Pretreatment of cellulosic waste and high-rate

biogas production

Solmaz Aslanzadeh
Copyright © Solmaz Aslanzadeh

School of Engineering
University of Borås
SE-501 90 Borås (Sweden)

Handle-ID http://hdl.handle.net/2320/12853
ISBN 978-91-87525-10-0 (Printed)
ISBN 978-91-87525-11-7 (pdf)
ISSN 0280-381X, Skrifter från Högskolan i Borås, nr. 47

Printed in Sweden by Ineko AB

Borås 2014

ii
 Abstract

The application of anaerobic digestion technology is growing worldwide, mainly because of its
environmental benefits. Nevertheless, anaerobic degradation is a rather slow and sensitive process.
One of the reasons is the recalcitrance nature of certain fractions of the substrate (e.g.,
lignocelluloses) used for microbial degradation; thus, the hydrolysis becomes the rate-limiting step.
The other reason is that the degradation of organic matter is based on a highly dynamic, multi-step
process of physicochemical and biochemical reactions. The reactions take place in a sequential and
parallel way under symbiotic interrelation of a variety of anaerobic microorganisms, which all
together make the process sensitive. The first stage of the decomposition of the organic matter is
performed by fast growing (hydrolytic and acid forming) microorganisms, while in the second stage
the organic acids produced are metabolized by the slow growing methanogens, which are more
sensitive than the acidogens; thus, methanogenesis becomes the rate-limiting step.
The first part of this work evaluates the effects of a pretreatment using an organic solvent, N-
methylmorpholine-N-oxide (NMMO), on cellulose-based materials in order to overcome the
challenge of biomass recalcitrance and to increase the rate of the hydrolysis. NMMO-pretreatment
of straw separated from the cattle and horse manure resulted in increased methane yields, by 53%
and 51%, respectively, in batch digestion tests. The same kind of pretreatment of the forest residues
led to an increase by 141% in the methane production during the following batch digestion assays.
The second part of this work evaluates the efficacy of a two-stage process to overcome the second
challenge with methanogenesis as the rate-limiting step, by using CSTR (continuous stirred tank
reactors) and UASB (up flow anaerobic sludge blanket) on a wide variety of different waste
fractions in order to decrease the time needed for the digestion process. In the two-stage semi-
continuous process, the NMMO-pretreatment of jeans increased the biogas yield due to a more
efficient hydrolysis compared to that of the untreated jeans. The results indicated that a higher
organic loading rate (OLR) and a lower retention time could be achieved if the material was easily
degradable. Comparing the two-stage and the single-stage process, treating the municipal solid
waste (MSW) and waste from several food processing industries (FPW), showed that the OLR
could be increased from 2 gVS/l/d to 10 gVS/l /d, and at the same time the HRT could be decreased
from 10 to 3 days, which is a significant improvement that could be beneficial from an industrial
point of view. The conventional single stage, on the other hand, could only handle an OLR of 3
gVS/l/d and HRT of 7 days.
Keywords: Biogas, Two-stage anaerobic digestion, N-methylmorpholine-N-oxide (NMMO)
pretreatment, Lignocelluloses, Textile waste

iii
iv
 List of Publications

This thesis is mainly based on the results presented in the following articles:

I. Aslanzadeh S, Taherzadeh MJ and Sárvári Horváth I. (2011): Pretreatment of straw fraction

of manure for improved biogas production. Bioresources 6: 5193-5205.
II. Aslanzadeh S, Berg A, Taherzadeh MJ and Sárvári Horváth I. (2014): Biogas production
from N-Methylmorpholine-N-oxide (NMMO) pretreated forest residues. Applied
Biochemistry and Biotechnology, in press.
III. Jeihanipour A, Aslanzadeh S, Rajendran K, Balasubramanian G and Taherzadeh MJ.
(2013): High-rate biogas production from waste textiles using a two-stage process.
Renewable Energy 52: 128-135.
IV. Aslanzadeh S, Rajendran K, Jeihanipour A and Taherzadeh MJ. (2013): The Effect of
Effluent Recirculation in a Semi-Continuous Two-Stage Anaerobic Digestion System.
Energies 6: 2966-2981
V. Aslanzadeh S, Rajendran K and Taherzadeh MJ. A comparative study between
conventional and two-stage anaerobic processes: Effect of organic loading rate and
hydraulic retention time (Submitted).

Statement of Contribution

Paper I: Performed the experimental work of the pretreatments and anaerobic digestion assays
and responsible for the data analyses and manuscript writing.

Paper II: Responsible for parts of the experimental work and data analyses. Active participant
in the preparation and organization of the manuscript.

Paper III: Responsible for parts of the experimental work and involved in the manuscript
preparation and its revision.

Paper IV: Responsible for parts of the experimental work and for the manuscript preparation.

Paper V: Responsible for major part of the experimental work and data analyses as well as the
manuscript preparation.

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 List of Publications not included in this thesis

Articles:

I. Rajendran K., Aslanzadeh S., Taherzadeh M.J. (2012): Household Biogas Digesters—A
Review. Energies 5, 2911-2942.

II. Rajendran K., Aslanzadeh S., Johansson F., Taherzadeh M.J. (2013): Experimental and
Economical Evaluation of a Novel Biogas Digester. Energy Conversion & Management 74:
183-191.

Book chapters:

I. Aslanzadeh S, Ishola MM, Richards T, Taherzadeh,MJ, (2014): An Overview of Existing

Individual Unit Operations in Biological and Thermal platforms of Biorefineries, In: N.
Qureshi, D. Hodge & A.V. Vertes (Eds): Biorefineries: Integrated Biochemical Processes
for Liquid Biofuels (Ethanol and Butanol), Elsevier, Chapter 1, in press

II. Aslanzadeh S, Rajendran K, Taherzadeh MJ. (2013): Pretreatment of Lignocelluloses for

Biogas and Ethanol Processes, In: Ram Sarup Singh, Ashok Pandey and Christian Larroche
(Eds): Advances in Industrial Biotechnology, Asiatech Publishers Inc, New Delhi, India,
Chapter 8, Pages 125-150.

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Table of content

Abstract ............................................................................................................................... iii

List of Publications .............................................................................................................. v
List of Publications not included in this thesis ................................................................ vi
Chapter 1. Introduction..................................................................................................... 1
Chapter 2. Anaerobic Digestion ....................................................................................... 5
2.1. Biogas industry: current status and challenges ....................................................................... 5
2.2. The AD process and its complexities ...................................................................................... 7
2.2.1. Factors influencing the AD process ............................................................................................. 10
2.3. Bottlenecks of anaerobic digestion ........................................................................................ 12
2.3.1. Organic loading rate ..................................................................................................................... 12
2.3.2. Retention time .............................................................................................................................. 13
2.4. Phase separation ................................................................................................................... 14
Chapter 3. Substrates for biogas production................................................................ 17
3.1. Substrate composition and its effect on AD ........................................................................... 17
3.1.1. Lignocellulosics-structural carbohydrates .................................................................................... 18
3.1.2. Textile waste-cellulose and synthetic fibers ................................................................................. 20
3.1.3. Starch-non structural carbohydrates ............................................................................................ 21
3.1.4. Organic fraction of municipal solid waste ..................................................................................... 22
3.2. Remarks on theoretical and experimental methods for determination of biogas potential ... 23
3.2.1. Theoretical methods .................................................................................................................... 23
3.2.2. Experimental methods ................................................................................................................. 25

Chapter 4. Approaching the challenge of biomass recalcitrance ................................ 27

4.1. Definition of substrate biodegradability .................................................................................. 27
4.2. Challenges with lignocellulosic recalcitrance ......................................................................... 27
4.3. Microbial strategy for lignocellulose recalcitrance: Cellulosome ........................................... 29
4.4. Goal of pretreatment .............................................................................................................. 30
4.5. Effect of pretreatment on biogas production .......................................................................... 30
4.6. Pretreatment technologies ..................................................................................................... 31
4.6.1. Physical pretreatment .................................................................................................................. 31
4.6.2. Physiochemical pretreatments ..................................................................................................... 31
4.6.3. Biological pretreatment ................................................................................................................ 32
4.6.4. Chemical pretreatments ............................................................................................................... 33

Chapter 5. High-rate anaerobic treatment systems ...................................................... 39

5.1. Background and Status ......................................................................................................... 39
5.2. Upflow anaerobic sludge blanket reactor .............................................................................. 40
5.2.1. Biogranulation of microorganisms ................................................................................................ 42
5.2.2. Factors influencing anaerobic granulation.................................................................................... 43

vii
 5.2.3. Characteristics of anaerobic granules .......................................................................................... 46
5.1. Two-stage process for high-rate methane production ........................................................... 47
5.1.1. Batch process- single vs. two-stage ............................................................................................. 48
5.1.2. Two-stage semi-continuous process ............................................................................................ 51
5.1.3. Two-stage- open system vs. closed system................................................................................. 54
5.1.4. Semi-continuous process- Single vs. two stage ........................................................................... 56

Concluding Remarks ......................................................................................................... 59

Future work ........................................................................................................................ 61
Nomenclature ..................................................................................................................... 64
Acknowledgments ............................................................................................................. 65
References ......................................................................................................................... 68

viii
Chapter 1.

Introduction

The ultimate aspiration of energy conversion systems is to achieve steady energy output at the
maximum possible conversion rate. Actually, in reality this is easier said than done because of the
recalcitrance nature of the substrate, in addition to the complexity of the anaerobic digestion (AD)
process. The rate of the biogas production is a function of the biochemical processes [1]. The
presence of difficult to degrade material fractions slows down the hydrolysis rate, which in turn
limits the rate of the overall anaerobic digestion process. However, for the easily degradable
materials the methanogenesis is considered as being the rate-limiting step due to the slow growth
rate of methanogens [2, 3].

Attaining the maximum biogas yield, by complete degradation of the substrate, would require a
long retention time of the substrate inside the digester and an equally large digester size. Putting this
into practice, the choice of a system design or of an applicable retention time is often based on a
compromise between receiving the highest achievable biogas yield and having a reasonable plant
economy [4]. In this regard, the organic load is a significant operational parameter, which indicates
how much organic dry matter can be fed into the digester, per volume and time unit. Today, the
total degradation time of the solid organic waste is normally about 30 days for the biogas process.
Nevertheless, it can be even longer depending on the specific substrate and the operational
temperature [4-6]. At lower HRTs (hydraulic retention times), the risk for a washout of certain
microorganisms is high. This makes it difficult to preserve the effective number of useful
microorganisms in the system. To maintain the population of anaerobes, large reactor volumes or
higher retention times is essential [7]. Today, this problem has been solved in the wastewater
treatment systems due to the introduction of the modern ―high-rate‖ reactors, in which the HRT is
decreased dramatically, usually to less than 1 day [8, 9]. Biomass immobilization is the key factor
for the successful applications of the high-rate anaerobic systems in wastewater treatment processes
[8]. However, the drawback of this technique is that it cannot handle a higher total solid content;

1
hence, only dissolved or soluble materials can be used as feed, which is the reason why this
technology has been successful in the wastewater treatment processes [10, 11]. On the other hand,
for the utilization of substrates with a high solid content, it is mandatory to divide the process into
two stages in order to take advantage of the high-rate reactors. Two-stage processes are divided into
two steps in order to optimize the conditions for different groups of microorganisms that are active
in the digestion process. The first step is a hydrolysis reactor, and the conditions are optimized there
to get the solid matter to be solubilized, while in the second step a high-rate reactor is used to
convert the solubilized material into biogas [11]. However, it should be mentioned that regardless of
the process configuration used, the rate of the biogas production will largely depend on the
composition of the substrate, and particularly, on its biodegradability. The hydrolysis of difficult to
degrade substrate fractions is one of the challenges the biogas industry is facing today. Although
materials, such as lignocelluloses, are available in large amounts and receive special attention for
utilization in the biogas production, they are prone to slow degradation; hence, they require some
kind of pretreatment to increase their degradation rate.

In this thesis, the potential of using an organic solvent N-methylmorpholine-N-oxide (NMMO) for
the pretreatment is studied in order to deal with the reluctant nature of lignocellulose- and cellulose-
based substrates and to increase the rate of hydrolysis during the following anaerobic digestion
process. The second part of this thesis focuses on a two-stage process and investigates the
performance at various organic loading rates and hydraulic retention times. For this propose, a wide
variety of substrates with a high total solid content and different degradability was used. The
following studies were performed:

o The effects of an organic solvent called N-methylmorpholine-N-oxide (NMMO)

used for the pretreatment of the straw fraction from the manure and forest residues
were evaluated by measuring the biogas potential during the following anaerobic
digestion process (paper I and II).
o The long-term effects of the best pretreatment conditions used for the forest residues
determined by batch digestion assays were also examined in a semi-continuous
anaerobic digestion system (paper II).
o Application of the NMMO–pretreatment on cellulose-based textile waste and their
subsequent digestion in a high-rate two-stage anaerobic digestion process was
examined at various organic loading rates and hydraulic retention times (paper III).
o The effect of effluent recirculation in a two-stage anaerobic process using
carbohydrate-based starch and cotton as the substrate at various organic loading rates
and hydraulic retention times was evaluated (paper IV).
2
o The effect of an organic loading rate and hydraulic retention time comparing single
stage and two stage processes using municipal solid waste and food processing waste
was evaluated (paper V).

3
4
Chapter 2.

Anaerobic Digestion

2.1. Biogas industry: current status and challenges

There is a variety of waste produced by human activities, and the amount of waste generated is on
the rise [12]. Anaerobic digestion of organic waste is of increasing interest as it offers an
opportunity to deal with some of the problems regarding the reduction of the amount of organic
waste, while diminishing the environmental impact and facilitating a sustainable development of the
energy supply [3, 13]. Long-term successful practice and understanding have made anaerobic
digestion to be one of the favorite treatment technologies for the organic fraction of MSW, applying
a range of technological approaches and systems [14].

Anaerobic digestion technology has been developed in the last 20 years. With a total of 244 plants
and a capacity of nearly 8 million tons of organic treatment capacity, anaerobic digestion is already
taking care of about 25% of the biological treatment in Europe [14]. By the year 2015, the
Netherlands and Belgium are expected to convert 80% of the composting plants into anaerobic
digestion as the primary treatment technology [14].

In comparison to other biofuels, in biogas production a wide range of substrates can be utilized as
long as they are biodegradable, which is one of the great advantages [13]. AD systems are
employed in a wide variety of wastewater treatment plants for sludge degradation and stabilization,
and are used in highly engineered anaerobic digesters to treat high-strength industrial and food
processing wastewaters before discharge. In addition, there are many cases of AD systems applied
in the agricultural sector at animal feeding operations and dairies to alleviate some of the impacts of
manure and for energy production [15]. The majority of these AD systems in operation are single
stage. The European market has shown a large inclination toward single-stage over two-stage
digesters [15]. The number of plants treating MSW using two-phase digestion has continued to

5
decline since the beginning of the 90s. It is predicted that no change is expected in this trend,
mainly due to the higher investment and operating costs of running two-stage processes [14]. There
are studies arguing that two-stage anaerobic digestion could provide great advantages over the
single-stage digestion due to a more rapid and more stable treatment achieved [16]. In practice,
however, it is argued that the two-stage digestion has not been able to validate its claimed
advantages in the market, and the added benefits in increasing the rate of hydrolysis and
methanization have not been confirmed [17]. Industrial applications, therefore, have displayed little
acceptance for the two-stage systems so far [18].

Anaerobic digestion systems are often appropriate for all wastewater treatment systems, given that
the solids can be introduced to the system at an acceptable concentration, which includes new
installations as well as retrofits. In fact, a great deal of the existing research on anaerobic digestion
is aimed at retrofitting multi-stage systems into facilities where single-stage processes are already
present. The most important factor in determining whether a multi-stage anaerobic digestion
process is achievable for a system is the concentration of the feed solids. Given that a multi-stage
process could be sensitive to variation in the feed solids, it might not be practicable if the
characteristics of the feed solids concentrations fluctuate extensively [19].

One cumulative Two cumulative Two stage % Single stage %

6000 100%
90%
5000
Cumulative (Kton/year)

80%
4000 70%
60%
3000 50%
40%
2000 30%
1000 20%
10%
0 0%
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010

Figure 1. Outline of the development and ratio of 1-phase and 2-phase digestion capacity in Europe. Adapted from [20,
21]

Figure 1 illustrates an overview of the development and the ratio of the one-phase and two-phase
digestion capacity in Europe, respectively. As noticeable, the vast majority is one-phase system [14,
22].

6
In order to get an overview of the status of anaerobic digestion of the organic fraction of MSW in
Europe, taking into account a wide variety of criteria, a quantitative analysis was performed on the
installed annual capacity up to the year 2014 [14]. It was estimated that the cumulative percentage
of the one-phase processes would add up to 93%, with only 7% remaining for the two-phase
capacity installed in 2014 [14].

Nonetheless, the future role of biogas in Europe is based on the availability of the substrates. The
technology development concerning biofuel production has opened up a larger substrate supply
base. On the other hand, for the same substrate, it generates more rivalry with the other related
technologies [23]. There is an abundant availability of cellulose-based waste, which could be
appropriate for biogas production e.g., lignocelluloses and waste textiles. These materials are
carbohydrate-rich and could be used as a substrate for biogas production. However, the reluctant
nature of these substrates makes them very difficult to digest, as their structure opposes microbial
hydrolysis in biogas production [12, 24]. Today, the application of lignocellulosic materials in
biogas production is limited and for waste textiles, it is nonexistent [12, 24].

The main goal of this thesis is to increase the rate of the biogas production as well as to investigate
the possibilities of difficult-to-degrade cellulose-based materials, utilized as a substrate for the
biogas production. In order to achieve this goal, one must first overcome the difficulties of the
degradation by using a pretreatment to make the material available for the following microbial
degradation, which was the focus in the first part of this thesis. Furthermore, the extent of the
increase in the organic loading and the decrease in the retention time while developing a two-stage
process, utilizing different waste fractions including cellulose-based materials, was evaluated in the
second part of this work.

2.2. The AD process and its complexities

Anaerobic digestion is often considered to be a complex process. The digestion itself is based on a
reduction process consisting of a number of biochemical reactions taking place under anoxic
conditions. By the actions of a variety of anaerobic and facultative anaerobic microorganisms, multi
molecular organic substances are degraded into simpler, chemically stabilized compounds, and the
final products are primarily methane and carbon dioxide and some smaller amounts of other gases,
such as hydrogen sulfide, hydrogen, carbon monoxide, nitrogen, ammonia NH3, and water [25, 26].
These reactions can be divided into four phases of degradation: hydrolysis, acidogenesis,

7
acetogenesis, and methanogenesis [25]. The AD process involves four fundamental steps, as
outlined in Figure 2. The individual phases are carried out in parallel; however, in each phase
different groups of microorganisms are involved, which partially stand in a syntrophic relation to
each other, with dissimilar requirements on the environment. Normally, the first and the second
phase are closely linked to each other while the third phase is closely connected to the fourth phase
[27].

Due to the small amount of energy available in methanogenic conversion, the microorganisms are
compelled to be part of a very complex, well-organized and efficient cooperation, which could be
the primary reason that this step is the final step to occur in the anaerobic digestion process [28].
The mutual reliance of the partner bacteria regarding energy limitations can go so far that neither
group of microorganisms can function without the other and that together they show a metabolic
activity that neither group could carry out on its own. This type of cooperation is called syntrophic
relationship [28]. Syntrophism is a special case of symbiotic collaboration between two
metabolically different types of microorganisms relying on each other, often for energetic reasons in
order to degrade a certain substrate. The term was created to express the close interrelation of fatty
acid oxidizing, fermenting bacteria with hydrogen oxidizing methanogens [28].

Hydrolysis / acidogenesis process

Undissolved compounds like carbohydrates, proteins, and fats are degraded into monomers, which
usually are water-soluble fragments, by exoenzymes. The microorganisms involved are facultative
and obligatorily anaerobic bacteria. In this phase, the covalent bonds are broken down with water
in a chemical reaction. The monomers produced in the hydrolytic phase are taken up by different
facultative and obligatorily anaerobic bacteria and are degraded further into short-chain organic
acids, such as butyric acid, propionic acid, acetic acid, alcohols, hydrogen, and carbon dioxide. The
concentration of the hydrogen formed as an intermediate product in this stage influences the type of
final products produced during the fermentation process. For example, if the partial pressure of the
hydrogen were too high, it would decrease the amount of reduced compounds (e.g., acetate). In
general, during this phase, simple sugars, fatty acids, and amino acids are converted into organic
acids and alcohols [29].

Acetogenesis /Methanogenesis
The products produced in the acidogenic phase are consumed as substrates for the other
microorganisms, active in the third phase. In the third phase, also called acetogenic phase, anaerobic

8
oxidations are performed. It is important that the organisms, which carry out the anaerobic
oxidation reactions, collaborate with the next group, the methane forming microorganisms. This
collaboration depends on the partial pressure of the hydrogen present in the system. Under
anaerobic oxidation, protons are used as the final electron acceptors, which lead to the production of
H2. However, these oxidation reactions can only occur if the partial pressure of H2 is low, which
explains why the collaboration with the methanogens is very important, since they will
continuously consume the H2 to produce methane. Hence, during this symbiotic relationship ―inter-
species hydrogen transfer‖ occurs [5, 27, 28, 30].

In the fourth phase, or the methanogenic phase, the methane is formed under strict anaerobic
conditions. These reactions are exergonic. The most important substrates for these microorganisms
are H2, CO2, and acetic acid. The methanogenic microorganisms can be divided into three main
groups:

(1) Acetoclastic methanogenesis Acetate → CH4 + CO2

(2) Hydogenotrophic methanogenesis H2 + CO2 → CH4

(3) Methylotrophic methanogenesis Methanol→ CH4 + H2O

Complex organic matter

100%

Biodegradable organic matter

Proteins Carbohydrates Lipids
Hydrolysis 21% 40% 5% 21%

Amino acids,
Sugars Fatty Acids
Acidogenesis
46% 20% 0% 34%

Intermediate products
Propionate, Butyrate, etc.
20%
12% 8%
35% 11% 23%
11%
Acetogenesis
Acetate H2, CO2

Methanogenesis Rate-limiting step 30%

70%

Methane, CO2

Figure 2. Schematic diagram of the anaerobic digestion process. Adapted from [31]
9
 2.2.1. Factors influencing the AD process

As is the case for all biological processes, the steadiness of the living conditions is of great
importance. Factors that affect the anaerobic digestion could be physical, chemical, or biological.
An alteration in the temperature, the composition, and/or amounts of substrates can have fatal
consequences for the gas production. The microbial metabolism processes are reliant on many
parameters. In order to achieve optimal conditions for the degradation process, apart from the
organic loading rate and the hydraulic retention time, various other parameters ought to be
considered and controlled. Given that the environmental requirements of the fermentative bacteria
vary from those of the methane forming microorganisms, the only way that the optimum
environmental conditions for all microorganisms involved can be achieved is in a two-stage system,
i.e., one stage for hydrolysis/acidification and one stage for acetogenesis/methanogenesis [27].
However, if the complete degradation process has to happen in the same reaction system (one-
phase), the requirements for methanogenesis must be prioritized, if not, it would be tough for the
methanogens to continue to be active within the mixed culture, due to their lower growth rate and
higher sensitivity to environmental factors.

Temperature
The time-span of the fermentation period is dependent on the temperature. The temperature of the
digester, even a few degrees, has an effect on nearly all the biological activities, especially on the
methane-forming archaea. The majority of the methane formers are active at two temperature
ranges: mesophilic range (30–35 °C) and the thermophilic range (50–60 °C) [27].
The methanogens are very responsive to thermal fluctuations. Thus, any rapid alterations in the
operating temperature should be avoided. In comparison to the psychrophilic and mesophilic
ranges, the thermophilic operation offers a shorter degradation time, better pathogens reduction,
higher gas production, and enhanced sludge separation. The drawback is that it is more difficult to
control the process [29]. The experiments in papers I and II were performed both at thermophilic
and mesophilic conditions, respectively. The operational temperature in the two-stage continuous
process, investigated in papers III, IV, and V, was in the thermophilic range in the first stage while
the second phase was under mesophilic conditions.

pH
pH is an important parameter in the AD process. It has an extensive influence on the performance
and growth of the various microorganisms involved in the different stages of the process [32, 33].
The pH of the digester can be maintained at a desired range (7.0–8.5) by feeding the system at an

10
optimal organic loading rate (OLR). A pH outside this range could cause disturbances to the system
by affecting most of the microorganisms including the methanogens. The pH of the system relies on
the rate of the intermediates formed (e.g., volatile fatty acids) during fermentation. Upon starting up
a biogas process, the pH in the digester can drop below 6.0 due to the production of volatile acids
during the first degradation steps. However, as methane-forming microorganisms consume the
volatile acids, the pH of the digester increases and then stabilizes [5, 34].

Volatile fatty acid

Volatile fatty acids (VFAs) are important intermediates of the anaerobic digestion process. They
exist in two forms: undissociated and dissociated. The dissociated form takes over at a high pH
level, whereas the undissociated fraction dominates at a lower pH [27]. An increase in the VFAs
leads to a drop in the pH; hence, the undissociated form of VFAs (free fatty acids) will dominate,
which in turn will inhibit the methanogenesis [27, 35]. Apart from the pH-value, the amount of
VFAs therefore is commonly used as an indicator of the performance of anaerobic digesters. It
should be noted that the level of inhibition of total VFA and individual VFAs differ from each other
[36, 37]. In order to monitor the stability of the process in papers II, III, IV, and V, the total volatile
fatty acid concentration was monitored. In paper V, the effect of the individual acid was analyzed as
well.

C/N ratio and ammonia

A C/N ratio in the range of 20 to 30 is considered to be an optimum level for anaerobic digestion
[32]. If the C/N ratio is too high, microorganisms will quickly consume the nitrogen in order to
meet their protein requirements and will no longer take care of the available carbon content of the
material, which would accordingly decrease the gas production. Conversely, if the C/N ratio is too
low, due to the degradation of the proteins and other nitrogenous materials, nitrogen will be
released and build up in the form of ammonium ion (NH4+) or ammonia (NH3) in the system [30,
38]. The chemical equilibrium between the ammonium and the ammonia is controlled by the
temperature and the pH. An increase in the temperature or the pH would shift this equilibrium more
toward NH3. The free ammonia could be a source of inhibition as it is capable of diffusing into the
cell, causing proton imbalance or leading to a potassium loss [39]. Moreover, it should be noted that
microorganisms are capable of adapting to higher levels [27]. The C/N ratio can be adjusted by
feeding the digester with a proper substrate mixture [30, 38]. In papers III and IV, NH4Cl was
added as an ammonium supplement to keep the C/N ratio at 25. In papers III, IV, and V the
concentration of ammonium was monitored.

11
Substrate
The biogas yield and composition are directly affected by the composition of the feed materials
with respect to carbohydrate, fat, and protein contents [5]. Moreover, physical and chemical
characteristics of the substrate used such as pH, moisture content, total and volatile solids (VS),
particle size, and biodegradability play a considerable role in the anaerobic digestion process.

2.3. Bottlenecks of anaerobic digestion

Controlling the anaerobic digestion process is a complicated task. Because of the complex mixed
microbial and substrate spectrum, advanced studies and development are necessary to eliminate
various bottlenecks in the degradation chain. However, practical experience shows that there are
several factors that can be attributed to the process failures in anaerobic digestion. These factors
include: microbiological limitations, affecting automatically the microbial community (e.g.,
ammonia inhibition, trace element insufficiency, etc.) or technical weaknesses of the equipment,
such as insufficient mixing caused by the inappropriate particle size or rheological limitations [40].

For a balanced and stable process, the reaction rate in both stages must be equivalent. If the rate of
the degradation in the first stage is too fast, the concentration of the acids increases, causing an
inhibition of methanogenic microorganisms in the second phase. On the other hand, if the second
phase runs too fast, the production rate of methane becomes limited by the hydrolytic stage [41].
Other bottlenecks related to the process performance include extended reactor start-up times and
process instability, as a result of the slow growth rates and sensitivity to changes in the
environmental conditions of the microorganisms involved in the process. Hence, monitoring the
process by measurements aiming to attain and maintain effective and robust microbial communities
are considered necessary to guarantee stable performance with high efficiencies [42]

2.3.1. Organic loading rate

Organic loading rate (OLR) is defined as the amount of substrate expressed as e.g., total solids (TS),
volatile solids (VS) or chemical oxygen demand (COD) fed to the system per unit volume per unit
time. It is a helpful criterion used for measuring the biological performance of the AD system [18],
since it is very sensitive to the organic loading rate (OLR) and the waste composition [43, 44].

12
It is well-known that easily degradable substrates can be quickly converted into volatile fatty acids
(VFA), which can cause the inhibition of methanogenesis as a consequence of the rapid hydrolysis
rate and accumulation of VFAs. At high OLRs, there is a risk for overloading the system/reactor,
particularly during the period of reactor start-up. In such cases, the feeding rate to the system should
be reduced [45, 46]. Higher OLRs can permit smaller reactor volumes, thus, reducing the capital
cost.

