20%) and small amount of other VFA's includes 3% (i-butyrate, valerate, caproate, icaproate, i-valerate and heptanoate) in the effluent. In addition, the main alcohol
composition was ethanol.
On the subject of the suspended solid concentration of the effluent, as shown in figure
(4.8 b), during first five days of the start up the concentration was high (~1.6 g/L). The
reason was the wash out of the dead microorganisms which could not tolerate the lack
of carbon source treatment. However, after 12 days the concentration of biomass in
effluent decreased to 0.435 g/L and become more stable. Since, synthetic wastewater
was used as feed; therefore, the difference between suspended solid (SS) and volatile
suspended solids (VSS) is small and that’s the reason in the graphs VSS has not been
shown.
At this stage, the Sludge Retention Time (SRT) was calculated as:
64
Chapter Four
Results and Discussion
Average SRT during startup
=
MLSS (mg / L) × V ( L)
13450(mg .L) × 10 L
=
= 8.58d
MLSS withdraw rate(mg / L.d ) 36( L / d ) × 435(mg / L)
(4.3)
Average SRT during the quasi steady state
=
MLSS (mg / L) × V ( L)
2659(mg.L) × 10 L
=
= 28.41 d
MLSS withdraw rate(mg / L.d ) 36( L / d ) × 260(mg / L)
(4.4)
The average MLSS of ~27 g/L of the reactor was due to the presence of biogranules
inside the reactor. In fact, this factor is a promising feature of the ASBR process that
granular biomass can be achieved, and in this way higher biomass can be maintained in
the reactor with efficient biomass setting time and a long solids retention time (SRT)
[114]. All the MLSS samples were taken during complete mixing of the reactor and
prior to effluent withdrawn.
4.5 Microbial Shift
As presented in section (4.3) and (4.4) hydrogen productivity increased in terms of gas
quality, after conducting each treatment. The possible reasons for a higher hydrogen
production after heat and lack of carbon source treatments could be because of
population shift or metabolic shift take place in the reactor [56]. A number of
experiments can be conducted to confirm the reason. The complex nature of consortia
in H2 producing microflora and the existence of population shift can be demonstrated
using genetics techniques. Regarding the metabolic shift, fermentation end products
vary within the same bacterium dependent on environmental conditions, and by
detecting VFA effluent concentrations, the metabolism pathways could be determined.
Therefore, to identify the reason of higher hydrogen productivity, genetic techniques
and microbial products (VFA and alcohols) could be detected by various methods.
65
Chapter Four
Results and Discussion
T-RFLP is one of the genetics techniques which had been conducted after heat
treatment and also after lack of carbon source treatment. The amplified fragments by
universal primer were digested with the restriction endonucleases Hha I (NEB, USA).
These results have demonstrated that the microbial population has shifted almost
completely after first treatment. However, similar species were observed before and
after lack of carbon source treatment and the population has changed.
66
Chapter Four
Results and Discussion
25000
a
20000
D y e S ig n a l
370.37
15000
72.39
10000
5000
266.76
369.47
71.38
600.69
581.74
471.24
0
0
50
100
150
200
250
300
350
Size (nt)
400
450
500
550
600
650
700
b
50000
45000
218.03
D y e S ig n a l
40000
35000
30000
25000
72.62
20000
15000
10000
62.99
225.54
566.75
5000
0
0
100
150
200
250
300
350
Size (nt)
400
450
500
550
600
650
c
30000
25000
D y e S ig n a l
50
63.03
20000
15000
10000
266.56
5000
72.69
573.09
550.45 581.79
218.18
0
0
50
100
150
200
250
300
350
Size (nt)
400
450
500
550
600
650
Figure 4.9 T-RFLP results, a) Before heat treatment, b) After heat treatment, c) After lack of
carbon source treatment
67
Chapter Four
Results and Discussion
4.6 Dynamic changes during one cycle of ASBR
The elucidation of the dynamic changes during one cycle is helpful in understanding
how the biological process occurs inside sequencing batch bioreactor. Figure (4.10)
shows the dynamic changes in organic matter (TOC), biomass (SS), VFA, gas quality
and gas production during one cycle in the system at HRT of 6.7 h with R/S ratio of
6.33.
The monitoring of the variation in substrate concentration showed that glucose was
completely degraded within first 15 min of operation. As the reaction phase started, the
total carbon and total organic carbon decreased and the total VFA concentration
increased as shown on figure (4.10).