2.3.2. Retention time

Another parameter that basically controls the rate of the substrate conversion into biogas is the
retention time [18]. It is an important parameter in terms of evaluating the conversion efficiency in
the process. Normally, shorter retention times are desired in order to reduce the system costs [30].
The retention time is usually expressed as: the hydraulic retention time (HRT), which states the
approximate time that the liquid sludge remains in the digester, and the solid retention time (SRT),
which is the time that the microorganisms /solids spend in the digester [47]. In general, HRT is
more important if the substrate is complex and slowly degradable, whereas SRT is significant for
easily degradable biomass [13]. In addition, at high OLRs, the retention times should be long
enough for the microorganisms to be able to utilize the substrate. Thus, there is a balance between
the OLR and HRT that must be determined in order to optimize the digestion efficiency and reactor
volume [48].

The different steps in the digestion process are directly connected to the SRT. Reducing the SRT
would decrease the extent of the reactions and vice versa. Whenever the sludge (mixture of biomass
solids and water) is removed from the digester, a portion of the bacterial population is also removed
[49]. Since methanogenic microorganisms have a significantly longer generation time compared to
hydrolytic and acid forming microorganisms, shorter HRTs would cause a washout of slow growing
biomass from the system, which would ultimately jeopardize the process stability and decrease the
conversion efficiency of the process [30]. Therefore, in order to avoid process failure and keep a
steady state condition, the rate of the cell growth must at least compensate the rate of the cell
removal [47, 49]. In general, the hydraulic retention times must be at least 10 – 15 days to avoid
washout from the system [27]. However, the risk for a washout of the microorganisms from the
system can be prevented with phase separation. In order to reach high cell densities of the slow
growing methanogenic microorganisms, the hydraulic and solid retention time should be uncoupled
in the second stage, and it is necessary to raise the solid content in the methanogenic reactor [18]. In
this way, the digestion rate can be increased for a given substrate and reactor volume, and the
13
conversion to methane can be achieved at shorter HRTs. Consequently, a greater amount of
substrate can be converted into methane in a given period of time, thus, increasing the productivity
[30].

2.4. Phase separation

Generally, in an anaerobic digestion process, the rate-limiting step can be defined as the step that
causes process failure under imposed kinetic stress. In other words, in a context of a continuous
culture, kinetic stress is defined as the imposition of a constantly reducing value of the SRT until it
is lower than its limiting value; hence, it will result in a washout of the microorganism [50].

The AD process can be divided into two phases as illustrated in Figure 3. The microorganisms
carrying out the degradation reactions in each of these phases differ widely regarding physiology,
nutritional needs, growth kinetics, and sensitivity to environment. Very often, it is difficult to keep a
delicate balance between these two groups: the acid forming and the methane forming
microorganisms, which lead to reactor instability and consequently low methane yield [51]. Poland
and Gosh [17] were the first to propose that two main groups of microorganisms could physically
be separated with the intention of making use of the difference in their growth kinetics. In order to
accomplish phase separation, several techniques have been employed such as membrane separation,
kinetic control, and pH control [52-56].

Suspended Dissolved Organic

Acetate Methane
Solids Solids Acids

Acid phase Gas (Methane) Phase

Liquification Acidification Acetification Methane Formation

Figure 3. Phase separation of the anaerobic digestion system. Adapted from [19]

A two-phase process allows for the selection and enrichment of the microorganisms corresponding
to each of the phases independently from each other. Thus, the first phase can operate at optimal
conditions for the growth of hydrolytic and acidogenic microorganisms, while the second phase can
be optimized for the acetate and methane formation [57].

14
The two-phase process has numerous potential advantages. First of all, it allows for a decrease in
the total reactor volume. Another advantage is the appropriate control of the acidification, which
improves the stability owing to the more heterogeneous bacterial population. The process would
tolerate organic and hydraulic overloading and fluctuations, as the first-phase will function as a
metabolic buffer. Toxic materials and substances that can affect the more sensitive methanogenic
microorganisms will possibly also be eliminated in the first phase [58]. Moreover, fast growing
acidogenic microorganisms may be disposed of, thus, avoiding the loss /washout of the
methanogens.

15
16
Chapter 3.

Substrates for biogas production

3.1. Substrate composition and its effect on AD

The substrate composition is extremely important for the microorganisms in the AD process, as it
affects the process stability, gas production, and composition. The substrate should meet the
nutritional requirements of the microorganisms, regarding the energy sources and various
components, vital for building new cells. The substrate should also include a wide variety of
components necessary for the activity of microbial enzyme systems, such as trace elements and
vitamins. When it comes to the decomposition of organic material in the AD process, the ratio of
carbon to nitrogen (C/N ratio) is also regarded to be of great importance [59]. Therefore, the
performance of the AD process is shown to be enhanced by using substrates from different sources
and with the right proportions. Investigations show that co-digestion of substrates from different
sources produce more gas than predicted compared to gas production from the individual substrates
[33, 60, 61]. Substrates that are complex and not too homogeneous encourage the growth of the
numerous types of microorganisms in the digester. A continuous process that is fed with a uniform
composition of substrate, for instance, carbohydrates, for a longer period will lead to a buildup of a
consortium of microorganisms, which will find it difficult to digest the proteins and fat, since most
of the organisms that had the ability to break down the fat and proteins have been washed out from
the process. Therefore, feeding the reactor with a diverse substrate is advantageous, as it amplifies
the build-up of diverse microbiological composition, hence, resulting in the possibility of a stable
and robust process [5].

17
 3.1.1. Lignocellulosics-structural carbohydrates

Lignocellulose is the most abundant renewable biomass worldwide [62] with an estimated annual
production between 10–50 billion tons [63]. Since both the cellulose and hemicelluloses are
polymers of sugars, they are potential sources of fermentable sugars. While the hemicelluloses can
be readily hydrolyzed, the cellulose fraction is more unwilling toward the hydrolysis due to the
presence of a lignin shield as well as its crystallinity. A more rigorous pretreatment, therefore, is
required to access the sugars [62]. Consequently, various pretreatment methods have been used to
improve the rate of the hydrolysis of lignocelluloses toward biogas production [24].

Lignocellulosic waste is produced by several sectors including industries, forestry, agriculture, and
municipalities [64]. A large fraction of animal manures consist of straw, which is used as bedding
material in animal cultivation. Straw is a lignocellulosic material; therefore, it makes these kinds of
manure fractions difficult to degrade. A pretreatment is needed to improve the rate as well as the
degree of enzymatic hydrolysis during the degradation process.

Forest residues, another example of lignocellulosic waste, have a potential for energy production.
Forest residues are the biomass material remaining in the forests that have been harvested for
timber, and are more or less identical in composition to forest thinning [65]. Forest residuals consist
of tops and branches, needles, bark, roots, logging residues, etc. It is estimated that in Sweden forest
residues have the energy potential of between 49–59 TWh/year [24]. Today, a major part of these
residues are not used for biogas production due to a high lignin content, which makes it hard to
digest.

In this thesis the effect of the NMMO-pretreatment on the straw fraction of manure (paper I) and
forest residues (paper II) and its subsequent effect on the hydrolysis and biogas production were
investigated.

Cellulose
Cellulose is the main structural component of plant cell walls. Typically, a plant cell wall consists
of up to 35 to 50% cellulose [66], which is a linear polysaccharide polymer of glucose molecule
linked together through β-1,4 glucosidic bonds. The character of the β-1, 4 glucosidic bonds allows
the polymer to build long and straight chains. The level of polymerization of the cellulose, which
refers to the number of glucose units making up one polymer molecule, can range from 800–10,000
units [67]. Cellulose occur in two forms: an unorganized amorphous form and an organized
crystalline form. Within the cell wall, however, the crystalline form of cellulose dominates, which
are less vulnerable to enzymatic degradation than the amorphous cellulose [30]. In crystalline
18
that are removed in order to achieve a high quality fiber for textile manufacturing, which means that
the cotton fibers in the waste textile can be considered to be more or less pure cellulose [12]. Still,
after extensive research, the use of cellulose as a platform for industrial production for different by
products has failed. The challenge of using cellulose is that it is highly crystalline, which opposes
microbial degradation, and a cost effective pretreatment method to overcome the crystallinity has up
to now been elusive. Jeihanipour et al. [81] showed the possibility for hydrolyzing the cotton using
enzymes or acids to achieve glucose and subsequently utilize it as a carbon source in the ethanol
fermentation.

Cotton based blue jeans is a textile waste, basically made of pure cellulose. In this thesis, the
possibility of using pure cotton and blue jeans with and without pretreatment as a substrate in two-
stage semi-continuous high-rate biogas production was investigated in papers III and IV.

3.1.3. Starch-non structural carbohydrates

Apart from sugars, starch is one of the most commonly found non-structural carbohydrates in
anaerobic digesters. Starch is present in food, coming mainly from grains, such as corn and wheat,
and tubers, such as yam and cassava. Starch comprises of two primary biopolymers: amylose,
which is a linear chain of α- 1,4-linked D-glucose units, and amylopectin, which is a chain of α-1,4-
linked D-glucose with branches of α-1,6-linked D-glucose [82]. Starch, which is partially water
soluble, is the primary polysaccharide for storing energy in higher plants. Some forms of starches
are insoluble and resistant to degradation (e.g., wheat breads), whereas others are partially
bioavailable [83]. In this thesis, pure starch was used as an easily degradable substrate to compare
with cotton in order to evaluate the semi-continuous two-stage process (paper IV).

Given that carbohydrates vary in their nature, they are degraded at different rates in the AD process.
Simple sugars and disaccharides are broken down easily and rapidly; this might appear
advantageous, but it can cause instability problems as a result of the accumulation of fatty acids as
intermediary degradation products [84-86]. In addition, carbohydrate-rich materials used to have
poor buffering capacity, and there is a risk of process instability due to a decrease in the alkalinity
of the system [51].

21
 3.1.4. Organic fraction of municipal solid waste

Municipal solid waste (MSW) is the waste generated from residential sources, for instance,
households and from institutional and commercial sources such as offices, schools, hotels, and other
sources. The main components of MSW are food, garden waste, paper, board, plastic, textile, metal,
and glass waste [87]. The global production of municipal solid waste (MSW) reached
1.3 billion tons /year in 2010, and it is predicted to increase to more than 2 billion tons/ year by
2025 [88, 89]. The disposal of this increasing volume of waste in a sustainable manner is a major
challenge. It is estimated that the major fraction of the global municipal solid waste consists of food
waste [88]. The application of the anaerobic digestion for the treatment of the organic fraction of
municipal solid waste (OFMSW) has been of interest because of its high content of fats /lipids and
proteins. The main obstacle in the treatment of this type of organic waste is its conversion, due to
the complexity of the organic material [90, 91].

Fats are a major part of the OFMSW and food processing waste (FPW). There are numerous
different lipids (fats, oils, greases), with a varying composition depending on their origin. Lipids are
distinguished by the length of their fatty acid chain, extent of chemical saturation, which refers to
the number of double bonds, and also their physical state, i.e., liquid or solid. Fats are classified as
saturated (found in meat and dairy products), monounsaturated (in vegetable oils and nuts), or
polyunsaturated fats (in fish and corn oil). Saturated fats are less biodegradable than unsaturated
fats. Triglycerides, the most common type of fat, are primarily hydrolyzed into glycerol and long
chain fatty acids (LCFAs) in the AD process[5, 92]. The degradation of fats is generally both easy
and fast [93]. However, while glycerol is rapidly converted into acetate by acidogenesis, the
degradation of LCFA is more complicated. The inhibitory effect of fats is usually connected to the
LCFAs [93, 94]. Fats are a very promising substrate for anaerobic digestion, since high methane
yields can be achieved.

Proteins are present in many organic materials such as OFMSW and FPW, which are rich in energy
and produce a relatively high amount of methane in the AD process. Proteins are linear polymers,
consisting of a string of subunits called amino acids. Proteins are primarily hydrolyzed into
individual amino acids or peptides by the action of an extracellular enzyme called protease [50].
Amino acids are then further broken down to amine groups while releasing ammonia (NH3) or
ammonium (NH4+) in the process. Ammonia and ammonium are in balance with each other, and the
form that would be present in the AD process is dependent on the pH and the temperature. At high
concentrations, ammonia (NH3) could cause inhibition in the AD process, as it can be lethal to
many microorganisms. Methane-producing archaea is the first to become inhibited, as the

22
concentration of ammonia begins to increase [86, 95, 96]. How this inhibition happens is not
completely understood yet. There are hypotheses that ammonia, as an uncharged compound, is
capable of entering the cell and changing the pH inside the cell leading to cell disruption [95]. The
rate and extent of protein degradation is dependent on many factors such as solubility, the category
of end group, pH, and tertiary structure. In general, the rate of protein hydrolysis under an anaerobic
environment is slower than the hydrolysis rate of carbohydrates [50, 97]. In this thesis, OFMSW
and waste from the FPW have been used as a substrate in a high-rate two-stage biogas production
system. Rapid processing was achieved by increasing the loading rate and decreasing the digestion
time (paper V).

3.2. Remarks on theoretical and experimental methods for

determination of biogas potential

3.2.1. Theoretical methods

Biogas production from the organic substrates engages internal redox reactions that convert organic
molecules into CH4 and CO2. The fraction of these two gases are defined by the composition as well
as the biodegradability of the substrates [98].

During the conversion of the carbohydrates, such as sugars, starch, or cellulose, an equal amount of
CH4 and CO2 is produced [27]:

C6H12O6  3 CH4+ 3 CO2 (1)

For proteins, the process can be described as follows:

C13H25O7N3S + 6 H2O  6.5 CH4 + 6.5 CO2 + 3 NH3+ H2S (2)

The degradation of fats and vegetable oils (triglycerides) can be summarized by the following
equation:

C12H24O6 + 3 H2O  7.5 CH4 + 4.5 CO2 (3)

The ideal stoichiometry, for a two-phase digestion, considering the simple case of carbohydrate
degradation, can theoretically be defined as follows:

First stage: C6H12O6 + 2 H2O  4 H2 + 2 C2H4O2 (acetic acid) + 2 CO2 (4)

23
Second stage: 2 C2H4O2  2 CH4 + 2 CO2 (5)

4 H2+CO2  CH4 + 2 H2O (6)

With the remaining sugars present in the substrate being converted into a more reduced form of
products such as propionic acid, butyric acid, ethanol, etc.:

First stage: C6H12O6  C4H8O2 (butyric acid) + 2 CO2 + 2 H2 (7)

Second stage: C4H8O2 + H2O  2.5 CH4 + 1.5 CO2 (8)

These simplified examples can vary according to the effects of numerous factors [27, 98]. For
instance, the reactions are often not complete e.g., up to half of the cellulose is refractory to
microbial degradation, and lignin is entirely inert. Part of the substrates is utilized by the
microorganisms for growth; consequently, there is also some biomass produced.

The theoretical methane potential in practice can be determined, for instance, by using the elemental
composition (C,H,O,S,N) and the Buswell formula (papers III and IV):

CcHhOoNnSs+ yH2O xCH4 + nNH3 + sH2S+ (c-x) CO2 (9)

Where: x= 1/8 (4c+h-2o-3n-2s)

The component composition e.g., carbohydrate, fat, and protein content of the substrate (papers I
and V) can also be used for the calculations according to the data presented in Table 1 below:

Table .1 Buswell’s formula for theoretical methane potential

Component Chemical formula Theoretical methane yield

(m3CH4 /kg VS)
Carbohydrates C6H10O5 0.42
Lipids/fats C57H104O6 0.50
Proteins C5H7O2N 1.01

For liquid substrates, such as wastewater, with low particulate organic content, the chemical oxygen
demand (COD) is followed by:

CH4 + 2 O2 CO2 + H2O (10)

24
One mole of CH4 needs two moles of O2 to oxidize carbon to carbon dioxide and water. Thus, one
kilogram COD is equal to 0.35 m3 CH4.

All these methods presented above are based on the assumption that the substrate is completely
degraded, and the utilization of the substrate for microbial growth is negligible [13, 27, 99].

3.2.2. Experimental methods

Theoretical methods assume complete degradation of the organic material. However, in practice the
actual digestibility is lower, which means that the calculated methane potential is usually higher
than the measured methane potential. The reason for that is that several factors, such as the presence
of inhibitors, the lack of a growth factor, nutrients, and suboptimal conditions during the actual
digestion process will limit the degradation and the biogas production will not reach its theoretically
calculated potential. Therefore, a digestion test of a substrate should be performed as a tool for the
actual biogas potential. Digestion tests can be performed in different scales and modes. In this
thesis, all the experiments were done in a lab scale. In batch single-stage and batch two-stage mode,
the biogas potential of the untreated and the pretreated materials were tested; in semi-continuous
single-stage and two-stage modes, the long-term effects of the digestion process were evaluated.

Batch digestion
Batch digestion assay is a method that is normally used for determining the methane potential and
for kinetic evaluation of the substrate. The substrate and the inoculums (different ratios) are placed
in the reactor, which is sealed thereafter. To provide an anaerobic environment, the head space is
flushed by a mixture of carbon dioxide and nitrogen and the reactor is then placed in an incubator at
a temperature depending on the inoculums optimum. Normally, these kinds of assays take 50 days
or more, to make sure there is a complete degradation of the substrate, as the anaerobic digestion is
a slow process [13]. Usually, many tests are performed in parallel with different substrates or to
compare different pretreatment methods and conditions.

Semi-continuous digestion
Semi-continuous systems are used to study the performance and stability of the anaerobic digesters,
where microbial populations are adapted for specific substrates; hence, product inhibition can be
accurately assessed over a long-term period with daily supervision. The testing period of the semi-
continuous process is usually several months. CSTR (Continuously stirred tank reactor) is the
extensively used technology in the lab as well as large-scale processes. In a single-stage semi-
25
continuous process, only a CSTR with a retention time between 10 – 50 days is used in order to
avoid washout of the slow growing microbial population[13]. In two-stage, the process is divided
into two parts: the first step digestion tank (CSTR) where the process is focused on hydrolysis and
fermentation. However, biogas is normally also produced, since a complete separation is not always
accomplished. In the second step, the fermentation and hydrolysis products are transferred to
another digestion tank that is adapted for methanogenesis. The high-rate reactors can be utilized in
the second step.
In this thesis, the CSTR reactors were used in the single-stage experiments (paper II); however, for
the two-stage experiments, the CSTR reactors were used in the first stage where fermentation and
hydrolysis occur while a high-rate upflow anaerobic sludge blanket (UASB) reactor using
granulated sludge was used in the second stage for methanogenesis. The two configurations, one
with effluent recirculation from the UASB to the CSTR (closed system) (papers III and V) and the
other without recirculation (open system), (paper IV) are illustrated in Figure 5.

Figure 5. The setup of the semi-continuous two-stage system. (Left) with recirculation (Closed system), and (Right)
without recirculation (Open system) (paper IV)

26
Chapter 4.

Approaching the challenge of biomass

recalcitrance

4.1. Definition of substrate biodegradability

Substrate biodegradability is typically described in terms of rate and degree of degradation. Rate is
the speed of substrate utilization (degradation), which under ideal and steady-state conditions in
absence of inhibition is directly related to the rate of intermediate or product(s) formation. The total
biodegradability stands for the maximum biological degradation, accomplished at solid retention
time equal to infinity. In batch conditions, the ultimate biodegradability is assumed to be attained
when the degradation rate moves towards zero, that is, the stabilization is considered to be
completed. Ultimate biodegradability of organic substrates is decided by physicochemical and
biochemical factors. The characteristics of the bio-molecules, of the influent material, and the
interaction between them, define the degree of complexity of the substrate and its surface area
accessible for enzymatic hydrolysis; therefore, constitute a physicochemical limitation for
biodegradability. Additionally, biochemical inhibition forms a significant factor that determines the
substrate biodegradability, by influencing the rate and finally the extent of any biologically-
mediated reaction taking place in the anaerobic digestion process [83].

4.2. Challenges with lignocellulosic recalcitrance

The plant cell wall offers mechanical strength, upholds the cell shape, controls cell expansion,
regulates transport, gives protection, and accumulates food reserves. Plant biomass has different
layers shielding its cellulose. Cell walls contain the cellulose microfibrils arranged together with

27
polymers, as hemicelluloses, lignin, and pectin. Cells are linked by lamellae, a lignin-rich layer.
Primary cell walls contain cellulosic microfibrils, which are randomly arranged. The secondary cell
wall is composed of three layers, including S1 (outer), S2 (middle), and S3 (inner) layers. S2 is the
thickest layer, comprising the major part of the cell wall. Additionally, the arrangements of these
layers are alternates of horizontal and vertical. S1 is arranged horizontally, while S2 is vertical
followed by S3, which is again horizontal. This arrangement is the reason for the mechanical
strength and the complexity of the plant cell walls [64, 100, 101].

Lignin is an extremely branched, hydrophobic polyphenolic aromatic compound, mainly placed in

the cell walls of vascular plants [102, 103]. Lignin provides rigidity to the plant cell walls and
resistance to biodegradation, and makes up the most significant factor restraining biodegradability
of lignocelluloses in anaerobic digestion systems [104]. Lignin is closely linked to hemicellulose,
which covers cellulose and constructs a physical barrier for hydrolytic enzymes [105]. In fact, the
biodegradability of hemicellulose is directly connected with that of cellulose and inversely related
to lignification [104, 106]. Lignin in itself is considered to be recalcitrant in anaerobic environments
[107]. However, earlier studies have revealed that the degradation of lignin is achievable under
anaerobic conditions, predominantly by the rumen microorganisms [102, 108].

Even after breaking down the lignin shield and exposing the cellulose to the microbial enzymes, a
second challenge appears, which is called cellulose crystallinity [64]. The rate-limiting step in the
hydrolysis of cellulose is not the cleavage of β-1,4 glucosidic bonds between the monomers, but
rather the interruption of a single chain of the substrate from its native crystalline matrix to facilitate
the contact with the active site of enzymes [109]. The cellulose microfibrils are twisted in their
native state. Cellulose is also able to take on a variety of crystalline forms of which two allomorphs
are most important: cellulose I and cellulose II. Cellulose II can be obtained from the cellulose I
after pretreatment, for example [110]. Cellulase enzymes easily hydrolyze the cellulose II portion
because of its lower crystallinity, making it more accessible, whereas the enzyme is not so efficient
when it comes to the crystalline part. It is, therefore, predictable that high-crystallinity cellulose will
be more opposing to enzymatic hydrolysis, and it is commonly acknowledged that decreasing the
crystallinity would enhance the digestibility of the lignocelluloses [64].

In order to improve the enzymatic hydrolysis, it is necessary to eliminate the lignin and
hemicelluloses to increase the accessible surface area of the cellulose. The first requirement for
degradation of the cellulose into simple sugars is the physical attachment of the cellulase enzymes
onto the surface of the cellulose. Thus, physical contact between the cellulytic enzyme and cellulose
is vital for enzymatic hydrolysis [64, 111].

28
Investigations show [111] that the rate of hydrolysis is generally very high at first, but decreases
later. The slowdown of the hydrolysis in the later stages is not because of the lack of available
surface area, but because of the difficulty in the hydrolysis of the crystalline part of the cellulose.
Consequently, a lower rate of hydrolysis might be expected after the hydrolysis of the amorphous
cellulose is completed [64, 111]. Accumulation of only small amounts of hydrolyzed products in the
reactor shows that the conversion of the cellulosic material into soluble products was the rate-
limiting step in the overall AD process [90].

4.3. Microbial strategy for lignocellulose recalcitrance:

Cellulosome

Degradation of cellulosic substrate in nature is accomplished by a variety of microorganisms. In

some cases, microorganisms aid higher animals e.g., ruminants, in transforming the polysaccharides
to more digestible components. Microbial degradation of cellulosic material is one of the most
significant processes in nature. Different microorganism e.g., bacteria and fungi approach the task
in their own specific ways. While aerobes normally produce copious amounts of relevant enzymes
e.g., cellulases and hemicellulases, the anaerobic microorganisms, on the other hand, are much
more frugal in their output of such enzymes. The energy yield per unit sugar hydrolyzed in the
aerobes is much higher than for anaerobes. As a result, the anaerobes tend to adopt other strategies
for degradation of the recalcitrant plant material. Among these strategies, the organization of
enzymes into cellulosome in anaerobic microorganisms is shown to be the most outstanding [112].

The cellulosome consists of an essential set of structural and some enzymatic multi-modular
components. A key non-catalytic subunit called scaffoldin locks different enzymatic subunits into a
complex, using the interaction between cohesin-dockerin. Therefore, the main scaffoldin needs a
series of functional modules, cohesins that are involved in the enzyme attachment. The scaffoldin
consists of the cellulose specific carbohydrate binding module (CBM) used for substrate targeting.
The different enzyme subunits, cellulases and hemicellulases, in particular, contain a specialized
doctrine module, which is complementary to the scaffoldin-based cohesins. The specificity of the
binding between the scaffoldin-based cohesin modules and the enzyme–borne dockerin domains
gives an idea about the supra-molecular construction of the cellulosome. These multi-enzyme
complexes anchor to the cell envelope and to the substrate, and mediate the proximity between the
cells and the cellulose. Binding to the scaffoldin stimulates the activity of each individual

29
component toward the crystalline substrate [113]. The range in cellulosome architecture among the
known cellulosome-producing microorganisms is dependent on the arrangement of their genes
[113].

4.4. Goal of pretreatment

The advantages of the pretreatment of lignocellulosic materials are widely recognized. The aim of
the pretreatment process is to get rid of the lignin and degrade the hemicellulose, decrease the level
of crystallinity in the cellulose, and enhance the porosity of the lignocellulosic materials.
Pretreatment should meet some important requirements [69]:

(1) Enhance the formation of sugars or facilitate the hydrolysis process after the pretreatment

(2) Avoid the degradation or loss of carbohydrates

(3) Not cause the formation of by products that could possibly give rise to inhibition in the
subsequent hydrolysis and fermentation processes

(4) Be economical

4.5. Effect of pretreatment on biogas production

It has already been mentioned that the degradation of complex materials is slow, and the AD
process is therefore usually limited by the long retention times [47]. The anaerobic digestion is rate-
limited by the hydrolysis step; pretreatment methods are therefore often used to support the
solubilization of organic matter. However, it was discovered that sometimes regardless of the high
solubilization after the pretreatment, the anaerobic conversion into methane did not improve. The
poor anaerobic biodegradability performances were accredited to the soluble molecules produced
after the pretreatment had been inhibitory to the anaerobic microorganisms [45]. There are authors
who argue that the rate of the hydrolysis of particulate organic matter is decided by the adsorption
of hydrolytic enzymes to the biodegradable surface sites [114]. Some substrates are either very
resistant against anaerobic digestion due to their compact, complex structure, or they contain
inhibitors [13, 64]. In some cases, the main idea of the pretreatment is to improve the degradation

30
rate and efficiency, as well as improve the bioavailability of the feedstock [64]. In other cases, the
goal is to eliminate the undesirable compounds such as inhibitors. Therefore, the selection of a
suitable pretreatment method should always go hand in hand with the properties of the substrate.

4.6. Pretreatment technologies

In general, pretreatment methods are divided into three distinct categories, namely, physical,
chemical, and biological pretreatments. Combination pretreatment, such as physicochemical
pretreatment by including two or more pretreatment techniques from the same or different
categories is quite common as well. However, combination pretreatment is not considered as an
individual pretreatment category [64, 115-117].

4.6.1. Physical pretreatment

The goal of physical pretreatments is to physically /mechanically decrease the particle size and
reduce the crystallinity. As a result, the accessible surface area and the pore sizes of the biomass
should be increased by these methods. Enhanced hydrolysis is achieved as crystallinity is reduced,
and mass transfer characteristics are improved due to the reduction of the particle size. Milling,
grinding, irradiation, ultrasound and hydrothermal pretreatments are some of the methods that
belong in this category [64, 118]. Forest residues (paper II), blue jeans (paper III), and cotton (paper
IV) were milled to 2 mm particle size prior to the pretreatment and digestion. The OFMSW was
crushed prior to the pretreatment (paper V).

4.6.2. Physiochemical pretreatments

In order to prevail over the recalcitrance of the lignocellulosic biomass, physiochemical

pretreatments, which combine chemical and physical processes, have emerged. The processes that
belong to this group are: ammonia fiber explosion (AFEX), CO2 explosion, SO2 explosion, steam
pretreatment/autohydrolysis, hydrothermolysis, and wet oxidation [13]. These pretreatment methods
have been successfully introduced prior to the biogas production.

31
 4.6.3. Biological pretreatment

Biological pretreatments make use of microorganisms’ (e.g., fungi) natural ability to degrade the
lignin and hemicellulose, leaving the cellulose intact [119-121]. The most studied microorganism is
white-rot fungi, which is considered to be promising due to the substrate specificity of its
ligninolytic enzymes [120]. Lignin is then degraded through the action of lignin degrading enzymes
secreted by the fungi. Although biological pretreatments involve mild conditions and are
economically beneficial, the drawbacks connected to these pretreatment methods, such as low rates
of hydrolysis and long pretreatment times, weigh against their advantages compared to other
technologies [122, 123]. However, there are studies combining the biological pretreatment with
other pretreatment technologies, as well as developing novel microorganisms for more rapid
hydrolysis [62, 118-120]. Oat straw treated with white-rot fungi during 28 days of incubation
increased the initial rate of hydrolysis during 10 days of digestion with and without a nutrient-rich
medium, while the total methane yield was slightly higher for the nutrient-rich medium after 28
days with white-rot fungi compared with those of the untreated straw (Figure 6). The total lignin
content decreased from almost 22% for the untreated oat straw to 17% for the treated straw (data
not published). The food processing waste (FPW) used in paper V was partially pretreated and pre-
hydrolyzed as well as acidified to some extent by the microorganisms present in the storage tank for
3–4 days.

RM0 RM7 RM14 RM28 PM28

0,45
0,4
0,35
Volume m3 CH4/kg VS

0,3
0,25
0,2
0,15
0,1
0,05
0
0 10 20 30 40 50 60
Days

Figure 6. Rm0=Rich medium (nutrient added) untreated, RM7=Rich medium 7 days pretreatment, RM14=Rich
medium 14 days pretreatment, RM28=Rich medium 28 days pretreatment, PM 28=Poor medium (without nutrient) 28
days pretreatment

32
 4.6.4. Chemical pretreatments

Chemical pretreatments involve chemical reactions for the disruption of the biomass structure [67].
Chemical pretreatment is comprised of alkali, dilute acid, alkaline peroxide, oxidizing agents, and
organic solvents [69]. Acid and alkaline pretreatments are the most commonly used chemical
pretreatments today. For the pretreatment of lignocellulosic material, strong acids e.g., sulfuric or
nitric acids are usually used for the removal of lignin and hemicelluloses. For the alkaline
pretreatment, sodium, potassium, calcium, and ammonium hydroxide are reused as a base causing
the degradation of the ester and glycosidic side chains, and thereby modifying the structure of the
lignin, as well as resulting in the cellulose swelling and its partial decrystallization [124]. In this
thesis, chemical pretreatment using N-methylmorpholine-N-oxide (NMMO), which is a cellulose
solvent, has been used for pretreatment prior to the biogas production (Papers I, II, and III).

NMMO- pretreatment
NMMO is a cyclic organic amine oxide, known as a non-derivatizing solvent for cellulose. It has
the ability to dissolve cellulose, while generating a solution with great rheological properties for
fiber spinning. This solvent is already commercially used in the textile industry for the production
of regenerated cellulosic fibers under different trade names, such as Tencel, Lyocell, and Newcell.
In addition to being almost recoverable as well as recyclable, NMMO is considered to be
environmentally friendly due to its non-toxicity and biodegradability potential [12, 125-127]. The
solubilization extent of cellulose in the NMMO-water mixture relies on the concentration of the
NMMO [128]. Optical microscopy investigations of the free floating fibers in the NMMO-water
mixtures using different concentrations of the NMMO showed four modes for the dissolution of
cotton fibers, depending on the concentration of the NMMO [129]:

(1) Fast dissolution by fragmentation with no major swelling (> 83% NMMO)

(2) Swelling by ballooning and dissolution (76–82% NMMO)

(3) Swelling by ballooning and partial dissolution (70–75% NMMO)

(4) Low homogenous swelling and no dissolution (below 65% NMMO)

The dissolution of cellulose entails breaking the hydrogen bonds. The crystallinity of cellulose is
also significant regarding the solubilization. The lower energy level of the crystalline form is more
difficult to dissolve than the amorphous form with a higher energy level. The reason is that the level

33
of solubility corresponds to the difference in the energy level involving the solid and the solution
state [130].

The effect of the NMMO-pretreatment on the rate of the pure cellulose solubilization in batch
anaerobic digestion has been performed earlier [131]. The swelling and ballooning mode showed a
complete degradation during 15 days of digestion. NMMO-pretreatment has been successfully
applied to lignocelluloses, aiming for improvements during the following biogas and bioethanol
production [24, 132-134]. NMMO degrades the intra-molecular hydrogen as well as van der Waals
interactions, and opens up the lignocellulosic structure and reduces its crystallinity [129];
consequently, it improves the methane and ethanol yield from different types of lignocellulosic
materials [24, 132]. Textile wastes, unlike waste from lignocellulosic materials, do not contain any
lignin or hemicelluloses but have a higher crystallinity; thus, the main goal of treating cotton-based
waste textile is simply to reduce its crystallinity [81]. Studies show that regenerated cellulose from
the NMMO-water-cellulose solution is three times more reactive in the hydrolysis reactions than the
untreated cellulose, as the result of the conversion of crystalline cellulose to amorphous cellulose
[135]. In this work, the use of the NMMO as a pretreatment method prior to the biogas production
has been applied on the straw fraction of manure (paper I), forest residues with high lignin content
(paper II) and textile wastes obtained from blue jeans (paper III).

Effect of NNMO-pretreatment on biogas production

The effect of the NMMO-pretreatment on the straw fraction of horse and cattle manure was
investigated (Paper I). The pretreatment was carried out for 5 and 15 h at 120 °C, with 85%
NMMO, and the effects were evaluated by batch digestion assays. The kinetic of the degradation
process was evaluated using the first-order kinetic model, according to Jiménez et al. [136]. The
results showed that the NMMO-pretreatment of the straw fraction of manure could improve the
degradation rate of the manure, and the specific rate constant, k0, was increased from 0.041 to 0.072
(d-1) for the cattle and from 0.071 to 0.086 (d-1) for the horse manure (Figure 7).

34
 1,0
(a)

0,8

ln [Gm/(Gm-G)]
0,6

0,4

0,2 Untreated
5h treatment
15h treatment
0,0

0 2 4 6 8 10 12 14

-0,2
Time(days)

1,2

(b)
1,0

0,8
ln[Gm/(Gm-G)]

0,6

0,4

0,2
untreated
5h treatment
15h treatment
0,0
0 2 4 6 8 10 12 14

-0,2
Time(days)

Figure 7. Variation of ln[Gm/ (Gm-G)] values for different retreatment conditions. A-cattle manure, b-horse manure. G =
ln[Gm/ (Gm-G)] =k0t. Where G (ml) is the volume of methane accumulated after a period of time t (days), Gm (ml) is the
maximum accumulated gas volume at an infinite digestion time, k0 (day-1) is the specific rate constant, and t (days) is
the digestion time (paper I)

Analysis of the pretreated straw showed that the structural lignin content decreased by
approximately 10% for both the samples. Furthermore, the structural changes caused by the
NMMO-pretreatment were confirmed by Fourier transform infrared spectroscopy (FTIR) (Figure
8). The weaker intra- and intermolecular hydrogen bonding as well as van der Waals interactions
could be the reason for improved gas production as increasing the accessible surface area for the
enzyme attachment. This is further confirmed by the increase in the carbohydrate levels after the
pretreatment, which was around 13% for the straw separated from cattle and 9% for the straw
separated from the horse manure. The crystallinity index, or the lateral order index (LOI), was
calculated as the absorbance ratio of the bands around 1,420 and 898 cm-1. The results showed that
the crystallinity of the cellulose was affected by the pretreatment, as it decreased with increasing
pretreatment time. Consequently, the NMMO-pretreatment for 15 h resulted in an increase of
methane yield by 53 and 51% for the cattle and horse manure, respectively (Paper I).

35
Figure 8. FTIR spectrum of treated and untreated straw in (a) cattle manure and (b) horse manure. Inside the spectrum
a-untreated, b-5 h treatment, c-15 h treatment

Previous studies showed that the water content in the NMMO affect the behaviour of wood and
cotton cellulose fibers [129]. The effect of NMMO concentration (75 and 85%), temperature (120
and 90 °C) and times (3h and 15 h) on the methane yield using forest residues as substrate with high
lignin content was investigated (Paper II). The forest residues were first milled to 0.5 – 2 mm in size
prior to the pretreatments. The following batch anaerobic digestion assays showed that all three
pretreatment conditions had positive effects on the initial reaction rates as well as the total methane
yields comparing to those of the untreated forest residues (Figure 9).

Anaerobic digestion of untreated forest residues resulted in 42 NmL CH4/gVSadded, and the initial
reaction rate of untreated forest residues obtained within the first 10 days of digestion was 0.83
Nml/gVS/d. The pretreatment with the highest NMMO concentration of 85%, temperature of 120°C
and duration time of 15h resulted in higher methane yield of 109 NmL CH4/gVSadded comparing to
that after the treatment with the same NMMO concentration but lower temperature of 90°C and
duration time of 3h (87 NmL CH4/gVSadded). Furthermore, the results indicate that regarding the
methane production the concentration and the pretreatment time are inversely proportional to each

36
other. For instance, the higher the concentration of NMMO 85%, the shorter the pretreatment time
3h and vice versa. This observation seem to be in accordance to previous study of NMMO-
pretreatment of lignocelluloses [132, 137].

120
untreated
100 90, 3h , 85 %
120, 3h, 85%
Volume Nml/gVS

80
120, 15h, 75%
60

40

20

0
0 10 20 30 40 50 60
Time (day)

Figure 9. Accumulated methane production of NMMO-pretreated and untreated forest residues at mesophilic batch
conditions. The pretreatment conditions are described in the figure

Based on the results from the batch experiment, the best pretreatment (75% NMMO at 120 °C for
15 h) regarding the methane production rate (4.27 NmL CH4/gVS/day) was further studied during
the semi-continuous digestion experiment (Paper II).

The results obtained in our study demonstrated that the NMMO-pretreated forest residues could be
used as a potential substrate in a continuous biogas process. However, forest residues are carbon-
rich substrates; hence, for a nutritional balance they can be utilized in the co-digestion with other
materials [138].

In textile wastes, the lignin or hemicelluloses are negligible; thus, the utilization of these materials
as a substrate for biogas production should be less complex compared to the lignocellulosic
materials. However, there are a wide variety of fibers and colors used in the textile waste that can
cause problems in the anaerobic digestion [12]. Therefore, the goal of a pretreatment is to reduce
the crystallinity, and to enhance the accessible surface area as well as get rid of colors during
washing after pretreatment. Previous studies have already confirmed the effects of the NMMO-
pretreatment on pure cellulose.

37
38
Chapter 5.

High-rate anaerobic treatment systems

5.1. Background and Status

Development of high-rate reactor systems demands a separation of the solid retention time from the
hydraulic retention time. This separation can be accomplished by different methods of sludge
retention, for instance, sedimentation, immobilization on a fixed matrix or moving carrier material,
and recycling of the biomass and granulation. Hence, high-rate systems can be divided into
suspended growth and attached-growth processes with expanded/fluidized bed reactors and fixed-
film processes. In an expanded/fluidized bed reactor, sand or porous inorganic particles are used to
build up an attached film. Fixed film processes count on the bacteria to attach to a fixed media, like
rocks, plastic rings, modular cross-flow media, etc. Some systems, such as the anaerobic hybrid
process, unite suspended- and attached-growth processes in a single reactor in order to make use of
the advantages of both types of biomass [139-141]. Current high-rate processes are anchored in the
concept of retaining high viable biomass. A variety of reactor designs has been developed in order
to achieve this goal [142]:

i) Development of biomass aggregates with high settling capabilities, e.g., UASB reactor and
anaerobic baffled reactor.

ii) Attachment of high density viable biomass to certain types of carrier materials e.g., fluidized bed
reactors and anaerobic expanded bed reactors.

iii) Entrapment of biomass aggregates between the packing materials provided for the reactor, e.g.,
down flow anaerobic filter and upflow anaerobic filter. Recent investigations on the development of
new techniques for cell immobilization by using specific capsules made of a membrane, permeable
to nutrients and metabolites with no leakage of the biomass, revealed a promising result towards
applications in the biogas production [143, 144].
39
The application of these high-rate anaerobic treatment systems has been successful in the treatment
of industrial wastewater. The development of this technology was crucial, since large volumes of
wastewater effluent needs to be treated in optimally designed bioreactors aiming to reduce the
treatment time and to increase the treatment efficiency [145-147]. High-rate reactors meet the
conditions for attaining the high retention of viable biomass under high organic loading rates, and
achieving high contact between the biomass and the incoming effluent, resulting in a reduced
reactor size and low process energy requirements [148, 149].

The most recognized sludge bed bioreactors are: Upflow Anaerobic Sludge Blanket (UASB)
Reactor, Expanded Granular Sludge - Bed (EGSB) Reactor, and Internal Circuit (IC) Reactor. The
IC reactor and the EGSB reactor are basically modified forms of the UASB [27]. The worldwide
number of plants in operation using these kinds of techniques between 1980–2007 was estimated to
be 2,266; however, this number declined to 610 between 2002–2007 (Figure 10) [150]. It should be
pointed out that the granular sludge based technologies (UASB, IC, and EGSB) were leading
technologies in the market during the past few decades.

1981-2007 2002-2007
FB 2% Af 1% LAGOON
HYBRID 3% FB 2% 1%
LAGOON HYBRID 2%
5% CSTR 4%
Af 6%
CSTR 7% UASB 34%
UASB 50% EGSB 22%

EGSB 12%

IC 15%
IC 33%

Figure 10. Anaerobic digestion technologies for industrial wastewater for different periods. UASB: upflow anaerobic
sludge blanket; EGSB: expanded granular sludge bed; Hybrid: combined system with sludge bed at the bottom part and
a filter in the top; IC: internal circulation reactor; AF: anaerobic filter; FB: fluidized bed reactor; CSTR: continuous
stirred tank reactor

5.2. Upflow anaerobic sludge blanket reactor

Regardless of its introduction early on, the awareness of anaerobic systems as the main biological
step in wastewater treatment was quite limited until the UASB reactor was developed by Dr. Gatze
Lettinga during the early 70s in the Netherlands, although a rather similar system, called the
40
―biolytic tank,‖ was already studied earlier in 1910 [151]. Today, the UASB reactor is widely used
for the treatment of several types of wastewater from different sources, such as distilleries, food
processing units, tanneries, and municipal wastewater [142, 148, 152, 153].

The reason that the UASB concept became successful is attributable to the establishment of a dense
sludge bed in the bottom of the reactor, where all biological processes occur. The bed is principally
created by the accumulation of incoming suspended solids and bacterial growth. In upflow
anaerobic systems, the microorganisms can aggregate naturally in the flocs and build granules under
specific conditions. These aggregates are quite dense and this characteristic gives the granules good
settling properties, which are not vulnerable to the wash-out from the system under practical reactor
conditions.

Retention of the active biomass, either granular or flocculent, inside the UASB reactor allows for a
good treatment performance at high organic loading rates. The flow of the influent at the bottom of
the UASB system and the biogas produced causes a natural turbulence providing a good contact
between the wastewater and the biomass [154].

One of the main advantages of the UASB technologies is that it has relatively less investment
requirements in comparison to the anaerobic filter or fluidized bed systems. It is worth mentioning
that a long start-up period or a necessity for an adequate amount of granular seed sludge for more
rapid start-up is considered to be the drawbacks of this system [153].

The UASB reactor is typically separated into four compartments: (i) the granular sludge bed, (ii) the
fluidized zone, (iii) the gas-solids separator, and (iv) the settling section (Figure 11). The granular
sludge bed is located in the bottom of the reactor. The wastewater is pumped in at the bottom of the
reactor and moved upward through the granular sludge bed. At this point the organic materials are
biologically degraded and biogas is produced. Just above the granular sludge bed, a fluidized zone
will develop, owing to the production of the biogas. Further biological degradation can occur at this
zone as well. The gas-liquid separator divides the biogas from the liquid. Strong granules with high
settling abilities will settle back to the granular sludge bed, whereas flocculated and dispersed
microorganisms are washed out of the reactor together with the effluent [155].

41
 Biogas outlet

Effluent
Tri-phase separator

Gas deflector

Sludge blanket

Sludge bed

Influent

Figure 11. Illustration of an Upflow Anaerobic Sludge Blanket reactor (UASB). Adapted from [156]

5.2.1. Biogranulation of microorganisms

There are numerous theories trying to shed light on the mechanisms of anaerobic sludge
granulation. These theories could be divided into three groups, that is, the physical, microbial, and
thermodynamical approaches, which are believed to be the main factors responsible for the granule
formation [157]. To put it in a simple expression, it is a conglomeration of biomass as a result of the
self-immobilization of the anaerobic microorganisms occurring under hydrodynamic conditions
[158]. However, these theories are not entirely firm as some theories have features that could fit in
to other classifications [157]. One of these theories for the initiation of the granulation process is
illustrated in Figure 12.

In general, it is considered that the extracellular polymeric substances (ECP) play an essential role
in the process of anaerobic biogranulation [159, 160]. Microbial adhesion, i.e., when a cell attaches
to a surface or another is conceded to be the reason for the cell granulation (Figure 22). Under a
proper physiological environment, ECP is excreted by the microbial cells and exposed on their
surfaces [155]. The ECP consists of a complex combination of polymeric substances excreted by
the microorganisms, lysis, and hydrolysis products, and adsorbed organic matter from the
surrounding environment. Proteins, polysaccharides, humic acids, uronic acids along with small
amount of lipids and nucleic acids are found to be the main components of ECP. So far, the exact
role of the components in the formation and the functions of ECP are not understood. Earlier
investigations pointed out that proteins, humic substances, and carbohydrates alter the cell surface
charge, hydrophobicity, and viscosity, which in turn will have an effect on the surface properties of
the bacterial flocs, which aid the adhesion of the flocculated sludge particles together [160].
42
Addition of polymers
One of the key factors for the granule development from non-granular sludge is the existence of a
nuclei or bio carriers for the microbial attachment. Synthetic and natural polymers have been
commonly used during the coagulation/flocculation processes. The addition of polymers, such as
water absorbing polymers (WAP), hybrid polymers, and cationic polymers, encourages particle
agglomeration and enhances the formation of the anaerobic granules considerably [161]. These
polymers behave as ECP substances in the aggregating process of anaerobic sludge. Polymeric
chains form a link between the cells and this encourages the formation of the initial microbial
nuclei, which in turn serves as the first step toward microbial granulation [160].

Addition of cations
There is strong proof that divalent and trivalent cations, such as Ca2+, Mg2+, Fe2+, and Fe3+ could
bind to negatively charged cells and aid the formation of a microbial nuclei [161]; hence, the
presence of cations can be a key factor in the granulation processes. Calcium-enhanced granulation
can be caused by physicochemical and biological effects. The Ca2+ binds with the ECP produced by
microorganisms, thus, promoting anaerobic granulation [161].

Reactor temperature
The performance of an anaerobic system is strongly associated with variations in the temperature.
Methanogenic archaea are the core microbial components of the UASB granules, hence, grow
slowly. There are studies that show that their generation time could range from 3 days at 35 °C to
50 days at 10 °C [162]. This suggests that temperatures below 30 °C would seriously cause an
inhibition in growth of the methanogens. This is the reason that the mesophilic UASB reactors
ought to be operated at a temperature range of 30–35 °C. Even though a relatively high temperature
encourages the growth of the microorganisms, extremely high temperatures would cause a loss of
metabolic activity [161, 163, 164].

Reactor pH
Investigations on the effects of the pH on anaerobic granulation explained that the strength of the
anaerobic granules decreased when the pH increased to a range of pH 8.5–11.0, indicating that high
pH conditions would weaken the granular structure. From pH 5.5 to 8.0, the strength of the granules
was unaffected and relatively stable. However, in the range of pH 5.0 to 3.0, a sharp decline in the
strength of the granule was observed [165]. These results suggest that a somewhat acidic condition

45
would assist the maintenance of the granular structure. As a result, the pH of the reactor needs to be
regularly monitored and kept stable at a very narrow pH range of 6.7–7.4 [161, 162].

5.2.3. Characteristics of anaerobic granules

The microstructure of the anaerobic granules are proposed to be a multi-layered structural model
with acidogenic bacteria dominating the outer layer, methanogenic archaea at the center, and H 2-
producing and H2-utilizing microorganisms in the middle layer [166, 167]. Filamentous
microorganisms were also found to be dominant not only on the surface of the granules but also in
the center.

Anaerobic granules in general have a black or dark brown color on their surface. When the OLR
and liquid upflow velocity are low, the granules are found to become lighter (gray or white) with a
hollow core, which makes them extremely soft and very weak under mechanical stress. On the other
hand, at a high OLR and liquid upflow velocity, granules were found to be dark black and had a
dense structure [168]. It has been suggested [168] that the color change and the hollowing of the
granules depends on different mechanisms. The "hollowing" of the granules is most likely
connected to the size of the granules. The feed may penetrate the granules simply by diffusion so
when the size of the granule goes beyond a certain limit, the concentration of the feed becomes too
small in the center of the granules, leading to starvation of the microbial population and
consequently autolysis to occur. Since the autolysis products are not as densely packed compared to
the viable cells, the gas produced will be captured inside the granules, reducing the density, which
would lead to the floatation of the granules. The change in color, on the other hand, is suggested to
be dependent both on the composition of the feed around the granules as well as on the hydro-
dynamic conditions in the reactor. When granules are packed densely at the bottom of the reactor,
nutrient deficiency may arise around the granules for a long period of time. This may cause
irreversible alteration in the granules’ composition and structure. This phenomenon occurs more
likely at low specific loading rates and at low upflow velocities. Higher loading rates and higher
upflow velocities increase the access of the granules to the nutrients and possibly slows down the
color change of the granules [168].

The density of the anaerobic granules stands for the compactness of the microbial community. A
higher density is linked to a faster settling velocity of sludge. The geometric dimension of the
granules has duel effects on the performance of the UASB system. A too small sized granule would
increase the possibility of a washout from the system and thus cause operational instability.
Conversely, for those large-size granules, the efficiency of mass transfer inside the granule would
46
be reduced. Furthermore, the size and density of the anaerobic granules are dependent on many
factors e.g., hydrodynamic conditions, OLR, and microbial species [169, 170]. The mechanical
strength of the granules influences the stability, and it reveals a more compact and stable structure
of the anaerobic granules. The higher the strength of the anaerobic granules, the more attractive
they become for large-scale industrial applications [161].

Figure.13 Size distribution of the granules from the UASB reactor digesting starch

5.1. Two-stage process for high-rate methane production

Besides introducing a pretreatment step for increasing the rate of the degradation, the rate of the
methane production can be accelerated by increasing the rate of conversion of the VFAs into
methane as well; this can be achieved by increasing the concentration of the methanogens in the
reactor. In this work, a two-stage system was developed, where a continuous stirred tank reactor
(CSTR) was used for the first stage and an UASB reactor filled with the granules was used for the
second stage (Papers III, IV, and V). The performance of this two-stage system was evaluated in
different configurations (Figure 5). The CSTR was operated at thermophilic conditions, while the
UASB at mesophilic conditions. The substrates used in the different two-stage processes were
blended fibers, viscose/ polyester, (60/40) and cotton/polyester (50/50) (Paper III); untreated and

47
NMMO-pretreated jeans (Paper III); cotton and starch (Paper IV); and organic fraction of municipal
solid waste (OFMSW), and food processing waste (FPW) (Paper V).

5.1.1. Batch process- single vs. two-stage

The differences between the digestion performances of two untreated cellulosic textile wastes, i.e.,
viscose and cotton blended with polyester, using a single stage CSTR or a two-stage (CSTR and
UASB) system, both in batch operation mode were investigated. In all of these experiments the
same initial cellulose concentration was applied. In the single stage process, cotton/polyester
showed a much longer lag phase compared to the viscose/polyester (Figure 14); furthermore, during
the first 10 days of digestion, 80% of the theoretical yield of methane could be achieved in the case
of viscose/polyester. An earlier batch study [171] showed that anaerobic batch digestion of
regenerated cellulose after the NMMO-pretreatment of viscose/polyester reached 53% of the
theoretical methane within 6 days of digestion; thus, viscose/polyester might not need any
pretreatment at all, as the rate of gas production was not different from the untreated material. In
contrast, for cotton/polyester, after a long lag phase period, only 17% of the theoretical yield of
methane was achieved. In an earlier study on the same material, only 5% of the theoretical yield
was observed after 6 days of digestion [171].

60
Viscose/polyester
50
Cotton/polyester
Methane (ml/gVS/day)

40

30

20

10

0
0 5 10 15 20 25 30

Days

Figure 14. Cumulative methane production in a single stage batch process - Viscose/polyester and Cotton/polyester
(paper III)

48
 Untreated Jeans

Share of reactor in methane production Share of reactor in methane production

100
500 90

Methane (mL/gVS/day)
80
400 70
60

(%)
300
50
40
200
30
100 20
10
0 0
Pretreated Jeans 100
500 90
Methane (mL/gVS/day)

80
400 70
60

(%)
300
50
40
200
30
100 20
10
0 0
0 10 20 30 40 50 60 70 80
Days

Figure 18. Methane volume in semi-continuous two-stage process at different OLR. ― methane volume, --- % share in
CSTR, and - - -% share in the UASB (paper III)

Untreated Jeans
7
1,6
Total VFA in UASB (g/L)
Total VFAin CSTR (g/L)

5 1,2

4
0,8
3

2
0,4
1

0 0

7 Pretreated Jeans
1,6
Total VFA in CSTR (g/L)

Total VFA in UASB (g/L)

5 1,2

4
0,8
3

2
0,4
1

0 0
0 10 20 30 40 50 60 70 80

Days

Figure 19. Total VFA concentration in (♦) the UASB and (▲) CSTR for the untreated jeans and the pretreated jeans
(paper III)

53
The digestion of starch, on the other hand, showed a much more stable methane production during
an OLR of 2 and 2.7 gVS/l/d and an HRT of 10 and 7 days, respectively (paper IV). However, a
sharp decrease in the methane yield was observed when the OLR was increased to 4gVS/l/d and the
HRT was decreased to 5 days. This is an indication that the hydrolysis process was not inhibited
like methanogenesis in the CSTR, and the volatile fatty acids could still be produced (Figure 22A),
but it was converted into methane in the UASB (Figure 22C) and therefore, the major share of the
methane production was shifted to the UASB without accumulating in the process and causing
failure. This shows that the microbial degradability and the structure of the substrate play a rather
significant role in handling a higher OLR and shorter retention times HRT.

450 Cotton 100

Share of methane production (%)

400 90
CH 4 volume (ml/gVS/day)

80
350
70
300
60
250
50
200
40
150
30
100 20
50 10
0 0

450 Starch 100

Share of methane production (%)

400 90
CH 4 volume (ml/gVS/day)

80
350
70
300
60
250
50
200
40
150
30
100 20
50 10
0 0
0 10 20 30 40 50 60 70 80 90 100

Days

Figure 20. Total methane production in the closed system for the cotton and starch with - - -% share in the CSTR and ---
% share in the UASB (paper IV)

5.1.3. Two-stage- open system vs. closed system

The degradation of starch and cotton was also investigated in the two-stage system in two different
configurations to study the effect of the process on accelerating the digestion. Two-stage processes,
one with recirculation (closed system) and the other without recirculation (open system), were run
in parallel (Figure 5). Starch and cotton were chosen as a substrate since both contain glucose as
monomers, but at opposite ends of the degradability scale. The goal was also to evaluate how these
two configurations would respond to the degradability of these substrates at different OLRs and
54
HRTs. The results of this study suggest that the recirculation has a positive effect regarding the
stability of the two-stage system, especially when higher OLRs and lower HRTs were applied. A
transition pattern observed in both the open and the closed systems showed that the major share of
the methane production shifted from the CSTR to the UASB (Figures 20 and 21). This shift,
however, emerges at earlier stages in the open system compared to the closed system. During this
transition stage, a decrease in the methane yield is observed, which is due to the increase in the
accumulation of the VFAs in the CSTR (Figure 22). The increase in the acids in the CSTR
decreases the pH; thereby, an inhibition of the methanogenic archaea occurs in the CSTR. All the
VFAs produced in the first stage are then converted into methane in the second stage or the
methanogenic phase. This shows that the recirculation could support the hydrolysis step as well as
avoid nutrient loss at a higher OLR, thus, improving the performance and the stability of the process
significantly [172].

450 Cotton 100

Share of methane production (%)

400 90
CH 4 volume (ml/gVS/day)

80
350
70
300
60
250
50
200
40
150
30
100 20
50 10
0 0

450 Starch 100

Share of methane production (%)

400 90
CH 4 volume (ml/gVS/day)

80
350
70
300
60
250
50
200
40
150
30
100 20
50 10
0 0
0 10 20 30 40 50 60 70 80 90 100

Days

Figure 21. Total methane production in the open system for the cotton and starch with --- % share in the CSTR and - - -
% share in the UASB (paper IV)

The capacity of the CSTR to hydrolyze cotton and starch is limited. In the open system, the cotton
did not handle more than 4 gVS/l/d while starch handled an OLR of 10 gVS/l/d, even though the
gas production decreased, but the process could continue until the methane production stopped.