68
Chapter Four
a
Results and Discussion
Concentration (mM)
80
70
60
50
40
30
20
10
0
0
20
40
60
80
100
120
140
160
140
160
Time (min)
Acetate
Gascoposition (%)
b
Butyrate
Ethanol
60
50
40
30
20
10
0
0
20
40
60
80
100
120
Time (min)
Hydrogen
Carbonconcentration(mg/L)
c
Methane
Carbon Dioxide
2500
2000
1500
TOC
TC
1000
500
0
0
20
40
60
80
Time (min)
GasProductionVolume(ml/min)
d
60
50
40
30
20
10
0
0
5
15
25
60
120
150
180
Time (min)
Figure 4.10 Evolution of (a) VFA concentrations, (b) Gas concentrations (%), (c) Carbon
Concentrations, (d) Gas production rates at each cycle operation
69
Chapter Four
Results and Discussion
During one cycle VFA includes of acetate (73%), Butyrate (23.6%), propionate
(1.5%), caproate (0.69%), valerate (0.58%), and very small amounts of i-caproate, ivalerate heptanoate, and i-butyrate. The concentration increased in the first 60minutes.
In addition, during one cycle the gas production concentrations (%) remain constant.
However the amount of hydrogen production rate varies during the cycle and the
maximum hydrogen production rate were during first 20minutes (53ml/min).
4.7 Characteristic of Enriched Acidogenic Granules
Throughout the enrichment process, physical, morphological and biological
characteristics of the acidogenic granules were modified. However, in this chapter the
main focus is on the characteristic of the enriched granule after both heat and lack of
carbon source treatment.
4.7.1 Size (Average granule Diameter) Distribution and Settling Velocity
The Hydrogen Producing Granule (HPG) had an average diameter of 1.7 ± 0.2mm. the
maximum granule diameter inside the reactor was 3.5mm. It exhibited an average
settling velocity of 43m/h, which was comparable to reported velocities for the
methanogenic granules. For each size three samples were examined and the average
size was taken.
R2 = 0.9422
100
Settling Velocity (m/h)
90
80
70
60
50
40
30
20
10
0
0
0.5
1
1.5
2
2.5
3
3.5
Diameter of the Granules (mm)
Figure 4.11 Granules settling velocity
70
Chapter Four
Results and Discussion
According to figure (4.11), there is a correlation between the diameter of the granule
and settling velocity rate. As shown in the figure 4.11 larger granules settle faster than
the smaller ones. Therefore, larger granules lead to shorter settling time and as a result
can improve the reactor performance.
4.7.2 Morphology and Microstructure of granules
Analysis of light microscopic shown that prior to treatments, granules were brown in
color; however, after enrichment the HPG became creamy white in color. The reason
of this change in color is due to suppression of sulfate reducing activity at pH 5.5.
Figure 4.12 Photos of Granules by light microscopic
Figure (4.13, 4.14, and 4.15), illustrates the SEM images of the hydrogen producing
acidogenic granules sampled after treatments. Different magnifications of 1000, 2000,
3500 and 5000 were used for morphological analysis.
71
Chapter Four
Results and Discussion
Figure (4.13) illustrates that the acidogenic granules had porous and multiple cracks on
the surface, which is different from that of the methanogenic granules. These pores
were likely to facilitate the passage of nutrients and substrate as well as the release of
hydrogen, which has a very low solubility 1.58 mg/L in water. As shown in figure
(4.14), the granules were sliced before observing under the SEM. In addition, for each
sliced granule 7 places (center and sides) were observed under the SEM to examine the
layered structure of the granule. Figure (4.16 and 4.17) illustrate that the hydrogen
producing bacteria were almost completely composed of the spore-forming, rod shaped
bacteria. Therefore, the dominant species might be clostridium. Comparing to the
studies [19 and 115] in which diverse morphology at the surface and inner layers of
the granule were observed, what was found in this study was different. In this study the
same morphological bacteria can be seen in both outer and inner structure of the
granule.
72
Chapter Four
Results and Discussion
a
b
c
d
e
f
Figure 4.13, Granule surface, a) sliced granule, magnification 75, b) magnification 5K, c)
magnification 1K, d) porous and multiple cracks on the surface, magnification 350, e)
magnification 5K, f) magnification 5K
73
Chapter Four
Results and Discussion
Figure 4.14 Sliced granules
Figure 4.15 Parts of sliced granule observed under SEM
74
Chapter Four
Results and Discussion
a
b
c
d
Figure 4.16 a) part 1, magnification 3.5K, b) part 3, magnification 3.5, c) part 3, magnification 2K,
d) part 7, magnification 3.5.