55
This also further confirms that the more degradable the substrate, the higher OLR and a lower HRT
can be applied.

10 A-CSTR-Closed system B-CSTR-Open system 10

8 8
Total VFA (g/L)

Total VFA (g/L)

6 6

4 4

2 2

1,2
0 0
C-UASB Closed system D-UASB -Open system
1 1

0,8 0,8

Total VFA (g/L)

Total VFA (g/L)

0,6 0,6

0,4 0,4

0,2 0,2

0 0
0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100

Days Days

Figure 22. Total VFA concentration.●- Starch ♦- Cotton in closed and open system

5.1.4. Semi-continuous process- Single vs. two stage

Anaerobic digestion (AD) under controlled conditions is one suitable technique for the treatment of
OFMSW. This technique is at present in use in large scale applications mostly in Europe [173].
Many attempts have been made to introduce anaerobic digestion processes for treating the organic
fraction of the industrial solid waste [174]. However, the main barrier in spreading this technology
is the lower biodegradation rate of the solid wastes, owing to the complexity of the organic material,
in comparison to the liquid ones [90]. Anaerobic digestion of the OFMSW usually requires a long
retention time of more than 20 days in conventional single stage digesters with a connected large
reactor volume requirements [175]. Pretreatment of the OFMSW to improve the hydrolysis can be
used to solubilize the organic matter prior to the digestion process in order to improve the
performance of the overall AD process, in terms of faster rates and degree of degradation, hence,
decreasing the HRT and increasing the methane production [176]. The application of the OFMSW
56
and FPW in a two-stage anaerobic digestion process was investigated (paper V). The objective was
to investigate the optimum OLR and HRTs that could be achieved using the OFMSW and FPW as
substrates in a single stage and two-stage process. The OLR was increased slowly from 2 gVS/l/d to
6 gVS/l/d and the HRT was decreased from 10 days to 3 days and then kept stable at 3 days to
provide a sufficient time for the breakdown of the organic matter in the CSTR, while the OLR was
further increased to 14 gVS/l/d.
The results of this study (Figure 24) show that the two-stage process could enable the possibility of
operating the anaerobic digestion at higher OLRs and lower HRTs. On the other hand, the single
stage reactors could handle an OLR of 3 gVS/l/d as the maximum; moreover, the HRT could not be
decreased to lower than 7 days. In contrast, the two-stage process was stable up to an OLR of 10
gVS/l/d and an HRT of 3 days for both substrates. The increase in the OLR to 12 and 14 gVS/l/d at
an HRT of 3 days showed a decrease in the gas production, but the process did not completely fail
as it did in the case of the single stage system.

one-stage MSW one-stage FPW two-stage MSW two-stage FPW

0,7
0,6
Volume CH4 m3/Kg VS

0,5
0,4
0,3
0,2
0,1
0
2gVS/l/d 3gVS/l/d 4g VS/l/d 5gVS/l/d 6gvs/l/d 8gVS/l/d 10gvs/l/d 12gvs/l/d 14gvs/l/d

10 d 7d 5d 3d 3d 3d 3d 3d 3d

OLR and HRT

Figure 24. Total methane production in the single stage and two-stage process at different organic loading rate and
hydraulic retention times for the organic fraction of municipal solid waste (OFMSW) and food processing waste (FPW)

57
58
 Concluding Remarks

In spite of its advantages, the potential of biogas technology is not fully harnessed, as certain
limitations are associated with it. Most common among these are the long degradation time of the
anaerobic digestion. One of the reasons is attributed to substrates with a recalcitrance structure that
oppose biological degradation and consequently increase the hydrolysis time, and thereby affect the
overall process; thus, hydrolysis becomes the limiting step. Lignin shield together with the compact
cellulose structure of the lignocellulosic material and high crystalline structure of the cellulose in
textile waste is a challenge in the AD process. These problems could be solved by the pretreatment
prior to the biogas production. In this thesis, the use of NMMO as a pretreatment improved the
hydrolysis of the straw fraction of manure and increased the methane yield by 53% for the cattle
and 51% for the horse manure. It also increased the methane yield of the forest residues by 141%
compared to the untreated material. The crystallinity of the cellulose was also affected by the
pretreatment, as it decreased with increasing pretreatment time. However, easily degradable
materials face another challenge in the anaerobic process. If the material is easily degradable, it
causes a problem by hydrolyzing too fast, however, slow growing methanogens are not able to
ferment the hydrolyzed products at the same rate as they are produced, which ultimately leads to the
accumulation of intermediate products, which results in a failed process; thus, the methanogenesis
becomes the limiting step. This means that a decrease in the retention time would lead to a biomass
washout. This challenge is solved by using high-rate systems by separating the process into two
phases. Two-stage processes make it possible to disconnect the dependence of the organic loading
and the retention time. Pretreatment of blue jeans showed that the hydrolysis rate was increased, but
the two-stage process could handle the intermediate products produced without the process failing.
The organic loading rate of the highly crystalline cotton and easily degradable starch could
successfully be increased to 4 gVS/l/d and to 10 gVS/l/d and the retention time could be decreased
to 5 and 2 days, respectively. Application of the two-stage process using the organic fraction of the
municipal solid waste (OFMSW) and the food processing waste (FPW) as inhomogeneous material
showed that the two-stage process could handle an organic loading rate between 8–10g VS /l/d and

59
a retention time of 3 days while the single stage could not handle more than 3g VS/l/d and a
retention time of 7 days.

60
 Future work

Research has demonstrated that a two-stage system has several advantages over a conventional
single stage system. However, most of these investigations have been carried out on a lab scale
process. The majority of the two-stage, full-scale, processes have been applied to wastewater
treatment systems with a low solid content. After decades of research, the advantages of the two-
phase anaerobic digestion are yet to be demonstrated.

More pretreatment methods should be investigated for biogas production in order to

decide which pretreatment method is most suitable for the biogas production.

There is still work to be done on the lab scale to investigate the textile waste with
blended fibers. The preliminary work in this thesis shows a potential for digesting
the types of material without pretreatment. However, further work is needed.
Furthermore, the feasibility of using textile waste in the anaerobic digestion should
also be evaluated further.

There is still little known about the application of high solid content materials to
two-stage process on both lab scale and pilot scale. Furthermore, to use the results of
the laboratory scale experiments, it is essential to know whether the results are
transferable to a pilot scale and whether the experiments are reproducible or not.

In reality, the most important benefit claimed for the two-phase digestion, that is, the
reduction in the overall tank sizes, have still not been confirmed. To confirm this
doubt, further investigation is needed in this area especially from an industrial point
of view.

61
 The economics of the two-stage process are also one of the obstacles that decrease
the interest in the commercialization of the two-stage process. Economic process
evaluation based on pilot scale data is necessary.

Biogas could be included within a broader category of biomass-related technologies,

and its possibilities will mainly depend on the availability of the biomass
(feedstock). The application of new feedstock (e.g., waste textile and the
lignocelluloses based material in this thesis) to this technology could result in it
having a better standing in the market.

More investigations and attention are needed regarding the by-products of the
anaerobic digestion process such as polyester in blended fibers and lignin left after
the digestion of forest residues in the process. This would be beneficial for the
economics of the process.

62
63
 Nomenclature

AD Anaerobic digestion

CSTR Continuous stirred tank reactor

UASB Upflow anaerobic sludge blanket

OLR Organic loading rate

HRT Hydraulic retention time

LCFAs Long-chain fatty acids

SRT Solid retention time

TS Total solids

VS Volatile solids

VFA Volatile fatty acids

COD Chemical oxygen demand

OFMSW Organic fraction of municipal solid waste

FPW Food processing waste

NMMO N-methylmorpholine-N-oxide

ECP Extracellular polymeric substances

64
 Acknowledgments

Pursuing my Ph.D. has been a long journey full of emotional ups and downs. Much has happened
and changed during these past four years that could have blocked my path of reaching the end of
this incredible journey. The support and encouragement of many people gave me the strength and
motivation to stay strong through the most difficult periods.

I would like to take this opportunity to acknowledge everyone whose contribution, help and
guidance made this thesis possible and a memorable experience for me in many ways.

I would like to express my deepest gratitude to my supervisor Professor Mohammad Taherzadeh

who gave me this opportunity, guided, and supported me throughout my studies. Thank you for
always being available at any time of the day, at any place in the world, which is one of many
positive qualities I realized about you as a supervisor. It has been a great privilege to have had your
guidance and support throughout this journey.

I wish to express my heartfelt appreciation and gratitude to my co-supervisor Dr. Ilona Sárvári
Horváth for being so kind and helpful. Thank you for your constant motivation, support, and advice,
both professionally and emotionally, especially through those difficult periods. You are an
invaluable friend.

I extend my acknowledgment to my examiner Professor Michael Skrivars, for supporting me in this

thesis.

To Karthik Rajedran, Massoud Salehi, and Håkan Romeborn: thank you for all your help and
support with this thesis. Karthik, your professional support, calming nature, and technical
knowledge as well as your kindhearted words “It will be OK!” during the most stressful times have
all been invaluable to me.

I would also like to declare my greatest appreciation to Dr. Gergely Forgács and Dr. Patrik
Lennartsson for their valuable scientific support during these four years. Thank you for giving me
your precious time and sharing your knowledge.

65
To my fellow lab-mates, and colleagues who supported me in one way or another, some whom I
have spent most of my time with during the past four years, Maryam, Jhosane, Päivi, Johan,
Supansa, Anna, Behnaz, Abbas, Farzad, Wikan, Jorge, Martin, Azam, Akram, Pour, Kamran,
Haike, Adib, Dan, Tomas, Tatiana, Isroi, Khamdan, Mofoluwake, and Julius. Thank you for
creating an environment that made me enjoy the long hours of working.

I also extend my gratitude to Jonas Hanson and Kristina Laurila, the past and the present lab
supervisor, for their technical and practical support in the lab.

I would like to express my profound gratitude to so many people at the Department of Engineering
at the University of Borås for their support and concern, specially the present and former Head of
the Department, Dr. Peter Axelberg and Dr. Hans Björk, Dr. Peter Therning, and Dr. Thomas
Wahnström. I am also very grateful to my teachers, Dr.Magnus Lundin and Dr.Elizabeth Feuk
Lagerstedt, for all the support and concern.

I also want to give great thanks to all the administrative staff, especially Susanne Borg, Sari
Sarhamo, Solveigh Klug, Louise Holmgren, and Thomas Södergren for their practical support.

I would like to extend a special thanks and gratitude to Borås Energy and Miljö AB for financing
this thesis, including Per Karlsson and Anna-Karin Schön. I am also very grateful to Rakel
Martinsson and Camilla Ölander for providing me with information, material, and resources
necessary for my studies.

Above and beyond all, I would like to thank the people that mean the world to me: my parents, my
brother, and my sister. I consider myself fortunate to have such an understanding and loving family.
I cannot imagine a life without your love and support.

66
67
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I
PEER-REVIEWED ARTICLE bioresources.com

PRETREATMENT OF STRAW FRACTION OF MANURE FOR

IMPROVED BIOGAS PRODUCTION

Solmaz Aslanzadeh,* Mohammad J. Taherzadeh, and Ilona Sárvári Horváth

Pretreatment of straw separated from cattle and horse manure using N-

methylmorpholine oxide (NMMO) was investigated. The pretreatment
conditions were for 5 h and 15 h at 120 °C, and the effects were
evaluated by batch digestion assays. Untreated cattle and horse manure,
both mixed with straw, resulted in 0.250 and 0.279 Nm3 CH4/kgVS
(volatile solids), respectively. Pretreatment with NMMO improved both
the methane yield and the degradation rate of these substrates, and the
effects were further amplified with more pretreatment time. Pretreatment
for 15 h resulted in an increase of methane yield by 53% and 51% for
cattle and horse manure, respectively. The specific rate constant, k0, was
increased from 0.041 to 0.072 (d-1) for the cattle and from 0.071 to 0.086
(d-1) for the horse manure. Analysis of the pretreated straw shows that
the structural lignin content decreased by approximately 10% for both
samples and the carbohydrate content increased by 13% for the straw
separated from the cattle and by 9% for that separated from the horse
manure. The crystallinity of straw samples analyzed by FTIR show a
decrease with increased time of NMMO pretreatment.

Keywords: Anaerobic digestion; Manure; Straw; Pretreatment; N-Methylmorpholine Oxide

Contact information: School of Engineering, University of Borås, 501 90, Borås, Sweden
*Corresponding author: Tel: +46 33 435 46 20, Fax: +46 33 435 40 08, E-mail:
solmaz.aslanzadeh@hb.se

INTRODUCTION

A reliance on fossil fuels as the main energy source has caused several
environmental and economical challenges (Budiyono et al. 2010). Thus, there is a
steadily rising worldwide interest in investigating renewable sources for energy
production (Amon et al. 2007). Anaerobic digestion (AD) is a technology generally used
for management of organic waste for biogas production, since it offers a renewable
source of energy and at the same time solves ecological and agrochemical problems
(Budiyono et al. 2010). A variety of raw materials, among others energy crops and
animal manure, can be utilized as organic matter for biogas production (Neves et al.
2009).
Methane is produced during the anaerobic degradation of the organic components
such as carbohydrates, proteins, and lipids present in the manure. The ultimate methane
yield is affected by several factors, such as the feed, species, breed, and growth stage of
the animals as well as the amount and type of the bedding material, together with the pre-
storage conditions prior to biogas production (Møller et al. 2004). The composition, i.e.,
the protein, fat, fiber, cellulose, hemicellulose, starch, and sugar content, are also
important factors that influence the methane yield (Comino et al. 2009).

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Straw, when used as bedding material in proper ratios or after appropriate pre-
treatment, can beneficially affect the methane yield by enabling a more advantageous
carbon to nitrogen (C/N) ratio for the substrate (Hashimoto 1983). Since straw belongs to
the class of difficult-to-degrade lignocellulosic materials, a pretreatment step is needed to
improve the rate and degree of enzymatic hydrolysis during the degradation process.
Lignocelluloses are composed of cellulose, hemicellulose, lignin, extractives, and several
inorganic materials. The cellulose and hemicellulose are sheltered by lignin, which
provides integrity and structural rigidity. The content and distribution of lignin is
responsible for the restricted enzymatic degradation of lignocelluloses, by limiting the
accessibility of enzymes (Taherzadeh and Karimi 2008). Therefore, to improve biogas
formation, often an effective and economically feasible pretreatment step is necessary.
However, most of the reported methods such as dilute acid, hot water, AFEX, ammonia
recycle percolation, and lime treatments are costly and have strong negative
environmental effects, while others such as biological pretreatments are time consuming
(Taherzadeh and Karimi 2008).
A study on the efficiency of biogas production of plant residues in co-digestion
with cattle manure (Hassan Dar and Tandon 1987) showed that pretreatment of plant
residues resulted in increased biogas yield by 31 to 42%. On the other hand, the rate of
the bioconversion was very slow. They have also reported that caustic soda pretreatment
has a promising effect on the delignification process compared to other pretreatments
using sulphuric acid, phosphoric acid, ammonia, sodium hypochlorate, and acetic acid.
However chemical pretreatments also can have strong negative environmental effects.
N-Methylmorpholine-N-oxide (NMMO) is one of the non-derivatizing solvents
that can break the intermolecular interactions in cellulose, and it is mainly used in the
textile industry for spinning of cellulose fibers (Lyocell process). It is considered to be
environmentally friendly, since it does not generate toxic pollutants and it is recyclable
with more than 98% recovery. Furthermore, NMMO is known to modify the highly
crystalline structure of cellulose, while leaving the composition of wood intact and
causing no hydrolysis of the hemicellulose (Lennartsson et al. 2011). Another study
(Shafiei et al. 2009) showed that NMMO pretreatment of oak and spruce resulted in an
increase of the digestibility during a following enzymatic hydrolysis.
The objective of this study was to investigate the effects of NMMO pretreatment
on biogas production from horse and cattle manures, both with a high content of straw.
The manure samples were first separated to obtain the straw fraction for the NMMO
pretreatment. The pretreated fractions were then mixed back with the rest of the samples,
and the biogas production was determined and compared with that of the untreated
samples using anaerobic batch digestion tests.

MATERIAL AND METHODS

Materials
Two different deep litter manures obtained from a horse farm and a cattle farm
outside Borås (Sweden) were investigated. The characterization of the substrates was

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PEER-REVIEWED ARTICLE bioresources.com
carried out by Analys- & Konsulatlaboratoreiet Borås, Sweden, according to standard
methods and the data are summarized in Table 1.

Table 1. Characterization of the Horse and Cattle Manures Mixed with Straw
Analyses Horse manure Cattle manure
Total Solids(wt%) 81.5 23.0
Volatile Solids(wt%) 61.8 18.1
Protein(wt%) 11.0 4.80
Kjeldahl Nitrogen(wt%) 1.70 0.76
Ammonium Nitrogen(wt%) 0.047 0.017
Fat Content(wt%) 1.60 0.30
Carbohydrates(wt%) 49.2 13.0

Pretreatment Procedure
The straw fraction of the manure was separated for each pretreatment condition
according to a procedure shown in Fig. 1, where 7.5 g of manure was washed with 150
mL hot tap water and then filtered using a coarse vacuum filter with 1 mm pore size. The
filtrate was collected and stored at -20ºC until further utilization.
The pretreatment of each straw fraction was carried out using a commercial grade
NMMO (50% w/w in aqueous solution) solution (BASF, Ludwig-Shafen, Germany). In
order to achieve a concentration of 85%, the NMMO solution was evaporated in a
vacuum evaporator. Then, propylgallate was added to a concentration of 0.6 g/L. It is an
antioxidant and prohibits oxidation and deterioration of the solvent during the following
pretreatment procedure (Shafiei et al. 2009). Each straw fraction was then pretreated with
92.5 g of 85% NMMO solvent in an oil bath at 120ºC for 5 h or 15 h. Under the 5 h
pretreatment, the suspension was mixed every 15 min, while during the 15 h
pretreatment, the suspension was left overnight without mixing. After the pretreatments,
the NMMO was separated by adding boiling tap water and then filtered through a coarse
vacuum filter. This washing and filtering step was repeated a few times, until the filtrate
was clear, indicating that the NMMO solvent was completely washed out. The filtrate
was then centrifuged (5 min, 5000 rpm) in order to obtain fine particles, which had
passed through the filter, and the supernatant was discarded. The pellet was also
repeatedly washed with hot boiling water and centrifuged until the supernatant was clear
and the NMMO solvent was completely washed out. The pretreated straw together with
the fine particles were dried in a freeze dryer and kept at 4ºC until use.

Anaerobic Batch Digestions

The anaerobic batch digestion experiments were carried out according to a
previously published method (Hansen et al. 2004). The digesters used were 118 mL glass
bottles closed with a rubber septum and aluminum caps. The inoculum was obtained from
a 3000-m3 municipal solid waste digester operating under thermophilic conditions (Borås
Energi och Miljö AB, Sweden), and was incubated and stabilized at 55ºC for three days
before use.

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Fig. 1. Schematic presentation of the pretreatment process

The filtrate collected after the separation of straw was centrifuged at 5000 rpm for
5 min. The supernatant was discarded, and the sediment was mixed with the NMMO-
pretreated straw fraction and used as substrate for the biogas production. Each reactor
contained 40 mL inoculum, 0.3 g volatile solid of pretreated or untreated manure straws,
and tap water to bring the total volume to 45 mL. In order to determine the methane
production from the inoculum itself, blanks containing only inoculum and tap water were
also examined in order to determine the biogas production from the substrate. In order to
facilitate anaerobic conditions and to prevent pH-change, the head space of each reactor
was finally flushed with a gas mixture of 80% N2 and 20% CO2. All experimental set-ups
were performed in triplicates, and the reactors were then incubated at 55ºC for 52 days.
During this experimental period, the reactors were shaken once per day.
The methane produced was measured by taking gas samples regularly from the
headspace, using a pressure-tight gas syringe. During the first two weeks, samplings and
measurements were carried out every third day, followed by weekly sampling for the rest
of the experimental period. The pH in the reactors was measured at the end of the
experiment.

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Kinetic Modeling
The kinetics of the degradation process were evaluated using the following first-
order kinetic model (Jiménez et al. 2004):

G = Gm [1íexp(ík0t)] (1)

or

ln[Gm/ (Gm-G)] =k0t (2)

where G (mL) is the volume of methane accumulated after a period of time t (days), Gm
(mL) is the maximum accumulated gas volume at an infinite digestion time, k0 (day-1) is
the specific rate constant, and t (days) is the digestion time. Plotting the calculated data of
ln[Gm/ (GmíG)] vs. time, t, gives a straight line with a slope equal to k0 with intercept of
zero. The value of Gm was considered equivalent to the volume of accumulated methane
at the end of the experiments.

Analytical Methods
The total solids (TS) and volatile solids (VS) were determined according to
Sluiter at al. (2005). Kjeldahl nitrogen and protein content were determined according to
Swedish standard method ISO 25663 (Swedish Standard Institute, 1984), in which the
materials are treated with a strong acid in order to release nitrogen, which can be then
determined by titration. Since the Kjeldahl method does not measure the protein content,
an average conversion factor of 6.4 is used to convert the measured nitrogen
concentration to a protein concentration. For determination of ammonium nitrogen, the
SIS 028134-1 method (Swedish Standard Institute 1976) was used. It is based on
sparging the samples with deionized water and mixed it with ammonium citrate and
reagents containing sodium nitroprusside, phenol, and sodium hypochlorite before
analysis. Fat content was determined according to Method no. 131 (Nordic Committee on
Food Analysis 1989). The method is based on treatment with hot concentrated hydro-
chloric acid to release fat bound to protein, prior to extraction of the fat with diethylether.
The structural carbohydrates and lignin content of the pretreated and untreated straw
fractions were determined using a two-step hydrolysis method that has been used for
lignocelluloses (Sluiter et al. 2008). The acid-soluble lignin was measured using a UV
spectrophotometer, while acid-insoluble lignin was determined after ignition of the
samples at 575ºC. The quantification of the sugars formed was performed by HPLC
(Waters 2695, Millipore and Milford, USA) equipped with a refractive index (RI)
detector (Waters 2414, Millipore and Milford, USA), using a Pb-based ion exchange
column (Aminex HPX-87P, Bio-Rad, USA) with 0.6 mL/min pure water at 85°C, or a H-
based ion exchange column (Aminex HPX-87H, Bio-Rad) at 60°C with 0.6 mL/min 5
mM H2SO4 as eluents.
The NMMO-pretreated straws were analyzed using a Fourier transform infrared
(FTIR) spectrometer (Impact, 410, Nicolet Instrument Corp., Madison, WI). The spectra
were achieved with an average of 32 scans and a resolution of 4 cm-1 in the range from
600 to 4000 cm-1 and controlled by Nicolet OMNIC 4.1 analyzing software (Jeihanipour,

Aslanzadeh et al. (2011). “Pretreatment for better biogas” BioResources 6(4), 5193-5205. 5197
PEER-REVIEWED ARTICLE bioresources.com
et al., 2009). The methane and carbon dioxide analyses were carried out using a gas
chromatograph (Auto System, Perkin Elmer, USA) equipped with a packed column
(Perkin Elmer, 6’ x 1.8” OD, 80/100, Mesh, USA) and a thermal conductivity detector
(Perkin Elmer) with inject temperature of 150°C. Nitrogen was used as carrier gas at
75ºC with a flow rate of 20 mL/min. For gas sampling, a 250μL pressure-tight syringe
(VICI, Precision Sampling Inc., USA) was used. The results are presented as gas volume
per kilogram volatile solids at standard conditions (0°C, atmospheric pressure).

RESULTS AND DISCUSSION

The straw fraction of horse and cattle manure was separated and pretreated with
85% NMMO for 5 and 15h at 120ºC in order to open up the lignin shield and make the
cellulose accessible for enzymatic degradation prior to biogas production. After the
pretreatment, the NMMO was washed out, and the pretreated straw samples were dried in
a freeze dryer and mixed with the rest of the manure samples and used for biogas
production. The effect of the pretreatment was evaluated using anaerobic batch digestion
assays. Moreover, the changes in the structure of the separated straw fraction due to the
pretreatment were investigated by FTIR analysis.

Biogas Production
The biogas potential of horse and cattle manures mixed with the fraction of straw,
before and after NMMO-pretreatment, was investigated in batch digestion experiments.
Figure 2 shows the average values of accumulated methane production of triplicate
samples measured during 52 days of incubation. The pretreatment improved the methane
potential of every pretreated material. The methane yield increased by 22% and 53%,
after the pretreatment of the straw fraction for 5 h and 15 h, respectively. The specific
methane production for untreated cattle manure was 0.250 Nm3 CH4/kgVS, which
increased to 0.305 Nm3 CH4/kgVS after 5 h pretreatment and further to 0.382 Nm3
CH4/kgVS after the 15 h pretreatment (Table 2). The same pattern was observed for the
horse manure. The specific methane production increased to 0.350 and 0.422 Nm3
CH4/kgVS after 5 h, respective, 15 h pretreatments, while the methane yield of the
untreated horse manure was 0.279 Nm3 CH4/kgVS. This means an increase in the
methane yields by 25% and 51% for 5 h and 15 h pretreatments, respectively (Table 2).
The theoretical methane yield for manure samples was calculated using the
general formula presented previously based on the fat, protein, and carbohydrate contents
of the substrate (Davidsson 2007). According to the data presented at Table 1, the
theoretical yield for cattle and horse manure was calculated to be 0.447 m3 CH4/kgVS
and 0.445 m3 CH4/kgVS, respectively. These results are in accordance with the
theoretical methane yield for dairy cattle manure of 0.469 m3 CH4/kgVS reported
previously (Møller et al. 2004). These authors also calculated the theoretical yield of
methane of manure mixed with straw, based on the composition of this mixture regarding
to carbohydrates, lipids, and proteins and concluded that in comparison with manure
without straw, 1 kg straw mixed with 100 kg manure would increase the yield of methane
by approximately 10% considering the compositional variation in such biomass.

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Table 2. Lignin and Carbohydrate Content, and Lateral Order Index (LOI) in IR Spectra
of Straw Separated from Horse and Cattle Manure before and after Pretreatments with
NMMO at 120ºC for 5 h and 15 h, Respectively. Specific methane yields and specific
rate constants (k0) were obtained during batch digestion of manure mixed with untreated
vs. pretreated straw.
Total
Lignin (% Total Specific Methane Specific rate
Sample of TS) Carbohydrates (%) LOIa Yield(Nm3/kgVS)b constant k0(day-1)
Horse manure    
untreated 32.92 44.68 2.68 0.279±0.002 0.071
5h-treatment 25.90 50.92 0.88 0.350±0.01 0.064
15h-treatment 22.72 53.84 0.66 0.422±0.05 0.086

Cattle manure    
untreated 39.53 25.44 5.36 0.250±0.09 0.041
5h-treatment 31.67 36.68 1.47 0.341±0.01 0.063
15h-treatment 29.75 38.68 1.26 0.382±0.08 0.072
a
Lateral order index A1420cm-1/A898cm-1
b
Accumulated methane per gram volatile solids produced after 52 days of incubation together
with two standard deviations on accumulated methane production
d
Specific rate constant during the first 12 days of incubation

Fig. 2. Methane yield obtained after 52 days of anaerobic batch digestion from untreated and
pretreated horse manure and cattle manure

A batch assay provides information about the methane yield from certain
substrates as well as the kinetics of the degradation process. The results showed that not
only the accumulated methane production, but also the degradation rate was improved as
a result of the treatments. Figures 3a and 3b illustrate the variation of specific rate
constant (k0) for treated vs. untreated horse and cattle manures, respectively.

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As shown in Fig. 3, k0 increased with the pretreatment time for both the cattle and
the horse manure samples (Table 2). After 15 h pretreatment, k0 increased from 0.071
(untreated) to 0.086 d-1 in the case of horse manure, while similar treatment conditions
resulted in an increase from 0.041 (untreated) to 0.072 d-1 for cattle manure. The pH was
around 7 at the end of each digestion setup.

1,0
(a)

0,8
ln [Gm/(Gm-G)]

0,6

0,4

0,2 Untreated
5h treatment
15h treatment
0,0

0 2 4 6 8 10 12 14

-0,2
Time(days)

1,2

(b)
1,0

0,8
ln[Gm/(Gm-G)]

0,6

0,4

0,2
untreated
5h treatment
15h treatment
0,0
0 2 4 6 8 10 12 14

-0,2
Time(days)
Fig. 3. Values of ln [Gm/ (Gm í G)] in function of time for (a) Untreated and pretreated cattle
manure. (b) Untreated and pretreated horse manure

The Effects of Pretreatment on the Composition and Structure of Straw

Separated from Manure
Lignin and carbohydrate contents of the untreated vs. pretreated straw fractions
are shown in Table 2. The total lignin content (acid-soluble and insoluble) for untreated
straw separated from cattle manure was 39.53%(w/w), which decreased to 31.67% and
29.75% following 5 h and 15 h pretreatment with NMMO, respectively. Consequently,
the total carbohydrates of the straw from untreated cattle manure increased from 25.44%
to 36.68% and 38.68% after the 5 and 15 h pretreatments, respectively. Similarly, with
investigations of the straw separated from horse manure, a decrease in the lignin content
was observed from 32.92 (wt %) to 25.90 (wt %) and to 22.72 (wt %) after 5 and 15 h

Aslanzadeh et al. (2011). “Pretreatment for better biogas” BioResources 6(4), 5193-5205. 5200
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pretreatments, respectively (Table 2). The total carbohydrate for untreated straw from
horse manure was 44.68%, which increased to 50.92% and 53.84% after the
pretreatments. This shows that the pretreatment reduced the structural lignin content by
approximately 10% for both the separated straw samples and increased the carbohydrate
content by 13% for straw separated from cattle manure and by 9% for that from horse
manure. Additionally, an increase in the pretreatment time made the delignification more
effective and further improved the following digestion process. This is because the
pretreatment opened up the lignin that shields the cellulose and hemicelluloses, which in
turn limits the accessibility of enzymes involved in further degradation during the
following digestion process.

Table 3. Assignments of FT-IR Absorption Bands (cm-1) with Related References

Bands (lit.) cm-1 Assignment Reference
3500-3100 OH –stretching vibrations (Denise S. Ruzene 2007)

2919-2925 Methyl, methylene, and (Lawther et al. 1996)

methine group vibrations
2850,2920 CH2-streching bands (Kristensen et al. 2008)

1727 Aliphatic carboxyl groups (Buta, Zadrazil et al 1989)

1665-1680 Carbonylgroup (C=O) (MacKay, O'Malley et al. 1997)

conjugated to aromatic ring
1595-1605 Aromatic skeletal vibrations (Buta, Zadrazil et al. 1989)

1595 Aromatic ring with C=O (MacKay, et al. 1997)

stretching
1505-1515 Aromatic skeletal vibrations (Buta, Zadrazil et al. 1989)

1510 Aromatic ring with C—O (MacKay et al. 1997)

stretching
1420 Aromatic skeletal vibrations (Buta, Zadrazil et al. 1989)

1245-1519 Guaiacyl and syringyl (Niu, Chen et al. 2009)

1040 Dialkylether linkages linking (MacKay, et al. 1997)

cinnamyl alcohol subunits
1035 polysaccharide vibrations (Lawther, Sun et al. 1996)

The change in the structure of the straws, caused by the pretreatment, was
investigated by FTIR analysis. The interesting bands studied are summarized in Table 3,
and the absorbance spectra are shown in Fig. 4. The comparison of these FTIR spectra
shows that NMMO pretreatment resulted in reducing the absorption band around 1420
cm-1 and in increasing the absorption band at 898 cm-1. These two bands are
characteristic for the crystalline cellulose I and amorphous cellulose II, respectively
(Nelson and O'Connor 1964). The crystallinity index, which is also called the lateral
order index (LOI), was calculated as the absorbance ratio of the bands around 1420 and
898 cm-1 (He et al. 2008; Zhao et al. 2009). The results in Table 2 show that the

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crystallinity index decreases as the time of the pretreatment increasesǤ This implies that
there is a breakdown in the structure of straw, and as a result more sugars are hydrolyzed
after the pretreatment, which improved the biogas production.

Fig. 4. FTIR spectrum of treated and untreated straw in (a) cattle manure and (b) horse manure
(a) untreated, (b) 5 h treatment, (c) 15 h treatment

The lignin IR spectra have a strong broad band between 3500 and 3100 cm-1,
which is related to OH stretching vibrations caused by the presence of alcoholic and
phenolic hydroxyl groups involved in hydrogen bonds (Adney et al. 2008). The OH
stretching band of the hydroxyl groups around 3300 cm-1 was changed to a higher
wavenumber and somewhat broadened as a result of the pretreatment, which is an
indication of weaker intra- and intermolecular hydrogen bonding and thereby a lower
crystallinity (Jeihanipour et al. 2009). This result confirms the analysis data showing that
the pretreatment reduced the structural lignin content in the straw (Table 2).

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An additional effect of the pretreatment was the elimination of waxes, which can
be observed from the reduced CH2- stretching bands at about 2850 and 2920 cm-1 for the
pretreated straw, suggesting a decrease in the amount of the aliphatic fractions of waxes
(Kristensen et al. 2008). Several other changes were also observed in the structure, as is
shown by changes in many other regions given in Table 3. These changes are more
obvious as the pretreatment time increases (Fig. 4).

CONCLUSIONS

1. The aim of this study was the pretreatment of straw fraction separated from cow
and horse manure, since the accumulation of this low digested lignocellulosic
material can cause problems when manure is utilized for biogas production, and
resulting in low methane yields.
2. NMMO pretreatment of straw separated from cattle and horse manure improved the
methane yield during the following digestion of both manure substrates and these
improvements were increased by increased the pretreatment times. Treating the
straw fraction for 15 h increased the methane yield by 53% and 51% for cattle and
horse manure, respectively, compared to that of when untreated straw was present
in the manure samples.
3. The kinetics of the degradation process were evaluated using a specific rate
constant, k0, which was also improved when the straw fractions separated from
both manure samples were pretreated for 15 h.
4. The effects of the pretreatment were evaluated by chemical and structural
characterizations of the separated straw fractions. The total lignin content
decreased by about 10% and the carbohydrate content increased by about 9% for
straw separated from horse manure and by 13% for straw separated from cattle
manure.
5. A reduction of crystallinity, obtained by FTIR, in the structure of the treated straw
fractions, indicates an increase of the accessible surface area on the lignocellulosic
material for further microbial degradation, improving the methane yield.

ACKNOWLEDGMENTS

This work was financially supported by the Swedish Rural Economy and
Agricultural Societies in Sjuhärad and Borås Energi & Miljö AB (Sweden).

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Article submitted: July 15, 2011; Peer review completed: September 24, 2011; Revised
version received and accepted: October 27, 2011; Published: November 1, 2011.

Aslanzadeh et al. (2011). “Pretreatment for better biogas” BioResources 6(4), 5193-5205. 5205
II
Biogas production from N-Methylmorpholine-N-

oxide (NMMO) pretreated forest residues

Solmaz Aslanzadeha*, Andreas Bergb, Mohammad J. Taherzadeha, Ilona Sárvári Horvátha

a
School of Engineering, University of Borås, Borås, Sweden

b
Scandinavian Biogas Fuels AB, Linköping, Sweden

Corresponding author: *E-mail: Solmaz.Aslanzadeh@hb.se

Keywords; Biogas, Anaerobic digestion, Forest residues, Batch experiment, Continuous

experiment

1
ABSTRACT

Lignocellulosic biomass represents a great potential for biogas production. However, a suitable

pretreatment is needed to improve their digestibility. This study investigates the effects of an

organic solvent, NMMO at temperatures of 120 °C and 90 °C, NMMO concentrations of 75%

and 85% and treatment times of 3h and 15 h on the methane yield. The long-term effects of the

treatment were determined by a semi-continuous experiment. The best results were obtained

using 75% NMMO at 120 °C for 15 h, resulting in 141% increase in the methane production.

These conditions led to a decrease by 9% and an increase by 8% in the lignin and in the

carbohydrate content, respectively. During the continuous digestion experiments a specific

biogas production rate of 92 NmL/gVS/day was achieved while the corresponding rate from the

untreated sample was 53 NmL/gVS/day. The operation conditions were set at 4.4 gVS/L/day

organic loading rate (OLR) and hydraulic retention time (HRT) of 20 days in both cases.

NMMO-pretreatment has substantially improved the digestibility of forest residues. The present

study shows the possibilities of this pre-treatment method, however an economic and technical

assessment of its industrial use needs to be performed in the future.

2
INTRODUCTION

As a consequence of the enormous wood exploitation, a large amount of forest residues, mainly

consisting of leaves, small branches, and bark, is generated. Up to date, the majority of these

forest residues are abandoned in situ, thus originating important environmental problems.

Among these problems, soil acidification (due to the accumulation of organic matter) and

increased risk of forest fires, especially during dry periods with high temperatures can be

mentioned (1).

Forest residues are one of the most abundant lignocellulosic waste streams in Sweden with 1.6

million tons total solids per year reported for 2008. This amount is predicted to more than double

by the year 2018 (2). This makes forest residues a potential biomass for biogas production and

an energy production on a scale of 59 TWh/year in Sweden (3). There is a large demand for

alternative fuels produced from renewable resources worldwide especially for the transport

sector, and biogas is one of the alternatives which can be used. However, in order to meet these

increasing requirements, new sources of substrates are needed to be utilized for biogas

production (4). Lignocellulosic biomass, such as forest residues, is primarily composed of

cellulose, hemicelluloses, and lignin. These kinds of materials can serve as an inexpensive

substrate for biofuel production, avoiding the moral dilemma connected with the utilization of

potential food resources (5). However, the enzymatic conversion of the cellulose and

hemicelluloses in lignocelluloses is slow if the biomass is not exposed to some kind of

pretreatment.

3
Several pretreatment methods have been investigated, including ammonia fiber explosion

(AFEX), wet oxidation, and liquid hot water (LHW), among others, which are shown to be more

successful for agricultural and forestry residues. However, all these pretreatments performed on

forest residues were carried out prior to ethanol production. Furthermore, none of these

pretreatment methods are considered to be enough efficient today (6).

Hendriks and Zeeman (7) reviewed the effect(s) of several pretreatment methods on the three

main parts of the lignocellulosic biomass to improve its digestibility. Steam pretreatment, lime

pretreatment, liquid hot water pretreatments, and ammonia-based pretreatments are concluded to

be pretreatments with a high potential. Their main effects are dissolving hemicellulose and

alteration in the lignin structure, providing an improved accessibility to the cellulose for

hydrolytic enzymes. However, it was also concluded that many of these methods give rise to

different inhibitory products, which especially in high concentrations can possibly be very

harmful to microorganisms in anaerobic digestion. On the other hand, it was also concluded that

during anaerobic digestion the microorganisms have a potential to adapt to these inhibitory

products, when presented at very low concentrations.

Recent studies (8-10) used an organic solvent N-methylmorpholine-N-oxide (NMMO) for

regeneration of cellulose in the industrial Lyocell process, and show that this solvent has a great

potential for the pretreatment of lignocelluloses. The melting point of this industrial solvent is

about 70 °C, while it decomposes at temperatures higher than 130 °C. Consequently, most of the

pretreatment studies with NMMO are performed between these temperatures (11).

4
Earlier studies focusing on NMMO-treatment of lignocellulosic materials aimed either to

improve the ethanol production rate (7-9) or to determine the effects of the treatment on biogas

production through anaerobic batch digestion assays (12, 10).

Previous studies showed that the behavior of wood and cotton cellulose fibers pretreated with

NMMO is highly affected by the water content in the solvent (13). In a recent study, it was

shown that pretreatment of cotton with NMMO corresponding to NMMO concentrations of 85%,

79%, and 73% resulted in dissolution, ballooning, and swelling of the cellulose fibers (14). All of

the experiments were carried out at both 90 °C and 120 °C during treatment times of 0.5 – 15 h.

The study showed that the dissolution (85%) mode had the best effect on the following

enzymatic hydrolysis of cellulose. However, the swelling (73%) and ballooning (79%) mode

resulted in the highest yields of methane production during the following anaerobic digestion.

Another study on NMMO-pretreatment of softwood spruce and hardwood oak with 85% at 90,

110, and 130 °C for 1–3 h showed that the temperatures as well as the treatment times had

significant effects on the performance of the following enzymatic hydrolysis (9). Furthermore,

pretreatment with 85% NMMO at 130 °C for 1–15 h on lignocellulosic materials, such as spruce

chips from the Swedish forests, triticale straw from the Swedish farmland, and rice straw from

the Indonesian fields indicated that increasing the pretreatment time can improve the methane

yield during the following anaerobic digestion (10).

All previous studies used batch digestion assays to determine the effects of different treatment

conditions on methane yield and methane production rate. This study was performed to

investigate the long-term effects of the NMMO-treatment using continuous digestion

experiments. The substrate utilized was forest residues in Sweden, which is a heterogeneous

5
material with high lignin content. Biogas production from the treated vs. untreated materials

were compared after different treatment conditions as well as at different operational conditions.

EXPERIMENTAL SECTION

Raw materials

The forest residues were delivered by Norrskog (Östersund, Sweden). It was an inhomogeneous

material consisting of a mixture of both spruce and pine with a high amount of bark. The

material was first milled to 0.5 – 2 mm in size using a laboratory mill (Retsch SM100, Retch,

Germany) prior to characterization, treatment, and digestion. The characterization of the

untreated material showed that it consisted of 45.75% lignin and 41.05% carbohydrates.

NMMO-pretreatment

NMMO-pretreatments at different conditions were carried out using an industrial grade, 50%

w/w, NMMO solution obtained from BASF (Ludwigshafen, Germany). In order to concentrate

the solution up to 75% and 85%, a rotary evaporator (Laborata 20 eco, Heidolph, Germany),

operating at a pressure of 0.10 bar and a maximum temperature of 130 °C was used. The

pretreatments were performed in 5 L beakers containing 6% forest residues in either 75% or 85%

NMMO solution. During the pretreatments, the reaction mixtures were heated in an oil bath at

120 or 90 °C for 3 and 15 h at atmospheric pressure, while mixing constantly with a mixer. After

the pretreatments, the reaction was stopped by adding 1 L of boiling water to the beakers. The

pretreated materials were then filtered and washed with hot tap water, which made it easier to

6
dissolve and wash away the NMMO until no traces of NMMO was observed in the filtrate. The

pretreated materials were stored at 6 °C until further use.

Biogas production

Batch digestion experiments

The anaerobic batch digestion experiments were carried out at mesophilic (37 °C) conditions

according to a method that was published earlier by Hansen et al (15). The inoculum used in

mesophilic experiments was obtained from a large scale digester treating municipal wastewater

sludge (Tekniska Verken, Linköping, Sweden). The batch assays were performed using sealed

serum glass bottles with a volume of 118 ml, and all experimental setups were prepared in

triplicates. All assay bottles contained 40 mL inoculum, pretreated or untreated forest residues as

substrate in amounts to achieve a VS ratio inoculum to substrate of 2:1 and tap water to give a

final volume of 45 mL. To determine the methane production from the inoculum itself, blanks

containing only inoculum and tap water without any substrate addition were also examined. In

order to create anaerobic conditions and to avoid pH-change, the headspace of each reactor was

finally flushed with a gas mixture containing 80% N2 and 20% CO2. During the experimental

period of 52 days, the reactors were shaken and moved around in the incubator once a day.

The production of methane was measured by taking gas samples regularly from the headspace,

using a pressure-tight gas syringe. During the first two weeks, samplings and measurements were

carried out in every third day, followed by once a week for the rest of the experimental period.

The pH in the reactors was measured at the end of the experiment.

7
Semi-continuous anaerobic digestion experiments

The semi-continuous experiments were carried out at mesophilic conditions (37 °C) in 2 L glass

digesters, equipped with plastic tubes protruding the top of the reactor and ending in the liquid

phase; one for addition and withdrawal and one for the impeller. A rubber stopper with an outlet

for gas covered the top of the reactor. Two experiments were performed in parallel; one with

milled forest residues and one with NMMO-treated milled forest residues. Both digesters were

fed with reject water and digested sludge obtained from a municipal wastewater treatment plant

(WWTP) (Linköping, Sweden). These experiments continued for 118 days and were performed

in two ways: fed batch during start up, and thereafter as semi-continuously fed digesters.

Initially, 540 mL of digester sludge from a municipal WWTP together with 260 mL of reject

water was added to a 2 L glass reactor. Forest residues (pretreated or untreated) together with

reject water and digester sludge were then added daily to the digesters until they reached a

working volume of 1500 mL on day 26. Thereafter, the digesters were kept at a constant

hydraulic retention time of 20 days by daily withdrawal of digester fluid and addition of

substrate, reject water, and digester sludge (90% of OLR from forest residues) as explained

above. The digesters were both initially loaded with 2.0 g VS/L/day. Every fifth day, the OLR

was increased with 0.5 g VS/L/d until reaching 4.2 g VS/L/day. Due to process disturbances, the

OLR was decreased to between 2.4 and 3.7 g VS/L during days 48–55. However, from day 56 to

118, the OLR was kept at 4.4 g VS/ L. Because of the process disturbance, 350 mL digester fluid

was removed from the digester and replaced with 100 mL digested sludge and 100 mL of reject

water from the same source as described above, on day 49. In addition, from day 49 onward, the

daily amount of reject water was decreased by 10 mL (to 21 mL) and the amount of digested

8
sludge was increased by 10 mL (to 26mL). Stirring was initially (up to day 12) performed with a

magnet and thereafter with a metal impeller at regular intervals, five times a day. Volatile fatty

acids (VFA’s), pH, Total solids (TS), and Volatile solids (VS) in the digestate residue were

measured weekly. The gas production was measured continuously using gas meters (own

design), working according to the gas displacement method. All gas volumes are given at

standard conditions.

Analytical methods

Total carbohydrate and lignin contents of the pretreated and untreated forest residues were

determined according to the NREL procedures (16). In these methods, a two-step acid hydrolysis

with concentrated and diluted sulfuric acid was performed to release the sugars from the

hemicellulose and cellulose fractions. The amount of different liberated sugars was measured

afterward by HPLC (Waters 2695, Millipore, Milford, U.S.A.) equipped with a refractive index

(RI) detector (Waters 2414, Millipore, Milford, U.S.A.) and an ion-exchange column (Aminex

HPX-87P, Bio-Rad, U.S.A.) at 85 °C, using ultra-pure water as eluent with a flow rate of 0.6

ml/min. The acid-soluble and acid-insoluble lignin contents were analyzed using UV

spectroscopy at 205 nm and after drying the material at 575 °C, respectively. All lignin and

carbohydrate analyses were carried out in triplicates.

The methane and carbon dioxide in the anaerobic batch digestion series were analyzed as

described by Teghammar et al (17). using a gas chromatograph (Auto System, Perkin-Elmer,

USA) equipped with a packed column (Perkin-Elmer, 6’x1.8’’ OD, 80/100 Mesh, USA) and a

thermal conductivity detector (Perkin-Elmer, U.S.A.) with the inject temperature of 150 °C. The

9
carrier gas was nitrogen, operated with a flow rate of 20 ml/min at 60 °C. A 250-µl pressure-tight

gas syringe (VICI, Precision Sampling, Inc., USA) was used for gas sampling. The overpressure

in the bottles caused by the excess gas was released through a needle following the gas analyses

in order to avoid overpressure higher than 2 bar in the head space of the flasks. The methane

content in the gas produced during the CSTR experiments was not measured due to the low

amount of gas being produced, which caused too large errors in the measurements. All the results

are presented as gas volume at normal conditions (0 °C and atmospheric pressure) per kilogram

volatile solids.

Total solids and volatile solids were determined by drying the samples to a constant weight at

105 °C and then igniting the dried material at 575°C (18).

The VFA’s obtained during the CSTR experiments was measured using GC-FID as described by

Jonsson and Borén (19). The pH was measured with a pH electrode (WTW Inolab, Germany).

RESULTS

Effects of NMMO-pretreatment on the composition of forest residues

The composition of forest residues before and after NMMO-pretreatments at different conditions

is presented in Table 1. The pretreatment with 75% NMMO at 120 °C for 15 h increased the total

carbohydrate content by 8% (from 41% to 49%) in comparison to the untreated forest residues.

On the other hand, the pretreatment with 85% NMMO for 3 h and at 120 °C increased the

carbohydrate content from 41 wt% for untreated material to approximately 45 wt% for the

treated materials. The decrease in the pretreatment temperature from 120 to 90 °C did not have a

10
considerable effect, so the carbohydrate content remained at the same level as it was obtained for

the untreated material. However, when the temperature was decreased from 120 to 90°C, while

all the other parameters for the pretreatment remained the same, i.e., 85% NMMO and 3 h, a

decrease by 3.5% in the carbohydrate content was observed.

The NMMO-pretreatment resulted in a decrease in total lignin content. The highest decrease in

lignin was achieved when the forest residues were pretreated with 75% NMMO at 120 °C for 15

h, reducing the lignin content by over 9% (from 45.75% to 36.48%). The pretreatment with 85%

NMMO for 3 h decreased the lignin content by 7% and by 5%, when the forest residue was

treated at 120 °C and at 90 °C, respectively.

Batch digestion of NMMO-pretreated vs. untreated forest residues

Anaerobic digestion of untreated forest residues resulted in 42 NmL CH4/gVSadded (Figure 1).

Furthermore, the initial reaction rate of untreated forest residues obtained within the first 10 days

of digestion was 0.83 Nml/gVS/d (Figure 1 and Table 1). The pretreatment at 120 °C with 85%

for 3 h and with 75% NMMO for 15 h had a positive effect on the methane production. The

methane yield increased more than twofold, achieving up to 109 NmL CH4/gVSadded, and 100

NmL CH4/gVSadded, respectively. The initial reaction rate was also improved, achieving 4.27

Nml/g VS/day and 3.65 Nml/g VS/day after 15h and 3h pretreatment, respectively. The material

pretreated with 85% NMMO for 3h and at 90 °C showed a slightly lower methane yield of 87

NmLCH4/g VSadded, and methane production rate of 2.75 Nml/g VS/day. The highest methane

production rate, i.e., 4.27 NmL CH4/gVS/day, was observed after pretreatment with 75%

11
NMMO at 120 °C for 15 h. Therefore the forest residues pretreated at these conditions were

further investigated during the CSTR experiments.

Anaerobic digestion forest residues in fed-batch and semi-continuous mode

The specific biogas production was 53 NmL /gVS /day day (n = 63; SD ± 7) for digester 1

(Figure 2), where untreated forest residues were included in the feed. In digester 2, where the

treated forest residues were digested, specific biogas production of 92 NmL /gVS /day (n = 51;

SD ± 24) was obtained (Figure 3). Both digesters were operating at maximum OLR of 4.4

gVS/L/day with HRT of 20 days. Under these conditions, the mean VS-reduction was 8% (SD ±

4) for digester 1 and 20% (SD ± 6) for digester 2. The pH was measured at between 7.4 – 7.8 in

reactor 1 and between 7.1 – 7.6 in reactor 2 during the experiments (Figure 3).

Acetate and propionate were the main VFAs detected, and the values varied both between the

processes and over time. Starting on day 41, the amount of acetate and propionate increased in

reactor 2, reaching at most 7.1 mM acetate and 1.3 mM propionate on day 49. On day 51, both

acetate and propionate started to decrease to concentrations lower than 0.6 mM. In reactor 1,

both acetate and propionate was below 0.6 mM during the experimental period.

DISCUSSION

In accordance with previous studies on NMMO-pretreated lignocellulosic materials, it was found

that the pretreatment had positive effects, resulting in increased methane yields during the

subsequent anaerobic batch digestion assays. A previous study on NMMO-pretreatment of

12
lignocellulosic materials such as spruce, rice straw, and triticale straw showed an increase

between 400 and 1200% in the methane production after the treatment (10). However, the

substrates that were used there were homogenous with lower lignin content (between 19 and 29

wt%) compared to the heterogeneous forest residues with much higher lignin content of 46 wt%.

The pretreatment (75% NMMO, 120 °C for 15h) decreased the total lignin content by more than

9% and consequently, increased the total carbohydrates by 8% resulting in an increase in

methane production by 141%.

The long-term effects of the most effective pretreatment conditions were further investigated in

semi-continuous anaerobic digestion using pretreated vs. untreated forest residues as substrates.

The results obtained in our study clearly demonstrate that a continuous biogas process can also

be based on NMMO-treated forest residues with a low addition of supplemental material to keep

the nutritional balance in the system. To our knowledge, this is the first stable continuous

digestion of NMMO-treated forest residues to be shown.

The biogas yield was also improved from 53 to 92 NmL/gVS/day, during continuous digestion

of untreated and treated forest residues, respectively (Figure 2). The rapid increase in VFA’s

obtained in digester 2 is probably due to the higher level of organic material available for

digestion in the NMMO-treated forest residues, which in turn inhibits hydrogenotrophic

methanogens due to a drop in pH, demanding a higher amount of active microorganisms and/or

enzyme activity (20, 21).

During continuous operation, an OLR of at most 4.4 g VS/ L/ day could be achieved. Around

90% of the OLR came from forest residues where 45% of VS is lignin. Since lignin is not

digested in the biogas process, the OLR calculated from the remaining 55% of the VS is 2.2 g

13
VS/ L/ day. This is a more moderate OLR and given the stable process shown in the present

study, it points toward the possibility of using a higher OLR, while still maintaining stable

conditions. A higher OLR would mean a better utilization of any given biogas plant.

Furthermore, with an OLR of 2.2 g VS/ L /day, the VS-reduction will increase from 8% and 20%

to 15% (SD ± 6) and 33% (SD ± 10) for digesters 1 and 2, respectively. Hence, NMMO-

treatment enables a doubling of the digestibility of forest residues. Nonetheless, there is still two-

thirds of the total digestible VS left in the digestate residue; thus, further work needs to be done

to increase the VS-reduction during the anaerobic digestion of forest residues. The lignin-rich

digestate residue contains a high heating value, and can be used as fuel for combustion in

combined heat and power (CHP) plants (22). The water content of the digetate should be

decreased to 45% TS prior to combusion (23).

In a previous study performed by Shafiei et al. (24), a techno-economic analysis for ethanol

production from wood based on NMMO pretreatment was developed and the process was

designed to utilize 200,000 tons of spruce wood per year. The wastewater from this process with

a large amount of unutilized pentoses was directed to an anaerobic digestion process for

production of biogas. According to this study the bioethanol production in combination with

biogas production using NMMO pretreated spruce as feedstock would be a feasible process. The

total energy output in form of ethanol, lignin, and methane were calculated to be 134 MW/year

and the share of the heat value generated from lignin residues is around 66 MW/year (24). The

total energy output based on the results in this present study can be calculated to about 85

MW/year, when 200,000 tons (dry weight) forest residues are utilized for biogas production in a

continuous process. The energy output from lignin is about 80 MW/year which is 21% higher

than for spruce 66 MW/year considering that spruce has higher carbohydrate content and lower

14
lignin content than the forest residues used in this study. The production of biogas from

lignocelluloses however would have a higher overall energy efficiency comparing to that for

ethanol production, hence pentoses can also be utilized in biogas production (25).

The methane yield obtained during the continuous process is approximately 60% lower than the

methane potential measured in the batch assay for both pretreated and untreated forest residues

(Figure 2 vs Figure 1). The lower yield could be due to the lower retention time of 20 days used

for the digestion of the substrate in the continuous process compared with 52 days digestion

period in the batch process. The accumulated methane yields observed after 20 days of digestion

time in the batch assay were about 25 Nml/g VS added for untreated and 64 Nml/g VS added for the

pretreated material (Figure 1). Comparing these data with the yield of biogas production of 53

and 92 NmL/gVS/day, during continuous digestion of untreated and treated forest residues,

respectively (Figure 2), and assuming 50% methane in the produced biogas from carbohydrates

(26), it can be concluded that the results from batch and continuous digestions are in accordance

with each other. This also explains the relatively low VS reduction obtained.

Moreover, addition of carbon-rich materials, such as lignocelluloses, to digesters treating waste

mixtures with low C/N ratios has previously shown to enhance the nutritional balance and

stabilize sensitive processes (27). It was also shown (28) that the material and the ratio, by which

the forest residues are co-digested with, would have a significant effect for the economy of the

process. Using OFMSW for co-digestion instead of sludge and decreasing the ratio of forest

residues in the mixture would increase the methane yield considerably, since OFMSW has higher

methane potential than sludge.

15
CONCLUSION

Today, forest residues are available for energy production in Sweden on a scale of 59 TWh/year

(3). However, the use of this feedstock for biogas production is limited due to the lack of an

efficient pre-treatment enabling digestion of the cellulose and hemi-cellulose in the forest

residue. The present study shows the possibility of one pre-treatment method; however, an

economic and technical assessment of its industrial use needs to be performed in the future. One

aspect not evaluated in this study is the quality of the digestate. Since 45% of the substrate is

lignin that is not degradable, hence remains in the digestate residue, which would after de-

watering have a potential value as a fuel for combustion.

16
NOMENCLATURE

NMMO = N-Methylmorpholine-N-oxide (NMMO)

OLR= Organic loading rate

HRT= Hydraulic retention time

VFA= volatile fatty acids

VS= volatile solids

TS= total solids

SD= Standard deviation

HPLC= High performance liquid chromatography

GC= Gas chromatography

CSTR= Continuous stirred tank reactor

WWTP= Wastewater treatment plant

17
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Bioenergy. 36, 116-120.
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Christensen, T. H. (2004) Waste Manage. (Oxford). 24, 393-400.
16. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D. and Crocker, D.
(2008) Determination of Structural Carbohydrates and Lignin in Biomass. Standard
Biomass Analytical Procedures. National Renewable Energy Laboratory.
17. Teghammar, A., Yngvesson, J., Lundin, M., Taherzadeh, M. J. and Sárvári Horváth, I.
(2010) Bioresource Technology. 101, 1206-1212.
18. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J. and Templeton, D. (2005)
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21. Schink, B. (1997) Microbiology and Molecular Biology Reviwes. 61, 262-280.

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(2013) Energy Fuels. 27, 277-284.
28. Teghammar, A., Forgács, G., Sárvári Horváth, I. and Taherzadeh, M. J. (2014) Applied
Energy. 116, 125-133.

19
FIGURES

120
untreated
100 90, 3h , 85 %
120, 3h, 85%
Volume Nml/gVS

80
120, 15h, 75%
60

40

20

0
0 10 20 30 40 50 60
Time (day)

Figure 1. Accumulated methane production on NMMO-pretreated and untreated forest residue at

mesophilic batch condition. The pretreatment conditions are described in the figure.