75
Chapter Four
Results and Discussion
a
b
c
d
Figure 4.17 a) part 4, magnification 10K, b) part 2, magnification 5K, c) part 5,
magnification 5K, d) part 6, magnification 5K
4.8 Biological Hydrogen Production Measured in UASB with Enriched
Granule
The enriched granule applied in an UASB reactor to evaluate the stability in a
continuous approach. As shown in figure (4.18) gas concentration (%) was stable
during 66 days of operation of UASB reactor and the average hydrogen was 61%.
76
Chapter Four
Results and Discussion
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
10
20
Hydrogen
30
40
Methane
50
60
70
Carbon dioxide
Figure 4.18 UASB gas concentration (%) during 66 days of operation
Since, the SRT is 29 days these data can be representative, however, further research
need to be done to confirm that the long-term stability can be maintained.
77
Chapter Five
Conclusions and Recommendations
Chapter Five: Conclusions and Recommendations
5.1 Application and Contribution of this Research
The results of this research show, for the first time, that the ASBR process can form
hydrogen producing granules. Hydrogen and methane production can easily be linked
using a two-stage process. That is, the ASBR process can be utilized for treatment of
high strength wastewater with short hydraulic detention times during first stage of a
two stage treatment. During the second stage of anaerobic treatment, methane can be
recovered from intermediate fatty acids and alcohols present in the effluent of the first
stage. This process would require longer hydraulic detention times and would produce
methane gas. Although, two-stage anaerobic treatment systems have been used, none
have yet been designed or operated at full-scale for hydrogen production and recovery
[116]. The present study has shown that acidogenic process which is the initial stage of
the complete process of anaerobic treatment can be stabilized in terms of hydrogen
concentration without additional costs for controlling methods such as "repeated" heat
treatment, sparging nitrogen gas and other kinds of treatments.
On the other hand, the results of this study have demonstrated that, completely mixed
cultures in wastewater treatment practice can be further enriched by several treatments
which lead to higher efficiency and stability.
5.2 Conclusions
Based on the experiments conducted here, the following conclusions can be drawn.
1. Granules were formed in an anaerobic sequencing batch reactor at acidic pH.
2. An increase in hydrogen productivity was observed after heat treatment and
lack of carbon source treatment.
78
Chapter Five
Conclusions and Recommendations
3. The ASBR process demonstrated to be efficient in terms of hydrogen
productivity and stability after treatments.
4. Sulfate reducing bacteria and methanogenic bacteria were completely inhibited
after treatments.
5. Based on microbial analysis, the enrichment of granules was successful and
accordingly the diversity of organisms changed significantly after performing
these treatments.
6. Granules morphological analysis showed multiple cracks on the surface of the
granule and the of interaction pathways inside the granule's structure.
Furthermore, these analyses verified the non-layered structure of acidogenic
granules.
7. Anaerobic degradation comprises a sequential process of hydrolysis,
acidogenesis, acetogenesis and methanogenesis. This study had showed that an
intermediate process like acidogenic/acetogenic can have stable hydrogen
production in terms of hydrogen concentration by selection of preferable
bacteria under typical operational conditions.
5.3 Recommendations for Further Research
1. Studies should be conducted to investigate same concept used in this research
in a two-stage wastewater treatment to understand the economical and technical
feasibility of this study. The first stage is designed for the initial
fermentative/acidogenic degradation by enriched biomass obtained in current
study. During these processes hydrogen could be recovered. The second stage
is for the subsequent acetogenic/methanogenic degradation of the intermediate
fatty acids to recover methane. This process would require longer hydraulic
79
Chapter Five
Conclusions and Recommendations
detention times and would produce methane gas. These two processes can be
separated by controlling pH and hydraulic detention times.
2. Studies should be conducted to identify microbial species in the enriched
granules.
3. Results from this study have shown that glucose was completely degraded
within first 15 min of operation. In addition, laboratory-scale works on simple
substrates have used HRTs as low as 4h with high hydrogen production [53 and
117]. In current study because of presence of granules lower HRTs can be
studied without washout.
4. Previous studies on granule structure indicated that outer layer of the granule is
mainly responsible for the acidogenic degradation lead to hydrogen production.