20
 140

120

100
Volume (Nml/gVS)

80

60

40

20

0
0 20 40 60 80 100 120 140
Time (Day)

Figure 2. Specific gas production for untreated forest residues (●...) and NMMO-treated forest

residue (o…)

21
 A
7,9 5

Organiic loading rate

7,8
7,7 4

[g VS/L & day]

7,6 3
pH

7,5
7,4 2
7,3 1
7,2
7,1 0
0 10 20 30 40 50 60 70 80 90 100 110

B
8 5

Organiic loading rate

7,8 4

[g VS/L & day]

7,6 3
pH

7,4 2
7,2 1
7 0
0 10 20 30 40 50 60 70 80 90 100 110 120 130

Time [Days]

Figure 3. pH (♦) and organic loading rate (▬) for the reactors fed with (A) untreated forest

residue and (B) NMMO-treated forest residues

22
 TABLES.

Table 1. Pretreatment condition, Methane production, Carbohydrates, lignin of untreated and

NMMO- Pretreated forest residue

Pretreatment conditions Methane production

Treatment Total Volatile Total Total Initial reaction
Temperature NMMO time solids Solids lignin carbohydrates rate Yield
°C (%) (h) (%) (%) (wt%) (%) (Nml/g VS/day)a (Nml/gVS added)b

120 °C 75 15 30.5 29.69 36.48 49.05 4.27 ± 1.5 100.5±16

120 °C 85 3 26.77 26.04 38.67 45.3 3.65 ± 1.0 109,5±20
90 °C 85 3 27.33 26.48 40.55 41.8 2.75 ± 0.6 87,68±17
Untreated - - 50.7 45.64 45.75 41.05 0.83 ± 0.4 41,53±3,0

a Reaction rate during the first 10 days with two standard deviations.
b Accumulated methane gas produced per gram volatile solids after 52 days of incubation with
two standard deviations

23
III
 Renewable Energy 52 (2013) 128e135

Contents lists available at SciVerse ScienceDirect

Renewable Energy
journal homepage: www.elsevier.com/locate/renene

High-rate biogas production from waste textiles using a two-stage process

Azam Jeihanipour a, Solmaz Aslanzadeh b, Karthik Rajendran b, *, Gopinath Balasubramanian b,
Mohammad J. Taherzadeh b
a
Department of Biotechnology, Faculty of Advanced Sciences and Technologies, University of Isfahan, Isfahan 81746-73441, Iran
b
School of Engineering, University of Borås, 50190 Borås, Sweden

a r t i c l e i n f o a b s t r a c t

Article history: The efficacy of a two-stage Continuously Stirred Tank Reactor (CSTR), modified as Stirred Batch Reactor
Received 3 July 2012 (SBR), and Upflow Anaerobic Sludge Blanket Bed (UASB) process in producing biogas from waste textiles
Accepted 30 October 2012 was investigated under batch and semi-continuous conditions. Single-stage and two-stage digestions
Available online 22 November 2012
were compared in batch reactors, where 20 g/L cellulose loading, as either viscose/polyester or cotton/
polyester textiles, was used. The results disclosed that the total gas production from viscose/polyester in
Keywords:
a two-stage process was comparable to the production in a single-stage SBR, and in less than two weeks,
Rapid digestion
more than 80% of the theoretical yield of methane was acquired. However, for cotton/polyester, the two-
Biogas
NMMO pretreatment
stage batch process was significantly superior to the single-stage; the maximum rate of methane
UASB production was increased to 80%, and the lag phase decreased from 15 days to 4 days. In the two-stage
NMMO semi-continuous process, where the substrate consisted of jeans textiles, the effect of N-methyl-
Waste textiles morpholine-N-oxide (NMMO) pretreatment was studied. In this experiment, digestion of untreated and
NMMO-treated jeans textiles resulted in 200 and 400 ml (respectively) methane/g volatile solids/day
(ml/g VS/day), with an organic loading rate (OLR) of 2 g VS/L reactor volume/day (g VS/L/day); under
these conditions, the NMMO pretreatment doubled the biogas yield, a significant improvement. The OLR
could successfully be increased to 2.7 g VS/L/day, but at a loading rate of 4 g VS/L/day, the rate of methane
production declined. By arranging a serial interconnection of the two reactors and their liquids in the
two-stage process, a closed system was obtained that converted waste textiles into biogas.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction production of different biofuels, such as biogas [2]. For instance, in

The annual global production of end-of-life waste textiles is
steadily increasing, causing an increasing concern regarding the
impact of the disposal of this enormous amount of waste on the
environment. In spite of textile waste in fact being a potentially rich
source of energy and materials, the current normal routine to
dispose of this waste is by incineration or as landfilling. Interest-
ingly, of the world’s total textile production, around 40% of the fiber
consumption comprises cellulose [1], the same percentage as the
average content of cellulose in lignocellulosic materials.
Waste textiles are mainly composed of cotton and viscose fibers,
and holds, thanks to their cellulose content, a significant potential for
Abbreviations: CSTR, continuously stirred tank reactor; SBR, stirred batch
reactor; UASB, upflow anaerobic sludge blanket bed; GC, gas chromatography; IC,
ion-exchange chromatography; HPLC, high performance liquid chromatography;
VFA, volatile fatty acid; AMPTS, automatic methane potential testing system.
* Corresponding author. Tel.: þ46 33 435 4855; fax: þ46 33 435 4008.
E-mail address: Karthik.Rajendran@hb.se (K. Rajendran).
2008, the influx of clothing and textiles to Sweden was 131,800 tons
[3]. Assuming that the total amount of waste textiles in Sweden nearly
equals the amount of imported clothing and textiles, and that 40% of
the textile fibers consists of cellulose, approximately 53,000 tons of
cellulose is wasted every year. A yield of 415 ml methane (at STP) per g
cellulose implies that the amount of waste textiles produced in
Sweden would suffice as substrate for producing more than 20
million Nm3 of methane, equaling in the region of 4 TWh power per
year; to be compared with 11 TWh estimated to be the biogas
potential of ley crops, straw, potato, and sugar beet tops in Sweden [4].
Fossil fuels are currently dominating the global energy market.
However, the growing world population along with diminishing
fossil fuel reserves have resulted in a global interest in gradually
shifting the energy source from fossil to alternative fuels [5,6]. In
addition, environmental pollution caused by e.g. the dumping of
waste materials in the environment, is one of the most important
issues the world is facing today. Biogas, produced by means of
anaerobic digestion of biological waste, is a renewable bioenergy
and a potential alternative to petroleum-based fuels [7]. In addition

0960-1481/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.renene.2012.10.042
 A. Jeihanipour et al. / Renewable Energy 52 (2013) 128e135
to the methane itself, biogas production holds the potential to
minimize the waste pollution, thus protecting the environment [8].
From the perspective of resource efficiency, biogas production has
a higher outputeinput energy ratio compared to, for example,
current ethanol production systems [9]. Furthermore, in terms of
emissions, biogas production might be better for the environment
than incineration of waste [10,11].
Methane-rich biogas has different applications. It may serve as
a source for heat, steam, and electricity, and can be further upgraded
to vehicle fuel, or for production of chemicals. It may also be used as
a household fuel for cooking and lighting, or in fuel cells. Taking all
these aspects into account, being a well-established technology for
generating bioenergy, biogas production is one of the most envi-
ronmentally beneficial processes for replacing fossil fuels [8,12].
Furthermore, development of new technologies has facilitated
biogas production for combined heat and power (CHP) systems in
small scale (100 KWe) [13]. Thanks to biogas production being an
uncomplicated process, which is a significant advantage, it can be
located near the place where waste is produced, and the waste
producers can be the end-users of the biogas, hence evading prob-
lems related to transport of both wastes and biogas. Such systems
(so-called on-farm biogas plants), have in Germany been commer-
cially installed in thousands [14], mainly using biomass from agri-
culture. Apart for the conventional waste streams such as municipal
solid wastes (MSW) and manure, the recent trend includes the
pretreated lignocellulosic biomass, wooden fractions and agricul-
tural residues are used for biogas production [15].
However, the potential of other available biological wastes, such
as cellulosic waste textiles, as substrate in small-scale biogas plants,
has not been adequately investigated. From the literature, there
was very little work that has been focused on the textile waste as
a substrate for anaerobic digestion. Previous work includes pre-
treating waste textile containing cellulosic blend fibers and high
crystalline cellulose fibers in a batch assay [16,17] in order to
increase the biogas production. Additionally, textile wastes were
never tested in a two-stage process.
The present study is focused on investigating the feasibility of
using waste cellulosic textile fibers for production of biogas,
employing different processes and comparing their efficacy.
2. Materials and methods
The first step was to examine a one-stage batch process (i.e. in an
SBR) and a two-stage (i.e. in an SBR and a UASB) anaerobic digestion,
using two different substrates (viscose and cotton fibers, both
blended with polyester fibers), without separating the cellulosic
fibers. In the two-stage process, textile was converted into biogas in
a closed system, which was obtained by arranging a serial inter-
connection of the liquids of both reactors. However, previous
attempts have been made to separate cellulose from mixed fibers
[18] by e.g. dissolution of cellulose [19] and subsequently regener-
ating it. Jeihanipour et al. [16] recently developed a process for
separating the cellulosic part from waste textiles, using an environ-
mental friendly cellulose solvent, i.e. N-methylmorpholine-N-oxide
(NMMO), in order to facilitate the production of biogas or bioethanol
from waste textiles [17]. Hence, the second step of the present study
comprised a semi-continuous two-stage anaerobic digestion
process, comparing biogas yield from NMMO-treated and untreated
cotton-based waste textiles, at different organic loading rates.
2.1. Materials and inoculums
The three waste textiles used in the present study were woven
textiles: orange (50% polyester, 50% cotton), blue (40% polyester, 60%
viscose), both provided by local shops in Borås (Sweden), and also
129
used blue jeans textiles (100% cotton). Prior to the experiments, the
first two textiles were cut into small pieces (approximately 2.5  2.5
cm2), while the jeans textiles were ground into fine materials. NMMO
was provided by BASF (Ludwigshafen, Germany) as a 50% water
solution.
The inoculum used in the CSTRs and SBRs was obtained from
a 3000-m3 municipal solid waste digester, operating under ther-
mophilic (55  C) conditions (Borås Energy & Environment AB,
Sweden). The granulated anaerobic sludge used as seed in the UASB
reactors was provided from a UASB reactor treating municipal
wastewater in Hammarby Sjöstad (Stockholm, Sweden).
2.2. Pretreatment procedure
For pretreatment of the ground jeans textiles, the NMMO
solution was concentrated to 85% in a rotary vacuum evaporator
(Laborota 20, Heidolph, Schwabach, Germany), equipped with
a vacuum pump (PC 3004 VARIO, Vacuubrand, Wertheim,
Germany). The concentrate was mixed with ground jeans textiles
(6% w/w dry matter) in an oil bath at 120  C for 3 h under atmo-
spheric conditions, using a mixer for continuous blending in order
to dissolve the cellulose [17]. The resulting celluloseeNMMO
solution was then added to boiling water while mixing continu-
ously, thereby regenerating the dissolved cellulose. Using a vacuum
filter, the regenerated cellulose was separated from the NMMOe
water solution, and washed with hot water. The washed cellulose
was stored wet at 4  C until used for anaerobic digestion.
2.3. Experimental setup
2.3.1. Reactors
Two types of reactors, a continuous flow stirred tank reactor
(CSTR), modified for batch process as stirred batch reactor (SBR)
and an upflow anaerobic sludge blanket bed (UASB) reactor, both
made of polymethylmethacrylate (PMMA), were used in different
configurations. The CSTR had a working volume of 3 L (an internal
diameter of 18.5 cm and a height of 18.5 cm), while the working
volume of the UASB was 2.25 L (an internal diameter of 6.4 cm and
a height of 70 cm). Temperature was set at 55  C for the CSTR and
SBR, and at 34  C for the UASB, using a temperature-controlled
water-bath with water recirculation through the reactor’s double
jacket. Both types of reactors were equipped with a feed inlet,
a liquid sampling point, an outlet, and a gas line to the gas
measuring system, which had a gas sampling port. The CSTR and
SBR were equipped with an impeller for continuous mixing of the
contents. The inlet of the UASB reactor had a mesh to avoid large
particles entering the system (Fig. 1B).
2.3.2. Reactor seeding and start up
The UASB reactors were seeded with 1.28 L of granular anaer-
obic sludge. The remaining volume of the reactor was filled with
water. Upon receipt, the inoculum for the CSTR was stored in an
incubator at 55  C for three days, to degrade easily degradable
organic matter still present in the inoculum, and to remove dis-
solved methane. The CSTR and SBR were filled with 2.5 L of inoc-
ulum and 0.5 L of nutrient solution to set the C:N:P:S ratio to
500:20:5:3, in accordance with the cellulose concentration in the
beginning of the experiment. The final nutrient concentrations for
the basic medium (1 g cellulose/L, containing inorganic macronu-
trients) were (in mg/L): NH4Cl (76.4), KH2PO4 (5.18), MgSO4$7H2O
(0.27), CaCl2$2H2O, (10.00), and trace nutrients, 1 ml/L [20].
2.3.3. Reactor configurations
In the present study, the efficacy of single-stage and two-stage
batch processes as well as a two-stage continuous process for
130 A. Jeihanipour et al. / Renewable Energy 52 (2013) 128e135
Fig. 1. Schematic diagram of the CSTReUASB combined system with internal recirculation. (A) Batch process equipped with internal filter in the SBR and (B) semi-continuous
process equipped with sedimentation tank.
anaerobic digestion of waste textiles was examined. The arrange-
ments of the reactor facilities are schematically illustrated in Fig. 1. In
the one-stage batch process, an SBR was used as a digester. In the
two-stage batch process, the SBR was serially connected with a UASB
reactor. Liquid effluent from the SBR was continuously pumped to
the UASB reactor at a rate of 3 L/day. At this flow rate, the hydraulic
retention times (HRT) in the SBR and the UASB reactors were ca. 24
and 18 h, respectively. A peristaltic pump with a tube diameter of
1.02 mm was used. The effluent of the UASB reactor was continu-
ously fed back to the SBR. The SBR of the two-stage batch process
was equipped with a cylindrical filter around the impeller, and the
textile wastes were placed inside the filter. The liquid outlet of the
SBR passed through the filter, while the textiles were retained within
the SBR. With this filter, the polyester part of the textile could be
recovered after the process was completed.
The configuration of the reactors in the two-stage continuous
process was quite similar to that in the two-stage batch process.
The difference was the removal of the internal filter in the SBR,
placing a sedimentation tank (with a volume of 100 ml) in liquid
line to the UASB, before the pump, to settle the large particles
(Fig. 1).
2.4. Experimental operations
The batch processes were conducted by feeding the SBRs with
cotton/polyester (50/50) and viscose/polyester (60/40) textiles, to
establish a cellulose concentration of 20 g/L. After 25 days, the
process was interrupted, and the remaining textiles were sepa-
rated, washed, and studied in a stereomicroscope. In the semi-
continuous processes, 2 two-stage systems were used to digest
ground jeans textiles and NMMO-treated jeans textiles. The OLR of
the process was increased stepwise from 2 up to 4 g VS/L/day. Once
a day, a certain amount of substrate was fed into the CSTR, in
accordance with the desired OLR. The HRT of UASB was controlled
by changing the speed of the pump in the beginning of each step.
Each OLR was continued for more than three HRTs in the CSTR,
when a steady state condition was attained. Table 1 describes the
conditions of the different steps during the process, including the
OLRs and their respective HRTs, flow rates, and durations.
No solids were withdrawn from the reactors during the exper-
imental period in neither the batch nor the continuous processes,
except when sampling for the analyses. Liquid and gas were
sampled twice a week throughout the running process, and the
Table 1
Organic loading rates (OLR), hydraulic retention times (HRT) in the CSTR and the
UASB reactor, and phase durations, determined at different stages.
Stage OLR (g VS/L/day) HRT in CSTR (day) HRT in UASB (day) Duration (day)
1 2.0 10.0 7.50 30.0
2 2.7 7.5 5.62 30.0
3 4.0 5.0 3.75 15.0
 A. Jeihanipour et al. / Renewable Energy 52 (2013) 128e135 131

volumes of produced gas were recorded. The gas samples were 60

analyzed directly by gas chromatography (GC), while the liquid Viscose/polyester
samples were stored in the freezer at 20  C for later analyses. 50

Methane (ml/gVS/day)
Cotton/polyester
40
2.5. Analytical methods
30

The cellulose content of the substrates was determined according 20

to the method provided by the National Renewable Energy Labora-
tory in the USA [21]. The gas production was recorded by using the 10
Automatic Methane Potential Testing System (AMPTS, Bioprocess
control AB, Lund, Sweden), whose function is based on water 0
displacement and buoyancy, with a measuring resolution of 13 ml. 0 5 10 15 20 25 30
The instrument was equipped with a laptop computer and the Days
volumes of produced gas vs. time were recorded for each reactor. The
composition of the biogas produced during anaerobic digestion was Fig. 2. Rate of methane production from (A) viscose/polyester and (C) cotton/poly-
measured using a gas chromatograph (Auto System Perkin Elmer, ester in the single-stage batch process.

Waltham, MA) equipped with a packed column (Perkin Elmer,

60  1.800 OD, 80/100, mesh) and a thermal conductivity detector
(Perkin Elmer) set to 200  C. The inject temperature was set to

Fig. 3. Stereomicroscopic pictures of viscose/polyester and cotton/polyester before and after single-stage and two-stage digestions.
132 A. Jeihanipour et al. / Renewable Energy 52 (2013) 128e135

Share of reactor in methane production

Viscose/polyester Untreated Jeans
100 7
1,6

Concentration in UASB (g/L)

Concentration in CSTR (g/L)
125 90 6
Methane (ml /gVS/day)

80
5 1,2
100
70
4
60
75 0,8

(%)
3
50
40 2
50 0,4
30 1

25 20 0 0
10 7 Pretreated Jeans

Concentration in CSTR (g/L)

Concentration in UASB (g/L)

1,6
0 0
6

Share of reactor in methane production

Cotton/polyester
100
5 1,2
125 90
Methane (ml/gVS/day)

4
80
100 0,8
70 3

60 2
75 0,4
(%)

50 1
40
50 0 0
30 0 10 20 30 40 50 60 70 80

25 20
Days
10
Fig. 6. Variation of VFA concentrations in (:) CSTR and (A) UASB during digestion of
0 0
untreated and pretreated jeans in the semi-continuous process.
0 5 10 15 20 25 30
Days

Fig. 4. Rate of methane production from (A) viscose/polyester and (C) cotton/poly-
150  C, and the oven temperature to 75  C. The carrier gas used was
ester in the two-stage batch process. The amount of methane, produced in each nitrogen, kept at a maintained pressure of 0.70 bar and a flow rate of
reactor, presented as (---) % share of the CSTR and (- - -) % share of the UASB. 40 ml/min at 60  C. A 250-ml pressure-tight gas syringe (VICI,
Precision Sampling Inc., LA) was used for the gas sampling.
Liquid samples were centrifuged at 10,000 rpm for 10 min, and
solid particles were removed by filtration through a 0.2-mm filter prior
Share of reactor in methane production

Untreated Jeans 100 to analyses for pH, soluble chemical oxygen demand (COD), and
500 90 volatile fatty acid (VFA) concentrations. The COD was measured using
Methane (mL/gVS/day)

80 an HACH apparatus equipped with a UVevis Spectrophotometer

400 70 (HACH, Germany), using Digestion Solution COD vials (operating
60 range 0e15,000 mg COD/L). The VFA concentrations, comprising
(%)

300 acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid,
50
40 and isovaleric acid, were analyzed by HPLC (Waters 2695, Waters
200 Corporation, Milford, MA, USA) with a UV detector (Waters 2414),
30
utilizing an ion-exchange column (Aminex HPX-87H Bio-Rad,
100 20
Hercules, CA) working at 60  C, and using 5 mM sulfuric acid as eluent,
10
with a flow of 0.6 ml/min. The macronutrients, ammonium and
0 0
potassium, were analyzed with an Ion Chromatograph (Metrohm,
Pretreated Jeans 100
Share of reactor in methane production

Herisau, Switzerland), using a cation column holding an eluent flow

500 90 rate of 1 ml/min; the pressure was set at 7e9 MPa, and the temper-
Methane (mL/gVS/day)

80 ature was 35e40  C. The eluent solution consisted of 4 mmol tartaric

400 70 acid and 0.75 mmol dipicolinic acid per L water. Samples were diluted
60 with the eluent, and pH was adjusted to 2e3. They were then
300
50 centrifuged at 10,000 rpm for 4 min, and filtered through a 0.45 mm
(%)

40 filter prior to injection. The texture of the textiles before and after
200
30 batch digestion was studied, using a NIKON stereomicroscope
(SMZ800, Tokyo, Japan) fitted with a C-DSD230 camera.
100 20
10
0 0 3. Results and discussion
0 10 20 30 40 50 60 70 80
Days 3.1. Batch digestion

Fig. 5. Rate of methane production from untreated and pretreated jeans textiles in the
semi-continuous process. The different line styles represent: () methane volume, (---) %
3.1.1. Single-stage anaerobic digestion in the SBR
share of methane produced in the CSTR and (- - -) % share of methane produced in the The cumulative methane produced during 25 days of single-
UASB. stage anaerobic digestion in the SBR is presented in Fig. 2. The
 A. Jeihanipour et al. / Renewable Energy 52 (2013) 128e135 133

Table 2
The COD, the ratio of methane to carbon dioxide in the CSTR and the UASB reactor, and the COD removal efficiency of the UASB reactor, during digestion of untreated and
pretreated jeans at different stages. The efficacy of the UASB-digesters (expressed as COD removal efficiency in percent) was calculated by dividing the difference between COD
inlet and outlet with the COD inlet.

Substrate OLR (g VS/L/day) COD (mg/L) COD removal efficiency (%) Ratio of methane to carbon
dioxide

CSTR UASB CSTR UASB

Untreated jeans 2.0 6169  1348 2027  626 66.8  9.7 1.46  0.09 5.33  0.55
2.7 4395  1234 1198  235 72.0  3.3 1.97  0.26 5.40  0.46
4.0 2873  562 1276  185 56.6  10.5 2.17  0.04 5.03  0.20
Pretreated jeans 2.0 4377  652 2019  638 53.4  14.4 1.92  0.29 4.72  0.53
2.7 3212  416 1822  239 42.2  11.5 2.14  0.13 4.18  0.15
4.0 2833  267 2220  305 22.9  14.5 2.18  0.06 3.63  0.31

reactor fed with viscose/polyester textiles had a 2-day-long lag
phase, whereas in the reactor fed with cotton/polyester, the cor-
responding lag phase before gas production was triggered was
about 15 days long. The longer lag phase may be due to the different
texture of cotton/polyester, providing a smaller contact surface area
for cellulolytic microorganisms to work on. Once the biogas
production started, the production rate from cotton/polyester was
slower than from viscose/polyester. The theoretical methane yield
was calculated according to Buswell formula [22], which is 415 ml/g
VS for cellulose. Within 12 days of gas production from viscose/
polyester, more than 80% of the theoretical yield of methane was
acquired, to be compared with the 17% yield from cotton/polyester,
gained during the 10 days following the lag period (Fig. 2). Differences
in contact surface area, molecular structure of cellulose, and chem-
istry of the dyes and reagents covering the cotton and viscose fibers,
are possible reasons for this huge difference in digestion outcome
between the viscose/polyester and cotton/polyester waste textiles
utilized in these experiments. The maximum rate of biogas produc-
tion from viscose/polyester reached 55 ml/g VS/day after 8 days.
The appearances of the textiles used in the batch process, as
shown in stereomicroscopy before and after digestion, are pre-
sented in Fig. 3. The microscopy revealed that viscose/polyester had
disintegrated fibers compared to cotton/polyester, consequently
facilitating the process of degradation of viscose/polyester by
microorganisms. Single-stage and two-stage digestion of viscose/
polyester (Fig. 3C and E) did not differ much, while degradation of
cotton/polyester was more successful in the two-stage process than
in the single-stage digestion (Fig. 3D and F).
3.1.2. Two-stage anaerobic digestion
The cumulative methane production acquired over 25 days, and
its share (in percentage) of the SBR and the UASB reactor, is pre-
sented in Fig. 4. Though the gas production from both textiles
(cotton/polyester and viscose/polyester) started after three days,
the initial rate of biogas production from viscose/polyester was
superior compared to the initial production rate of cotton/poly-
ester, where it was low in the single-stage digestion as well. The
total gas production from viscose/polyester did not differ between
the single-stage process and the two-stage process.
Jeihanipour et al. [16] reported that under batch conditions,
methane yield from untreated cotton/polyester was lower than
from viscose/polyester; after six days of digestion, only about 4.95%
of the theoretical yield was acquired from cotton/polyester, while
36.28% was produced from viscose/polyester. In the present study,
however, biogas production from cotton/polyester reached 40% of
the theoretical yield after 10 days of digestion, while 80% of the
theoretical yield was attained from viscose/polyester after 12 days
of digestion. The maximum rate of methane production from

2800 A C 700
Ammonium (mg/L)

Potassium (mg/L)

2400 600
2000 500
1600 400
1200 300
800 200
400 100

0 0
2800 B D 700
Potassium (mg/L)

2400 600
Ammonium (mg/L)

2000 500
1600 400

1200 300

800 200

400 100

0 0
0 10 20 30 40 50 60 70 80 0 10 20 30 40 50 60 70 80
Days Days

Fig. 7. Variation of ammonium and potassium concentrations in the CSTR (A and C) and the UASB (B and D) during digestion of (A) untreated and (-) pretreated jeans in the semi-
continuous process.
134 A. Jeihanipour et al. / Renewable Energy 52 (2013) 128e135
cotton/polyester in the two-stage process reached 30.6 ml/g VS/day
on day 8, and in the single-stage process, this textile produced
a maximum methane volume of merely 17 ml/g VS/day, which was
achieved only on day 17. This implies an 80% yield increase when
using the two-stage process rather than the single-stage SBR
(during this time period). This efficacy increase may be due to
a more efficient conversion of VFA into methane in the UASB
process than in a single-stage process. The SBR produced the major
share of gas from both textiles, as compared to the UASB reactor.
Since the textiles were neither milled nor pretreated, the contact
surface area available for the microorganisms’ degradation of the
textiles was probably low.
3.2. Semi-continuous two-stage anaerobic digestion
3.2.1. Gas production
The accumulated volume of methane produced per gram VS per
day from untreated jeans and NMMO-pretreated jeans are presented
in Fig. 5, which also illustrates the share of methane production in the
CSTR and the UASB reactor, expressed as percentage. The volume of
methane produced per gram VS per day increased with an increased
OLR. Comparing biogas production from untreated and treated jeans
revealed that an OLR of 2 g cellulose/L/day (stage 1) produced 200 ml/
g VS/day from untreated jeans, but more than 400 ml/g VS/day from
treated jeans, i.e. pretreatment increased methane production with
100%. Furthermore, an accumulation of VFA in the CSTR with
untreated jeans evidently resulted in a lower methane production
during the experimental period (Fig. 6). When increasing the OLR
from 2.0 to 2.7 g VS/L/day, the microorganisms adapted to the
conditions, resulting in acquiring 91% of the theoretical methane yield
from untreated jeans and 96% from treated jeans. However, increasing
the OLR to 4.0 g VS/L/day did not improve the methane production
any further. The CSTR was responsible for the largest share of the total
methane production (w90%) from treated jeans, most likely as
a result of the enzymatic degradation of the cellulosic part of the
textiles being facilitated by the pretreatment.
The only comparable information found was the application of
rumen microorganisms in combination with a high-rate UASB using
filter paper cellulose as substrate produced 438 ml/g VS/day, which
is equivalent to 98% of the theoretical yield. This slightly higher yield
compared to present study could be explained by presence of
ruminant bacteria which have high efficiency to hydrolyze even the
cellulose based material with high-crystallinity [23].
3.2.2. COD and COD removal efficiency
The chemical oxygen demand (COD) during the operation,
measured from the influent and effluent of the UASB reactor, is
illustrated in Table 2. The efficacy of the UASB digestion of
untreated jeans textiles increased from 66.8% to 72.3% when
increasing the OLR from 2.0 to 2.7 g VS/L/day and decreasing the
HRT from 10 to 7.5 days. A further increase in the OLR to 4.0 g VS/L/
day decreased, however, the COD removal efficiency to 56.6% when
processing untreated jeans in the UASB reactor. When treated jeans
textiles were used, a decrease trend (from 53.4% to 22.9%) in the
COD removal efficiency was observed when the OLR was increased
from 2.0 to 4.0 g VS/L/day. The COD in the CSTR decreased with
increasing OLR and decreasing HRT, when processing untreated as
well as pretreated jeans textiles. Furthermore, during the entire
process, the COD in the UASB reactor processing untreated jeans
decreased from 2027 mg/L to 1276 mg/L while a more stable COD
around 2000 mg/L was established when digesting pretreated
jeans. An increase in the OLR decreased the COD in the CSTR,
regardless of textiles having undergone pretreatment or not.
However, an increase in OLR resulted in a decreasing efficiency of
the COD removal in the UASB reactor.
Mahmoud et al. [24] studied the COD removal efficiency of
a single-stage UASB reactor and a combined UASB-digester system,
and found that the COD removal efficiency was higher in the
combined system (30%) compared to the single-stage system
(about 5%). In the present study, for the complete process, the
average COD removal efficiency was 65.1% for untreated jeans and
39.5% for treated jeans.
3.2.3. Effect of nutrients
The concentrations of the macronutrients (ammonium and
potassium) during the process, are illustrated in Fig. 7. The nutrient
concentration decreased with time in both digesters for both
textiles which was due to activity of the cells to remove COD,
produce biogas and of course some biomass. The final ammonium
concentrations (Fig. 7A and B) in the CSTR and the UASB were in the
range of 600e800 mg/L, while the potassium concentration (Fig. 7C
and D) decreased to around 150 and 200 mg/L in the CSTR and the
UASB, respectively. After decreasing the nutrients to a minimum
level, in spite of no nutrients or water being added to or removed
from the system, the two-stage process was still able to produce
biogas with a good yield. This observation may be because of
endogenous metabolism which causes autohydrolysis of some of
the biomass present in the reactor.
3.2.4. Ratio of methane to carbon dioxide
The ratio of methane to carbon dioxide in each stage is illus-
trated in Table 2. By increasing the OLR from 2.0 to 4.0 g VS/L/day,
the ratio in the CSTR increased from 1.46 to 2.17 and from 1.92 to
2.18 for treated jeans and untreated jeans, respectively. However,
the ratio for untreated jeans in the UASB reactor was stable at
around 5 throughout the experimental period, while the ratio for
treated jeans during the same period, decreased from 4.72 until
3.63. Accumulation of VFA in the CSTR with untreated jeans,
increased the ratio of methane to carbon dioxide in the UASB
reactor. Pretreatment with NMMO decreased the crystalline
structure, and increased the digestibility of the material. Conse-
quently, during digestion of treated jeans, the ratio of methane to
carbon dioxide increased in the CSTR but decreased in the UASB
reactor. The treated jeans textiles were easily degraded to methane
in the CSTR with no accumulation of VFA.
4. Conclusions
The comparison of single-stage and two-stage batch digestion
processes for producing biogas from cotton/polyester and viscose/
polyester with no pretreatment or milling revealed that gas
production efficacy is highly affected by the molecular structure of
the textile. In the semi-continuous process, pretreatment of textiles
had a significant effect on the biogas production, due to a more
accessible surface area for the degradation of cellulose fibers.
Despite the complex structure of cotton/polyester, the initial rate of
biogas production was higher and the lag phase shorter in the two-
stage batch process, in comparison with the single-stage CSTR. It
was furthermore concluded that when digesting treated or
untreated jeans textiles, the semi-continuous two-stage process
was able to handle a high OLR with a shorter HRT, in the CSTR as
well as in the UASB reactor.
Acknowledgement
This work was financed by Borås Energy & Environment AB and
the Sparbank foundation in Sjuhärad (Sweden). The authors would
like to acknowledge Adib Kalantar Mehrjerdi for the design and
construction of the reactors and Michael Lacintra for technical
support in the laboratory.
 A. Jeihanipour et al. / Renewable Energy 52 (2013) 128e135 135

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IV
Energies 2013, 6, 2966-2981; doi:10.3390/en6062966
OPEN ACCESS

energies
ISSN 1996-1073
www.mdpi.com/journal/energies
Article

The Effect of Effluent Recirculation in a Semi-Continuous

Two-Stage Anaerobic Digestion System
Solmaz Aslanzadeh 1,*, Karthik Rajendran 1, Azam Jeihanipour 2
and Mohammad J. Taherzadeh 1
1
School of Engineering, University of Borås, Borås 501 90, Sweden;
E-Mails: karthik.rajendran@hb.se (K.R.); mohammad.taherzadeh@hb.se (M.J.T.)
2
Department of Biotechnology, Faculty of Advanced Sciences and Technologies,
University of Isfahan, Isfahan 81746-73441, Iran; E-Mail: a.jeihanipour@gmail.com

* Author to whom correspondence should be addressed; E-Mail: solmaz.aslanzadeh@hb.se;

Tel.: +46-33-435-4620; Fax: +46-33-435-4008.

Received: 5 May 2013; in revised form: 5 June 2013 / Accepted: 9 June 2013 /
Published: 17 June 2013

Abstract: The effect of recirculation in increasing organic loading rate (OLR) and
decreasing hydraulic retention time (HRT) in a semi-continuous two-stage anaerobic
digestion system using stirred tank reactor (CSTR) and an upflow anaerobic sludge bed
(UASB) was evaluated. Two-parallel processes were in operation for 100 days, one with
recirculation (closed system) and the other without recirculation (open system). For this
purpose, two structurally different carbohydrate-based substrates were used; starch and
cotton. The digestion of starch and cotton in the closed system resulted in production of
91% and 80% of the theoretical methane yield during the first 60 days. In contrast, in the
open system the methane yield was decreased to 82% and 56% of the theoretical value, for
starch and cotton, respectively. The OLR could successfully be increased to 4 gVS/L/day
for cotton and 10 gVS/L/day for starch. It is concluded that the recirculation supports the
microorganisms for effective hydrolysis of polyhydrocarbons in CSTR and to preserve the
nutrients in the system at higher OLRs, thereby improving the overall performance and
stability of the process.

Keywords: two-stage anaerobic digestion; recirculation effect; UASB; CSTR;

cotton; starch
Energies 2013, 6 2967

1. Introduction

Anaerobic digestion is gaining more attention nowadays, both as a solution to environmental

concerns, and also as an energy resource for today’s energy-demanding life style [1]. Biogas is a
product of anaerobic digestion processes, which is produced by a consortium of microorganisms. The
anaerobic digestion process is highly dependent on a variety of different factors such as pH,
temperature, HRT, carbon to nitrogen ratio, etc., [2–4]. However, the anaerobic degradation process is
a quite slow and sensitive process, which is highly affected by environmental stress and alterations in
operating conditions [5], that would lead to a disturbance of the balance in the microbial community.
The consequence of this imbalance is usually process failure. Stability of an anaerobic process,
especially in an industrial scale, is thus a vital factor for evaluation [6].
The microbial community in an anaerobic digestion comprise of fermentative, acetogenic and
methanogenic microorganisms. In general, methanogens have slower growth rates compared to
hydrolytic and acetogenic organisms, and are more sensitive to environmental stress. Efficient
anaerobic digestion requires the development and maintenance of a large, stable and viable population
of methane-forming microorganisms [5]. The most common reactor configuration used for anaerobic
digestion is the continuously stirred tank reactor (CSTR), in which the active biomass is constantly
removed from the system. These conventional systems usually have long retention times. This
drawback has been overcome using a high rate system, which is basically based on immobilization of
the active biomass which enables short retention times. It is because the sludge retention time is more
or less independent of the hydraulic retention time [7–9]. The microorganisms in the upflow anaerobic
sludge blanket (UASB) reactors are kept in the reactor by their ability to flocculate and produce
granules and thereby give the sludge good settling properties [9–11].
Two-stage anaerobic digestion process is considered to be effective when the rate limiting step in
the process is hydrolysis and liquefaction [12]. It consists of two separate reactors; one for
hydrolysis/acidogenesis and one for acetogenesis/methanogenesis. This physical separation makes it
possible to overcome the problem of the differences in the optimum conditions of the microorganisms’
activity and their growth kinetics [12] by optimizing conditions that are favorable to the growth of
each group of microorganisms in each reactor, such as short HRT and low pH for acid formers, which
is inhibitory for methanogens [13]. This type of phase separation would increase the stability of the
process, which is not possible in a conventional anaerobic process, where these two groups of
microorganisms are kept together in a single phase in a delicate balance [14]. Ever since the phase
separation was introduced into anaerobic digestion technology in 1970s, a significant number of papers
and reports have been published on the benefits of treating a variety of wastes at mesophilic as well as
thermophilic conditions such as treating fruits and vegetables [15,16], urban wastewaters [17],
industrial wastes [18], grass [19], coffee pulp juice [20], food wastes [21], cane-molasses alcohol
stillage [22], spent tea leaves [23], dairy wastewater [24–27], olive mill oil [28], and abattoir
wastes [29]. However, two-stage digestion processes have been used for treatment of wastes with very
low solid content [30–33]. The drawback of UASB is that this technology is not able to handle high
solid content [34]. Apart from this, data concerning the optimization of operating conditions, operation
and performance of two-phase configuration are inadequate as well. There is a lack of investigations
Energies 2013, 6 2968

on how effluent recycling affects the two stage process with high solid content and high organic
loading rate [35].
This paper investigates the effect of the effluent recirculation in a high rate semi-continuous two
stage anaerobic process using carbohydrate-based starch and cotton as substrate with high solid content
at various organic loading rates and hydraulic retention times.

2. Materials and Methods

2.1. Materials and Inoculums

The substrates used in this study were pure cotton and starch provided from local shops in Borås
(Sweden). The cotton was ground into fine materials before using them. The volatile solid of the cotton
and starch was 96% and 75%, respectively. The COD of the both materials were 1.19 kgCOD/kg of
the materials [36]. The inoculum used in the CSTR bioreactors was obtained from a 3000-m3 digester
treating municipal solid waste and working under thermophilic (55 °C) condition (Borås Energy &
Environment AB, Borås, Sweden). The UASB reactors were seeded using granulated anaerobic
sludge, which was provided from a pilot scale UASB reactor treating municipal wastewater at
Hammarby Sjöstad (Stockholm, Sweden) operating at 37 °C.

2.2. Experimental Set-Up

2.2.1. Reactors

The CSTR and UASB reactors were made of polymethylmethacrylate (PMMA), and used in
different configurations. The CSTR had a working volume of 3 L with an inner diameter of 18.5 cm
and a height of 18.5 cm, while the working volume of the UASB was 2.25 L with an internal diameter
of 6.4 cm and a height of 70 cm. Temperature of the reactors was sustained at 55 °C for CSTR and
34 °C for UASB by a thermal water-bath with water recirculation through the reactor’s water jacket
during the whole digestion process. Both reactors were equipped with a feed inlet, a liquid sampling
point, an effluent outlet, and a gas line to the gas measuring system which contained a gas sampling
port. The CSTR had an impeller for continuous mixing. The inlet from the bottom of the UASB reactor
was equipped with a net trap to prevent the large particles away from entering the reactor (Figure 1).

2.2.2. Reactors Seeding and Start Up

The UASB reactors were seeded with 1.3 L of granular anaerobic sludge and the remaining volume
of the reactors were filled with water. The inoculum for the CSTR was incubated at 55 °C for three
days in order to get stabilized before use, and remove the dissolved methane. The CSTR’s were filled
with 2.5 L of inoculum and 0.5 L of nutrient solution in which the C:N:P:S ratio was adjusted at
500:20:5:3 at the beginning of the experiment. The nutrient concentration for 1 g cellulose/L contained
basal medium with inorganic macro nutrient (in mg/L): NH4Cl (76.4), KH2PO4 (5.18), MgSO4·7H2O
(0.27), CaCl2·2H2O (10), and 1 mL/L of trace nutrients according to [37].
Energies 2013, 6 2969

Figure 1. Schematic figure of the semi-continuous two-stage system. (A) with

recirculation (Closed system), and (B) without recirculation (Open system).

2.2.3. Reactors Configuration

The arrangement of the two stage closed system and the two stage open system is presented
schematically in Figure 1. The configuration of the closed system and open system continuous process
was quite similar. The difference was that in the closed system the effluent of the UASB reactor was
continuously recirculated back to the CSTR, while the open system did not have any effluent
recirculation from UASB [38]. The recirculation rate of the liquid in the closed system was 91% ± 3%.
The recirculation rate of the liquid is based on the HRT in each OLR, which is controlled by the flow
rate in the pump. In order to separate particulate matter from the CSTR effluent, the outlet of the
Energies 2013, 6 2970

CSTR was equipped with a sedimentation tank consisting of a 100 mL glass bottle, to separate and
settle the large particles before pumping the liquid to the UASB. The feeding to both systems was once
and twice a day depending on the OLR.

2.2.4. Experimental Procedure

The semi-continuous digestions (open and closed) were carried out by feeding the bioreactors with
OLRs increasing from 2 up to 20 gVS/L/day in several steps. Once a day, depending on the OLR, the
substrate was fed into the CSTR. The HRT of UASB was controlled by adjusting the speed of the
pump prior to each step. Each OLR was maintained for more than three HRTs in the CSTR in order to
achieve a steady state condition. The steady state condition in each OLR refers to the constant loading
rate and gas production, which was achieved during three HRT periods. The process conditions;
including the OLR and their respective HRT, flow rate and duration are summarized in Table 1.
During the experiments, no solids/biomass was withdrawn from the reactors, except for the sample
analyses. The volume of biogas produced was recorded continuously by Automatic Methane Potential
Testing System (AMPTS, Bioprocess Control AB, Lund, Sweden) and gas chromatography. The liquid
and gas sampling were performed twice a week during the initial state of the process and increased to
every day from stage 4–6 due to short retention times. The liquid samples were kept at −20 °C until the
analyses were performed.

Table 1. The process conditions including the OLR and their respective HRT, flow rate
and duration in each stage of the experimental period.
Stage OLR (gVS/L/day) HRT in CSTR (day) HRT in UASB (day) Duration (day)
1 2.0 10.0 7.50 30.0
2 2.7 7.5 5.62 30.0
3 4.0 5.0 3.75 15.0
4 8.0 2.5 1.88 8.0
5 10 2.0 1.50 6.0
6 20 1.0 0.75 6.0

2.2.5. Analytical Methods

The production of biogas was recorded using AMPTS, operating based on water displacement. It
was equipped with a computer to record the biogas volume from each reactor. The composition of the
biogas produced during anaerobic digestion was measured using a gas chromatograph (Auto System
Perkin Elmer, Waltham, MA, USA), equipped with a packed column (Perkin Elmer, 6’ × 1.8’’OD,
80/100 Mesh) and a thermal conductivity detector (Perkin Elmer) with an inject temperature of 150 °C,
detection temperature of 200 °C, and oven temperature of 75 °C. The carrier gas used was
nitrogen-operated at a maintained pressure of 0.70 bar and a flow rate of 40 mL/min at 60 °C. A
250 µL pressure-tight gas syringe (VICI, Precision Sampling Inc., Baton Rouge, LA, USA) was used
for the gas sampling.
Liquid samples were analyzed for pH, soluble chemical oxygen demand (COD), and VFA
concentrations after centrifugation at 17,000 g for 10 min and subsequent filtration through a 0.2-µm
Energies 2013, 6 2971

filter to remove solid particles. The COD was measured using a HACH apparatus equipped with a
UV–Vis Spectrophotometer (HACH, Düsseldorf, Germany), with Digestion Solution COD vials
(operating range 0–15,000 mg COD/L). The VFA concentrations, including acetic acid, propionic acid,
butyric acid, isobutyric acid, valeric acid and isovaleric acid, were analyzed by HPLC (Waters 2695,
Waters Corporation, Milford, MA, USA), equipped with an ion-exchange column (Aminex HPX-87H
Bio-Rad, Hercules, CA, USA), working at 60 °C using 5 mM sulfuric acid as eluent with a flow of
0.6 mL/min, and a UV detector (Waters 2414, Milford, MA, USA). The macronutrients, including
ammonium and potassium were analyzed using an Ion Chromatography (Metrohm, Herisau,
Switzerland) working with a cation column at an eluent flow rate of 1 mL/min, pressure of 7–9 MPa,
and temperature of 35–40 °C. The eluent solution was composed of 4 mM/L tartaric acid and
0.75 mM/L dipicolinic acid in water. Before injection, the samples were diluted with eluent, the pH
was adjusted to 2–3, were then centrifuged at 17,000 g for 4 min and filtered through a 0.45 µm filter.

3. Results

Cellulose and starch were used as a substrate in a semi-continuous two-stage anaerobic digestion
process for biogas production. The substrates were digested separately, in two CSTRs with an OLR of
2 gVS/L/day, which was then increased stepwise up to 20 gVS/L/day for starch and 4 gVS/L/day for
cotton. The HRT was decreased in each step, and the reactors were continuously operated for three
consecutive HRTs to obtain steady state condition. The total methane with its percentage share of
methane production in CSTR and UASB produced per gram VS per day for the operational period of
90 days of digestion is presented in Figure 2.

Figure 2. Total methane production in open system for cotton and starch with --- % share
in CSTR and - - - % share in UASB.

450 Cotton 100

Share of methane production (%)

400 90
CH 4 volume (ml/gVS/day)

80
350
70
300
60
250
50
200
40
150
30
100 20
50 10
0 0

450 Starch 100

Share of methane production (%)

400 90
CH 4 volume (ml/gVS/day)

80
350
70
300
60
250
50
200
40
150
30
100 20
50 10
0 0
0 10 20 30 40 50 60 70 80 90 100

Days
Energies 2013, 6 2972

3.1. Gas Production

3.1.1. Biogas Production in Open System (without Effluent Recirculation)

During the startup for cotton, the maximum methane production reached 363 gVS/L/day at day 12,
and then kept stable for 8 days before it started to decrease to 150 mL/gVS/d. Even after the increase
in OLR up to 2.7 gVS/L/day, the methane production remained stable at 150 mL/gVS/d for 10 days.
However, after day 40, the gas production decreased to less than 100 mL/gVS/d. An additional
increase in OLR to 4 gVS/L/day produced only 84 mL/gVS/d until day 75. Further increase in OLR to
8 gVS/L/day resulted in reactor failure, and the experiment was stopped.
On the other hand for starch, the process could be continued up until OLR 20 gVS/L/day for
95 days, while adding 2 gVS/L/day OLR, 340 mL/gVS/d of methane was produced. However, further
increase in OLR to 2.7 gVS/L/day resulted in decreased biogas production and just 45% of the
theoretical methane yield was achieved. A significant shift in the share of methane production from
CSTR to UASB could be observed at OLR 2.7 gVS/L/day. During the OLRs 4–10 gVS/L/day, the
theoretical methane yield was constant around 55%–60%. Furthermore, the accumulation of VFA in
CSTR was increased to more than 8 g/L during the same period (Figure 3C). In addition, the methane
production decreased from 230 mL/gVS/d to 128 mL/gVS/d when OLR was increased from
10 to 20 gVS/L/day. Starch had a more stable process during the first stage compared to cotton, which
could not reach steady state conditions even at low OLRs.

Figure 3. Volatile fatty acid concentration during the experimental period. ●- Starch;
♦- Cotton. (A) CSTR closed system; (B) UASB closed system; (C) CSTR open system;
(D) UASB open system.

10 A B 10

8 8
Total VFA (g/L)

VFA (g/L)

6 6
Total

4 4

2 2

0 0
C D
1 1
Total VFA (g/L)

0,8 0,8
VFA (g/L)

0,6 0,6
Total

0,4 0,4

0,2 0,2

0 0
0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100

Days Days
Energies 2013, 6 2973

3.1.2. Biogas Production in Closed System (with Effluent Recirculation)

The two-stage process in closed system was more stable compared to the open system. The
accumulated methane volume produced per gram VS per day for cotton and starch in the closed system
are presented in Figure 4. The percentage share of methane production in CSTR and UASB are also
marked in the same figure.
For cotton, the OLR could be increased from 2 to 2.7 gVS/L/day, and it resulted in a theoretical
methane yield of 85% and 76%, respectively. Further increase of OLR to 4 g VS/L/d caused a rapid
decline in the total gas production and the process was stopped.
In the case of starch, the theoretical methane yield was higher than 90% in the OLRs of 2 and
2.7 gVS/L/day. Additional decrease in HRT and increase in OLR up to 10 gVS/L/day stepwise
resulted in a theoretical methane yield of 50%–60%. The transition of the major share of methane
production from CSTR to UASB was observed at OLR 8 gVS/L/day. Though, the methane yield
between the OLR 4 to 10 gVS/L/day, the closed system possessed an overall stability. However, when
the OLR increased to 20 gVS/L/day, the gas production declined rapidly and the process was stopped.

Figure 4. Total methane production in closed system for cotton and starch with --- % share
in CSTR and - - - % share in UASB.

450 Cotton 100

Share of methane production (%)

400 90
CH 4 volume (ml/gVS/day)

80
350
70
300
60
250
50
200
40
150
30
100 20
50 10
0 0

450 Starch 100

Share of methane production (%)

400 90
CH 4 volume (ml/gVS/day)

80
350
70
300
60
250
50
200
40
150
30
100 20
50 10
0 0
0 10 20 30 40 50 60 70 80 90 100

Days
Energies 2013, 6 2974

3.2. COD and Its Removal

The COD was analyzed from the influent and effluent of the UASB during the operation. The
UASB digesters performance was examined using COD removal efficiency, calculated by dividing the
differences between COD inlet and outlet of UASB by the COD inlet to UASB. The results are
presented in Table 2. Equation (1) shows the calculation of the COD removal efficiency:
CODin − CODout
COD removal efficiency = ×100 (1)
CODin

3.2.1. Open System

The COD removal efficiency was greater than 95% throughout the process for starch. The COD
was increased from 4300 to 27,700 mg/L in the CSTR fed with starch by increasing the OLR from
2 to 10 gVS/L/day. Furthermore, when the OLR was increased further to 20 g VS/L/d for starch, the
COD was decreased to approximately 24,000 mg/L. The COD in the UASB on the other hand, kept
stable throughout the entire process between 3000 and 4000 mg/L.

Table 2. The ratio of methane to carbon dioxide, concentration of COD and the COD
removal efficiency for cotton and starch in UASB and CSTR during different organic
loading rates.
COD (mg/L) COD (mg/L)
Substate

OLR Open system COD removal Closed system COD removal

(gVS/L/day) efficiency (%) efficiency (%)
CSTR UASB CSTR UASB
2 3,259 ± 638 1,194 ± 95 61.9 ± 16.3 4,204 ± 742 1,958 ± 791 49.2 ± 21.6
Cotton

2.7 3,699 ± 844 229 ± 82 93.3 ± 3.5 2,651 ± 572 1,525 ± 172 39.4 ± 16.5
4 3,241 ± 545 196 ± 33 93.9 ± 1.8 2,571 ± 204 1,476 ± 357 45.6 ± 15.9
2 4,324 ± 1,345 1,308 ± 335 69.7 ± 14.2 5,041 ± 430 3,273 ± 674 35.0 ± 12.8
2.7 9,034 ± 1,127 317 ± 143 96.49 ± 1.2 4,463 ± 626 3,353 ± 374 24.8 ± 16.2
Starch

4 19,500 ± 2,493 350 ± 165 98.2 ± 2.1 4,396 ± 565 3,051 ± 494 30.5 ± 7.8
8 21,450 ± 2,185 708 ± 186 96.6 ± 2.7 17,865 ± 2,767 3,091 ± 423 82.69 ± 3.8
10 27,716 ± 1,606 876 ± 270 96.8 ± 1.3 25,833 ± 3,333 3,995 ± 873 84.5 ± 4.2
20 23,800 ± 2,347 898 ± 98 96.2 ± 2.5 12,516 ± 2,171 3,256 ± 658 73.9 ± 9.5

In contrast, for cotton in open system, the COD removal efficiency was as high as 93%.
Interestingly, in the open system the increase in OLR from 2 to 4 gVS/L/day, did not significantly
change the COD of the CSTR fed with cotton which was stable around 3500 mg/L. The COD
concentration in the UASB on the other hand, decreased from 1194 mg/L to 196 mg/L during the
same period.
Energies 2013, 6 2975

3.2.2. Closed System

In the closed system digesting starch, the COD removal efficiency of UASB reactor performance of
starch increased from 35% to 84.5% with increasing in OLR from 2 to 10 g VS/L/d and decreasing in
HRT from 7.5 to 1.5 days. A further increase in OLR up to 20 gVS/L/day decreased the COD removal
efficiency of starch in UASB with more than 10%. The effluent COD out of UASB of starch during
the entire process in closed system was stable, even though the OLR increased and the HRT decreased
compared to the open system, which were more stable between 3000 and 4000 mg/L. A decreasing
trend was observed for the COD removal efficiency in cotton in closed system, in which a reduction
from 49.2% to 45.6% was occurred by increasing the OLR from 2 to 4 gVS/L/day.

3.3. Effect of Nutrients

The effects of macronutrients, including ammonium and potassium were studied and the results of
ammonium and potassium concentration during the entire experimental period are illustrated in
Figures 5 and 6.
A decreasing trend of nutrient concentration was observed for cotton and starch in both the open
and closed systems. In the closed system, the final ammonium concentration in CSTR and UASB was
four times higher than in the open system at OLR of 8 to 10 gVS/L/day for both the substrates. An
interesting observation was obtained in OLR between 10 and 20 gVS/L/day. The concentration of
ammonium show a sudden increase in the CSTR fed with starch in the closed system from 300 mg/L
to more than 1300 mg/L (Figure 5A).

Figure 5. The ammonium concentration during the experimental period. ●- Starch;

♦- Cotton. (A) CSTR closed system; (B) UASB closed system; (C) CSTR open system;
(D) UASB open system.

3000
A-CSTR C-CSTR 3000

2500 2500
Ammonium (mg/L)
Ammonium (mg/L)

2000 2000

1500 1500

1000 1000

500 500

0 0

3000 B-UASB D-UASB 3000

2500 2500
Ammonium (mg/L)
Ammonium (mg/L)

2000 2000

1500 1500

1000 1000

500 500

0 0
0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100

Days Days
Energies 2013, 6 2976

The same trend could also be observed in the potassium concentration for both starch and cotton as
the potassium concentration declined in CSTR in both systems. However, in the closed system, the
concentration was maintained between 100 and 500 mg/L (Figures 6A,C). On the other hand, in open
system the concentration of potassium decreased to less 100 mg/L in both CSTR and UASB
(Figure 6B,D).

Figure 6. The potassium concentration during the experimental period.●- Starch;

♦- Cotton. (A) CSTR closed system; (B) UASB closed system; (C) CSTR open system;
(D) UASB open system.

800 A-CSTR C-CSTR 800

700 700

Potassium (mg/L)
Potassium (mg/L)

600 600

500 500

400 400

300 300

200 200

100 100

0 0
B-UASB D-UASB
600 600

500 500
Potassium (mg/L)

Potassium (mg/L)
400 400

300 300

200 200

100 100

0 0
0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100
Days Days

3.4. Ratio of Methane to Carbon Dioxide

Anaerobic digestion of the carbohydrate in starch and cotton results in 50% methane
(CH4/CO2 = 1 mol/mol) in the biogas formed. However, partial dissolution of carbon dioxide in water
can lead to higher content of methane in the formed biogas. On the other hand, if the earlier steps in
the digestion process (e.g., hydrolysis and acidogenesis) occur and methanogenic bacteria fail to
produce methane, this CH4/CO2 ratio approaches to zero, since CO2 is still produced.
The ratio of methane to carbon dioxide in each stage of the experiments in this work is illustrated in
Table 3. The increase in OLR had a significant effect on the methane to carbon dioxide ratio for both
open and closed systems in CSTR.
In the CSTR open system for starch, the ratio CH4/CO2 ratio decreased from 2 to almost 0.1 at OLR
10 gVS/L/day. During the same period, the closed system could maintain a high ratio around 0.8. In
UASB, in contrast the ratio was stable throughout the process for both systems. However, while
adding 20 gVS/L/day OLR, the ratio was decreased for starch in the open system.
Energies 2013, 6 2977

The CH4/CO2 ratio for cotton in CSTR was very stable in both open and closed system and did not
show any significant change as the OLR increased from 2 to 4 gVS/L/day, being stable around 4.9.
The CH4/CO2 ratio for cotton in UASB was somewhat lower than in CSTR for both systems, being
around 2, and remained stable and during the entire process.