Therefore, granule structure can play an important rule on hydrogen
production. Shear forces (e.g. resulted from stirring) affect granule size and
structure. Studied can be conducted to investigate effects of shear forces on
granule performance in terms of hydrogen production.
5. A one-off cessation of feed supply for few hours or regular feed interruption
gave shifts in product formation, thought to be related to a population shift
away from butyrate/H2 producing spore formers and towards propionate
producing non-spore formers. The semi-continuous feeding mode such as
ASBR used by some workers at laboratory scale could thus give poor
performance [56]. Therefore, the continuous approach by UASB can be further
studied by enriched granules to confirm the stable hydrogen production.
80
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[...]... technology is widely applied in industry, especially in the food and beverage and pulp and paper industry, particularly for wastewater treatment Throughout time, studies and experiments has shown various advantages and disadvantages of anaerobic treatment The rationale and interest in the use of an anaerobic treatment process can be explained by considering the advantages and disadvantages of this system... reaching the steady state After each treatment, the performance of the reactor is determined and the data collected from the reactor running, is analyzed for hydrogen productivity Finally, the hydrogen production stability of the enriched culture is compared in a continuous approach by UASB and a batch approach by an ASBR for stable hydrogen production 7 Chapter One Introduction Hydrogen Production by. .. mole of glucose in the production of acetic acid (maximum theoretical hydrogen) , and 2 mole of hydrogen in the production of butyric acid However, several volatile fatty acids and alcohols such as propionate, hexanoate, ethanol, and butanol can be as fermentation products during hydrogen production process H2 can readily be produced from a range of biomass materials However, without substantial improvement,... limitation of hydrogen production are both energy consuming and costly Therefore, the aim of this study was to avoid these controlling methods and as result obtain sustainable and economical hydrogen production The proposed idea in this research was supported by four theoretical facts as follows: 1 Sludge can be treated to form hydrogen- productive granules [16] 2 Granules can be retained because of... released by the degraded compound remains in the fermented products This process normally occurs in an anaerobic digester, but it may also take place in sewer systems, a secondary clarifier, or a thickener Fermentation can be completed by both facultative anaerobic bacteria and strict anaerobic bacteria and can occur by many different pathways with many different products The types of organic compounds... during this process are dependent on the type of bacteria and the existing operational conditions 18 Chapter Two Literature Review In many of the fermentative pathways, hydrogen is produced The production of hydrogen gas is important in anaerobic digesters because, hydrogen is one the main substrates for the production of methane and also hydrogen pressure may inhibit the acetogenic bacteria Strict anaerobic... be practically useful As an example, Antonopouluo et al (2007) had produced 10.4l hydrogen per kg of sorghum biomass [31] The demand of hydrogen as a new clean energy source is rapidly increasing Therefore, low-cost technology for bio -production of hydrogen is being developed in many countries Improving bio -hydrogen- producing capacity and reducing cost is the key to achieve industrialization The microbiological... bio-solids needing disposal by 50-80 percent In addition, during anaerobic treatment, methane or hydrogen is produced which has energy value, and the residual bio-solids can be safely used as a humus-rich compost if it is low in heavy metal content 2.2.3 Hydrogen Production Feasibility Biomass obtained from different sources can have various hydrogen productions depending on the kind and concentration... research on fermentation microorganisms in 1861 [33] In 1881, Mouras' Automatic Scavenger used anaerobic microorganisms to treat waste for the first time [34 and35] Currently, anaerobic digestion is an established technology for the treatment of wastes and wastewater Anaerobic treatment is applicable for a wide range of users, from industry to farming, waste-treating companies, water boards and individual... settling characteristics [17 and18] and since, low HRT favors hydrogen productivity; therefore this characteristic of granules can be beneficial for hydrogen production 3 Hydrogen producing Clostridium and bacillus are able to produce endospores in harsh conditions such as heat, chemical toxicity, lack of carbon source, ultraviolet, ionizing radiation, Acid/base conditions, and Desiccation [19] 4 Granules .. .HYDROGEN PRODUCTION BY ENRICHMENT GRANULES IN AN ACIDOGENIC FERMENTATION PROCESS SHIVA SADAT SHAYEGAN SALEK (B.Sc, Tehran University) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING... approach by an ASBR for stable hydrogen production Chapter One Introduction Hydrogen Production by Enrichment Granules in an Acidogenic Fermentation Process Seed from anaerobic digestion treatment... advantages and disadvantages of anaerobic treatment The rationale and interest in the use of an anaerobic treatment process can be explained by considering the advantages and disadvantages of this
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