Table 3. The Ratio of methane to carbon dioxide, in open and closed system, for cotton
and starch in UASB and CSTR.
Ratio of methane to carbon dioxide
Substrate OLR (gVS/L/day) Open system Closed system
CSTR UASB CSTR UASB
2 1.8 ± 0.2 4.4 ± 0.8 1.9 ± 0.2 4.4 ± 0.4
Cotton 2.7 1.7 ± 0.1 4.9 ± 0.6 2.0 ± 0.1 4.2 ± 0.2
4 2.0 ± 0.0 4.9 ± 0.2 2.1 ± 0.0 4.1 ± 0.1
2 2.0 ± 1.0 4.0 ± 0.5 2.1 ± 0.2 4.1 ± 0.6
2.7 1.3 ± 0.3 4.5 ± 0.6 2.2 ± 0.0 3.4 ± 0.2
4 1.0 ± 0.1 4.9 ± 0.2 1.8 ± 0.5 3.4 ± 0.1
Starch
8 0.3 ± 0.2 4.4 ± 0.1 0.9 ± 0.1 4.2 ± 0.3
10 0.1 ± 0.0 4.2 ± 0.2 0.8 ± 0.1 4.7 ± 0.4
20 0.07 ± 0.04 3.5 ± 0.8 0.7 ± 0.1 4.3 ± 0.8

4. Discussion

The results of this comparative study suggest that the recirculation in the closed system increases
the stability and the performance of a two stage system, using substrates at high OLRs. A higher
methane production was also achieved in the closed system comparing to open system for both
substrates. The major share of methane production seems also to be higher in CSTR at lower OLRs
rather than UASB for both processes and substrates. However, an interesting transition pattern is
observed in both systems as the major share of methane production is shifted from CSTR to UASB
Figures 2 and 3. This shift, however appear to occur at earlier stages in the open system comparing to
the closed system.
During OLR 2–2.7 gVS/L/day in the closed system the major share of the methane, produced in the
UASB was around 90% in CSTR for both cotton and starch. However, the increase in OLR to
4 gVS/L/day decreased the total methane yield and the transition of the major share of methane
production from CSTR to UASB begins. Additional increase of OLR to 8 gVS/L/day in closed system
digesting starch shifted the major share of methane produced shifted from CSTR to UASB and it
continued to increase with increasing OLR.
In the open system this transition was also observed, but at lower OLR (2.7 gVS/L/day) for both
substrates. The decrease in methane yield, which is the starting point of the transition, could be
explained by the accumulation of VFA in CSTR from less than 1 g/L to more than 8 g/L. The pH was
more stable in the closed system, which could be due to the effect of effluent recirculation from UASB
with pH around 8 to the CSTR and thereby stabilizing and keeping a stable pH over 6 in CSTR (data
not shown). Furthermore, the VFA produced in the CSTR was converted to biogas in the
UASB without accumulating in the first phase and reaching inhibitory levels for the acidogenesis
Energies 2013, 6 2978

process, which consequently contributes to the stability of the closed system in comparison to the open
system. This transition on the other hand, was never reached in the closed system digesting cotton. A
combination of the composition and efficient hydrolysis and the conversion of the intermediates to
methane in the CSTR due to the effect of recirculation causing higher and stable pH can be the
possible explanation [39].
The COD removal efficiency and the COD concentration in the CSTR were also affected by
recirculation as it started to increase during the same time as the transition occurs. The COD
concentration in the CSTR is highly dependent on the hydrolysis of the organic material to VFA and
follows more or less the same trend. The COD removal efficiency was higher, around 95%, in the open
system comparing to closed system which started for starch at the OLR 8 gVS/L/day to almost 85%.
However the COD efficiency in closed system could also be increased at higher OLR comparing to
open system. This also shows that UASB is more efficient at higher COD concentrations and could
handle high OLRs.
In contrast, the concentration of COD in the UASB decreased as the OLR increased in the open
system compared to closed system. The COD concentration in the closed system stayed stable between
1500 and 2000 mg/L for cotton and around 3000 mg/L for starch. It could be because of some
solubilized material kept recirculating in the system, and thereby keeping the COD both higher and
stable in closed system for both substrates, without having any considerable effects on the process.
Furthermore, when the OLR was increased further to 20 g VS/L/d for starch in open system, the COD
concentration in CSTR was decreased to approximately 1000 mg/L. This observation indicates that the
capacity of the CSTR to hydrolyze cotton and starch is limited. This capacity was obtained as less than
4 gVS/L/day for cotton, and 10–20 gVS/L/day for starch.
The ratio of methane to carbon dioxide was increased in UASB and decreased in CSTR in the
closed system. The increased CH4/CO2 ratio in the UASB could be due to dissolution of some part of
the produced carbon dioxide in the UASB and the capacity of the media in the UASB to capture and
further convert the carbon dioxide to methane by methanogens [39]. In the CSTR open system for
starch, the CH4/CO2 ratio decreased from 2 to almost 0.1 at OLR 10 gVS/L/day. During the same
period, the closed system could maintain a high ratio around 0.8. As the pH falls in the CSTR, the
more CO2 is dissolved to compensate as buffering system. A too strong acidification, consumes the
entire CO2 produced to keep the pH stable, which consequently inhibits the methanogens [40], and
hence, lower CH4/CO2 ratio in the CSTR open system comparing to the CSTR closed system. This is
an indication that recirculation could be able to support the microorganisms for effective hydrolysis in
CSTR. In UASB, the ratio was stable throughout the process for both systems.
In the closed system, the final ammonium concentration in CSTR and UASB was four times higher
than in the open system for both starch and cotton. The closed system supported the maintenance of
the nutrients in the system, compared to the open system, where fresh nutrients were added every day.
Since no liquid was removed or added to the system, the nutrients kept recycling in a closed cycle in
the process, leading to negligible loss of nutrients compared to the open system. An interesting
observation was obtained in OLR between 10 and 20 gVS/L/day in CSTR closed system. The
concentration of ammonium show a sudden increase at OLR 10–20 gVS/L/day in CSTR closed system
fed with starch (Figure 5A). This could be explained by the fact that shorter HRT, which is
accomplished by the increase in flow rate, causes high upflow velocities and thereby turbulence in the
Energies 2013, 6 2979

UASB. The consequence of this high flow rate is granule disintegration as the effect of shearing. The
resulting fragments are then washed out of the reactor [41] and are migrated to the CSTR by
recirculation. The subsequent degradation of the biomass and the release of the proteins into the
medium in CSTR cause an increase in the ammonium concentration [42].

5. Conclusions

The effect of recirculation in a semi-continuous two-stage anaerobic digestion combining CSTR

and UASB was studied using starch and cotton as substrate. The comparison of the closed system with
open system revealed that higher theoretical yield of methane could be achieved in the closed system
compared to the open system. Furthermore, it can be concluded that the recirculation could support the
hydrolysis step as well as avoiding nutrient loss at higher OLR and thus improving the performance
and the stability of the process a great deal.

Acknowledgements

This work was financially supported by Sparbank foundation in Sjuhärad (Sweden) and Borås
Energy and Environment AB (Sweden). The authors acknowledge Gopinath Balasubramanian for
experimental, technical and analytical support.

Conflicts of Interest

The authors declare no conflict of interest.

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© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).
V
 1

2 A comparative study between single and two-stage anaerobic digestion

3 processes: Effect of organic loading rate and hydraulic retention time

5 Solmaz Aslanzadeh*, Karthik Rajendran, Mohammad J. Taherzadeh

7 School of Engineering, University of Borås, Borås, Sweden

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9

10

11 * Corresponding author:

12 Phone: +46-33 435 4620

13 Fax: +46-33 435 4008

14 E-mail: Solmaz.Aslanzadeh@hb.se

15

16

17

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1
29 Abstract
30
31 The effect of organic loading rate (OLR) and hydraulic retention time (HRT) was evaluated
32 by comparing single-stage and two-stage anaerobic digestion processes. Waste from food
33 processing industry (FPW) and organic fraction of municipal solid waste (OFMSW) were
34 used as substrates. The OLR was increased at each step from 2 gVS/l/d to 14 gVS/l/d and the
35 HRT was decreased from 10 days to 3 days. The highest theoretical methane yield achieved in
36 the single-stage process was about 84% for FPW during OLR 3 gVS/l/d at HRT of 7 days and
37 67 % for OFMSW at OLR of 2 gVS/l/d and HRT of 10 days. The single-stage process could
38 not handle further increase in OLR and decrease in HRT and the process was stopped. A more
39 stable operation was observed at higher OLRs and lower HRTs in the two-stage system. The
40 OLR could be increased to 8 gVS/l/d for FPW and to 12 gVS/l/d for OFMSW, operating at a
41 HRT of 3 days. The results show a conclusion of 26 % and 65 % less reactor volume for two-
42 stage processes compared to single-stage processes for FPW and OFMSW, respectively.
43 Key words: Anaerobic digestion, Single stage process, Two-stage process, Hydraulic
44 retention time (HRT), Organic loading rate (OLR), Food waste

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62

2
63 1 Introduction

64

65 The global energy demand is currently being met by coal and oil, which are depleting sources,
66 not to mention the rise in environmental problems that the use of the fossil fuel is causing. It
67 is estimated that the energy demand will increase by a factor between two and three during
68 this century (Weiland, 2010). Biogas from wastes could be a part of the solution in the
69 security of energy supply. According to Forgács (2012), about 10,000 biogas plants are
70 currently operating in Europe and the number of the plants is expected to increase by a factor
71 of five within 10 years.
72 The methane production process of organic wastes such as organic fraction of municipal solid
73 waste (OFMSW), and industrial waste is today carried out by a sequence of biochemical
74 transformations. The process can be generally separated into a first step where hydrolysis,
75 acidification and liquefaction occur and a second step where acetate, hydrogen and carbon
76 dioxide are converted into methane. All these reactions occur simultaneously in a single
77 reactor (Forster-Carneiro et al., 2008). A balanced anaerobic digestion process demands that
78 in both phases, the rates of degradation must be equivalent in size (Angelidaki et al., 1999).
79 Numerous studies have shown to improve the efficiency of single-stage reactors (Cecchi et
80 al., 1991; Climenhaga and Banks, 2008; Forster-Carneiro et al., 2008; Heo et al., 2004). High
81 methane yields have already been achieved during digestion at total solids content less than
82 5% total solid content (Cho et al., 1995; Heo et al., 2004; Verrier et al., 1987; Zhang et al.,
83 2007). However, in the single-stage processes, the organic loading rate (OLR) still remains
84 unsatisfactory, at 1–4 kgVS/m3/day (Cho et al., 1995; Heo et al., 2004; Verrier et al., 1987;
85 Zhang et al., 2007). The most important reason for this limitation is that higher OLRs cause
86 inhibition because of accumulated volatile fatty acids (VFAs) (Ahring et al., 1995). In
87 addition, at high OLRs, retention times (HRTs) should be sufficient for the microorganisms to
88 have enough time to degrade the substrate. Thus, there is a balance between OLR and HRT
89 that must be determined in order to optimize digestion efficiency and reactor volume
90 (Demirer and Chen, 2005).
91
92 The development of high rate reactors was based on immobilization of biomass in wastewater
93 treatment systems, which improved the degradation rate of anaerobic treatment systems by
94 decreasing the retention time. However, a drawback of these systems is that they are usually
95 suitable for dilute waste water streams, which contain around 3% total suspended solids with
96 a particle size less than 0.75 mm (Revitt et al., 2010). This means that substrate with high
97 solid content should be solublized before it can be entered in these high rate systems.
98 Therefore, a two-phase system is required in order to achieve a rapid digestion and more
99 stable operation and a higher organic loading capacity. However, there are very little
100 investigations on the application of substrate with high total solid content in two-stage
101 processes.
102
103 Our earlier studies on evaluating the two-stage process, based on pretreated and untreated
104 waste textile (Jeihanipour et al., 2013) and cotton and starch (Aslanzadeh et al., 2013) showed
105 that the two-stage process can be beneficial using substrate that are rather unconventional
106 substrate. These studies indicate that the structure and the degradability of the material is one
107 of the factors deciding the OLR and HRT.
108

3
109 In this work, the effects of OLR and HRT in conventional one stage and two-stage systems
110 using organic fraction of municipal waste (OFMSW) and food processing waste (FPW) with
111 high total solid content were compared. These two substrates are among the most common
112 waste streams that are currently used for biogas production, which principally relies on single-
113 stage systems.
114
115 2 Materials and Methods
116

117 2.1 Substrates and inoculums

118 The substrates used in this study includes organic fraction of municipal solid waste
119 (OFMSW), waste from food processing industry (FPW). The inoculum used in the continuous
120 stirred tank reactor (CSTR) were obtained from a 3000-m3 biogas plant (Borås Energy &
121 Miljö AB, Borås, Sweden), treating municipal solid waste at thermophilic (55 °C) conditions.
122 The UASB reactors were seeded using granulated anaerobic sludge, which was provided from
123 a pilot plant using upflow anaerobic sludge blanket (UASB) reactor treating municipal
124 wastewater at Hammarby Sjöstad (Stockholm, Sweden). The FPW was obtained from storage
125 tank with a retention time of 3-4 days, before it was fed to the digester.
126

127 2.2 Experimental set up

128 2.2.1 Reactors

129 The reactors, both CSTR and UASB were built in house from thermoplastic material
130 polymethylmethacrylate (PMMA). The CSTR had a working volume of 3 l with an inner
131 diameter of 18.5 cm and a height of 18.5 cm, whereas the working volume of the UASB was
132 2.25 l with an internal diameter of 6.4 cm and a height of 70 cm. Temperature of the reactors
133 were kept constant at 55 °C for CSTR and 34 °C for UASB by a thermal water-bath with
134 water recirculation through the reactor’s water jacket during the entire digestion process. The
135 reactors were equipped with a feed inlet, a liquid sampling point, an effluent outlet, and a gas
136 line connected to the gas measuring system containing a gas sampling port. The CSTR was
137 equipped with an impeller for continuous mixing. The inlet from the bottom of the UASB
138 reactor was provided with a mesh to stop the large particles from entering the reactor.
139 2.2.2 Reactor seeding and start up
140 The CSTR inoculums were incubated at 55 °C for three days in order to stabilize it before use.
141 The CSTR’s were filled with 3 l of inoculums at the beginning of the experiment. The UASB
142 reactors were inoculated with 1.3 l of granular anaerobic sludge and the remaining volume of
143 the reactors were filled with water.
144
145 2.2.3 Reactors configuration
146 In the single-phase, digestion a CSTR was employed. The two-stage continuous process, on
147 the other hand consisted of a CSTR connected to a UASB reactor. The liquid of the CSTR
148 was pumped continuously to the bottom of the UASB and the effluent of the UASB reactor
149 was continuously recirculated back to the CSTR. In order to separate particulate matter from
150 the CSTR effluent, the outlet of the CSTR was connected to a sedimentation tank consisting
151 of a 100 ml glass bottle, to separate and settle the large particles before pumping the liquid to
152 the UASB. The reactor configurations are illustrated in Figure 1. Both systems were fed once
153 a day.

4
154
155 2.3 Experimental procedure

156 The semi-continuous digestions were carried out by feeding the bioreactors during the start up
157 period by increasing the OLR by 0.5gVS/l/d until reaching the desired OLR (2gVS/l/d). The
158 HRT of UASB in the two-phase process was controlled by adjusting the speed of the pump
159 prior to each step. To achieve a steady state condition, each OLR was sustained for more than
160 three HRTs in the CSTR. There were no solids withdrawn from the reactors except for the
161 sample analyses during the entire experimental setup. The volume of biogas produced was
162 recorded continuously by Automatic Methane Potential Testing System (AMPTS, Bioprocess
163 Control AB, Sweden) and the composition of the gas was measured by gas chromatography.
164 The liquid and gas sampling were performed 3 times a week during the start of the process. At
165 higher OLRs and lower HRTs, however, the sampling was performed every day. The liquid
166 samples were kept at -20 °C until the analyses were performed.
167
168 2.4 Analytical method

169 The total solid (TS) and the volatile solid (VS) content of the substrates were determined
170 based on drying the samples to constant weight at 105 ºC and 575 ºC respectively (Sluiter et
171 al., 2005). The characterization of the substrate (Table 1) was carried out by Analys-&
172 Konsulat labouratoreiet (AK labbet, Borås, Sweden). The Kjeldahl nitrogen and protein
173 content of the substrates were determined according to Swedish standard method ss-en
174 25663/NMKL 6-4 (Swedish Standard Institute, 1984). Ammonium concentration was
175 measured according to method SIS 028134-1 (Swedish Standard Institute, 1976) and fat
176 content was determined by NMKL method 131 (Nordic Commity on Food Analysis, 1989).
177
178 The biogas production was measured using AMPTS, working based on water displacement. It
179 was equipped with a computer to record the volume of the biogas produced from each reactor.
180 The composition of the biogas produced was determined by a gas chromatograph (Auto
181 System Perkin Elmer, Waltham, MA), equipped with a packed column (Perkin Elmer,
182 6’x1,8’’OD, 80/100 Mesh) was used. The gas chromatograph was set at a thermal
183 conductivity detector (Perkin Elmer) with an inject temperature of 150 °C, detection
184 temperature of 200 °C, and oven temperature of 75 °C. The carrier gas used was nitrogen-
185 operated at a pressure of 0.70 bar and a flow rate of 40 ml/min at 60°C. A pressure-tight gas
186 syringe with a volume of 250 μl (VICI, Precision Sampling Inc., LA) was used for the gas
187 sampling.
188 Liquid samples were analyzed for pH, alkalinity, TS, VS, COD (soluble chemical oxygen
189 demand) and VFA. Prior to analysis of alkalinity, COD, VFA, the samples were centrifuged
190 at 17,000 g for 10 min and filtered through a 0.2-µm filter to remove solid particles. The COD
191 and ammonium concentration was measured using a HACH apparatus equipped with a UV–
192 Vis Spectrophotometer (HACH, Germany), with digestion solution COD and ammonium
193 vials operating range 0–15,000 mg COD/l and 0-80 mg/l ammonium. The total VFA
194 concentrations, based on acetic acid, propionic acid, butyric acid and valeric acid were
195 analyzed by HPLC (Waters 2695, Waters Corporation, Milford, MA, USA), operating with an
196 ion-exchange column (Aminex HPX-87H Bio-Rad, Hercules, CA), working at 60 °C using 5
197 mM sulfuric acid as eluent with a flow of 0.6 ml/min, and a UV detector (Waters 2414). The
198 total alkalinity was measured according to Forgács et al. (2012) based on an end point
199 potentiometric titration with 0.05 mol/l HCl to pH 4.0.
200

5
201 3 Results and Discussions

202 The effects of increasing OLR and decreasing HRT on the performance of a single-stage and
203 a two-stage process digesting FPW and OFMSW were evaluated. The OLR was increased
204 from 2 gVS/l/d and the HRT was decreased gradually from 10 days in 9 steps to OLR 14
205 gVS/l/d and HRT of 3 days.
206
207 3.1 Methane production

208 The average total methane produced at various OLRs and HRTs in the single-stage is
209 presented in Figure 2A. The average total methane production for FPW increased from 0.42
210 m3/kgVS to 0.44 m3/kgVS corresponding to 81 % and 84 % of the theoretical methane yield
211 at step 1 (OLR 2 gVS/l/d, HRT 10 days) and step 2 (OLR 3 gVS/l/d, HRT 7 days)
212 respectively. During the same period, the methane production for OFMSW decreased from
213 0.33 m3/kgVS to 0.24 m3/kgVS, which is 67 % and 48 % of theoretical methane yield,
214 respectively. Further increase to step 3 (OLR 3 gVS/l/d, HRT 5 days) failed the process for
215 both substrates.

216 The two-stage process, on the other hand showed higher overall stability compared to the
217 single-stage process at higher OLRs and lower HRTs (Figure 2B & C). The overall total
218 methane production for the CSTR digesting FPW showed a relatively stable performance up
219 to step 6 (OLR 8 gVS/l/d, HRT 3 days). During this period the theoretical methane yield
220 fluctuated between 82 % and 93 %. Additional increase in OLR decreased the methane yield
221 successively at each step. The digestion of OFMSW between on the other hand showed a
222 relative stable methane production until step 8 (OLR 12 gVS/l/d, HRT 3 days) with a
223 theoretical yield fluctuating between 60 % and 78 %. The process could not handle a further
224 increase in OLR to step 9 (OLR 14 gVS/l/d, HRT 3 days) and the methane yield was
225 decreased. The major share of methane was produced in CSTR in the two-stage process for
226 both substrates. However, this major share of methane production was shifted to UASB at the
227 last step for both FPW and OFMSW.

228 Compared to single-stage process, in two-stage process the OLR was increased by 167 % i.e.,
229 (from 3 gVS/l/d to 8 gVS/l/d) for FPW. Similarly, the HRT was decreased by 57 % (i.e. from
230 7 days to 3 days). The overall increase in OLR and the decrease in HRT have result in the
231 need of total reactor volume by 26% less than a single-stage process for FPW. Likewise for
232 OFMSW, the two-stage process could handle higher OLR by 333 % compared to single-stage
233 process (from 2 gVS/l/d to 12 gVS/l/d). The HRT and the overall reactor volume were
234 decreased by 70 % and 65 %, respectively.
235 The process fed with FPW showed less fluctuation and was easier to handle the changes in the
236 operational condition than OFMSW as in both single and two-stage processes. This could be
237 due to the combination of two factors; difference between the substrate and the solubilization
238 efficiency in the single-stage and two-stage processes. A previous study (Jash and Ghosh,
239 1996) shows that the maximum amount of methane produced in anaerobic digestion of solid
240 organic residue depend on the extent of solubilization of the organic material. The FPW was
241 prehydrolyzed and contained readily solubilized material in the feed thus much more stable
242 methane production was observed compared to OFMSW.
243

6
244 3.2 Volatile fatty acid and Alkalinity

245 The concentration of total VFA and individual fatty acids is presented in Figures 3 and 4 for
246 single-stage and two-stage process, respectively. The concentration of total VFA in single-
247 stage process at initial step (OLR 2gVS/l/d, HRT 10 days) was high around 4.4 g/l and 3.9 g/l
248 for FPW and OFMSW respectively. However, after the adaptation period the VFA
249 concentration decreased and continued to be stable until the last step (OLR 4 gVS/l/d, HRT 5
250 days) which it started to increase in the CSTR digesting OFMSW started to increase while the
251 CSTR digesting FPW kept stable under 1 g/l. The individual organic acid dominating in the
252 total VFA, with the highest concentration first was; valeric acid, for both substrates and
253 increased at the initial step to 2.2 g/l and 2.3 g/l in CSTR digesting OFMSW and FPW
254 respectively.
255 In the two-stage process on the other hand, the VFA concentration was stable between 0.3 g/l
256 to 0.5 g/l in the CSTR for both substrates until step 4 (OLR 5 gVS/l/d, HRT 3 days).
257 However, the concentration started to increase in the CSTR for both substrates at the end of
258 step 4 (OLR 5 gVS/l/d, HRT 4 days) and continued to increase with each step until the end of
259 the experiment and reached above 14 g/l and 16 g/l in CSTR digesting FPW and OFMSW
260 respectively. The concentration of total VFA in the UASB followed the same trend as CSTR
261 and was stable in the initial steps (OLR 2-5 gVS/l/d, HRT 10-3 days) under 0.2 g/l for both
262 substrates. Even though the concentration was raised in parallel at each step it kept under 1.6
263 g/l for both substrates until the end of the experimental period.
264 The valeric acid was also the dominating acid in the two-stage process as well and shows a
265 sudden increase to 1.3 g/l for in CSTR digesting FPW and to more than 3 g/l for CSTR
266 digesting OFMSW at step 5 (OLR 6 gVs/l/d, HRT 3) for both substrates. The other acids start
267 to rise during the last 3 steps (OLR 10-14 gVS/l/d, HRT 3 days) of the process for both
268 substrates.
269 The high fluctuation of total VFA concentration at the initial step in the single-stage process
270 indicates that the acidification is reversible at lower OLR and higher HRT. The increase in
271 VFA concentration reflects a kinetic disconnection between acid producers and consumers
272 and is characteristic for stress situation (Ahring et al., 1995). In the two-stage process, the
273 total VFA started to increase almost to the same level as single-stage but at much higher OLR
274 and lower HRT for both substrates. Even at higher VFA level than observed in single-stage
275 the methanogenesis activity was still high in the CSTR and the increase in VFA could be
276 handled. In a two-stage process, short HRT could be beneficial, as the optimum conditions of
277 the first stage should aim for the maximum rate of acid production. For a given substrate
278 concentration, the acid production is maximized for short HRTs (Dinopoulou et al., 1988).
279 The total alkalinity is an indication of buffering capacity of the system and was monitored
280 during the entire experimental period in both single and two stage processes and the results
281 are illustrated in Table 2 and Table 3. In the single-stage process, the total alkalinity shows a
282 decreasing trend during entire experimental period for both substrates from 3,188 mg/l to
283 1,695 mg/l in the CSTR digesting FPW and from 3,130 mg/l to 2,354 mg/l in the CSTR
284 digesting OFMSW (Table 1).
285 In the two-stage process the same trend was observed as single-stage for both substrates. In
286 the CSTR the alkalinity was fluctuated between 9,000 and 12,000 mg/l with the highest in the
287 earlier steps of the process, while in UASB it was stable between 11,000 and 12,000 mg/l
288 thorough all steps during the entire experimental period. In the CSTR digesting OFMSW was
289 slightly lower than FPW and fluctuated between 4,000 mg/l and 10,000 mg/l with the highest
290 alkalinity at the initial steps of the process which suggests that enhancement of the OLR

7
291 further would reduce the methanogenesis activity in the CSTR due to accumulation of VFA in
292 the CSTR for both single-stage and two-stage processes. Furthermore, in the UASB the
293 alkalinity decreased from 11,000 mg/l in step 1 to 6,248 mg/l at last step (OLR 14 gVS/l/d,
294 HRT 3 days), which indicates that the methanogenesis were inhibited. The reason for this
295 inhibition could be due to the high concentration of valeric acid. In the two-stage process, the
296 valeric acid concentration in the CSTR, digesting OFMSW, was higher than the concentration
297 in the CSTR for FPW. Previously, it has been shown that at higher concentration of butyrate
298 or valerate can inhibit the methanogenic activity (Ahring et al., 1995). This might be the
299 reason that the alkalinity was reduced at higher OLR and shorter HRTs in the UASB for
300 OFMSW.
301
302 3.3 Volatile solid reduction, Chemical oxygen demand (COD) and COD removal efficiency
303 and Ammonium
304 The VS-reduction in The CSTR was measured at each stage of the process for both substrates
305 In the single-stage process, the VS reduction of 95 % and 93 % was achieved at step 1 (OLR
306 2 gVS/l/d, HRT 10 days) for the CSTR digesting FPW and OFMSW, respectively (Table 2).
307 Further increase in OLR and decrease in HRT at step 2 did not affect the VS reduction
308 considerably, and it was stable about 94 % for both substrates, and increased to 97 % at the
309 last step in the CSTR digesting FPW.
310 The VS reduction was higher at earlier steps of the process in the two-stage process, when the
311 organic loading rate was lower and the HRT was higher (Table 3). The VS reduction for FPW
312 fluctuated between 86.2 % and 95.7 % between steps 1 and 5 (OLR 2-6 gVS/l/d, HRT 10-3
313 days). However, it starts to decrease from step 6 (OLR 8 gVS/l/d, HRT 3 days) with 86.2 % to
314 step 9 (OLR 14 gVS/l/d, HRT 3 days) with 57.7 %. The CSTR digesting OFMSW between
315 step 1 and step 7 (2-10 gVS/l/d, HRT 10-3 days) of the process the VS reduction fluctuated
316 between 95.1 % and 85.42 % with the highest VS reduction at the earlier steps of the process.
317 The VS reduction which is an indication of the treatment efficiency obtained in this study is in
318 accordance to previous study on two-stage process using fruit and vegetable waste which was
319 about 96% (Verrier et al., 1987). The VS reductions were high and stable the single-stage
320 process. In the two-stage process it observed to be higher at earlier steps with lower OLR and
321 higher HRT. The low HRT of 3 days did not affect the VS reduction, however the OLR
322 between 8 and 10 gVS/l/d seem to be the limitation for the system in the two-stage process.
323 The high VS reduction shows that the process is able to dissolve the particulate organic
324 material, which is confirmed by the increase in COD content in the CSTR and the COD
325 removal efficiency of the process.
326 In the single-stage process between step 1 (OLR 2 gVS/l/d, HRT 10 days) and step 3 (OLR 4
327 gVS/l/d, HRT 5 days) the COD concentration showed a slight decrease, but stable between
328 3,000 and 5,000 mg/l FPW. The COD concentration for the CSTR digesting OFMSW
329 decreased from 3,700 mg/l to 3,433 mg/l at step 1 and 2 respectively. In two-stage process,
330 the COD concentration of both CSTR and the UASB was monitored and the COD removal
331 efficiency was calculated based on the difference between the COD content of the influent
332 and the effluent of the UASB (Table 3).
333 The COD removal efficiency rose in the two-stage process with each step both in CSTR and
334 UASB. The COD efficiency fluctuated but observed to be increasing during the entire
335 experimental period for both substrate from approximately 42.3 % and 74 % for FPW and
336 from 21.3 % to 83 % for OFMSW. The COD content in the CSTR during the last 3 steps of
337 the experiment display a significant increase from approximately 6,800 mg/l to 15,000 mg/l
338 while it was stable around 3,000 mg/l in the UASB for both substrates. Earlier studies on the

8
339 hydrolytic and acidogenic process confirm that shorter HRTs slightly improves total
340 solubilization yield (De La Rubia et al., 2009). Increase in COD removal efficiency shows
341 that the methanogenesis in the CSTR is inhibited while the intermediate products were
342 digested in the UASB without accumulating in the system, increasing efficiency of the UASB
343 (Aslanzadeh et al., 2013).
344 The ammonium concentration showed a decreasing trend with increasing OLR and decreasing
345 HRT in both single as well as two-stage process in the single-stage process (Figure 5). In the
346 single-stage process, the concentration of ammonium decreased from 885 mg/l in the initial
347 step to 200 mg/l at the last step for the CSTR digesting OFMSW and from 990 mg/l to 350
348 mg/l for FPW during the same period (Figure 5A). In the two-stage process, the same trend
349 was observed as single-stage process (Figure 5B and 5C). The ammonium concentration
350 decreased slightly in CSTR from 1,860 mg/l to 1,500 mg/l for FPW while for OFMSW 1,830
351 mg/l to 800 mg/l. The ammonium concentration in the UASB was stable between 500 mg/l
352 and 700 mg/l throughout the entire process for both substrates. The decrease in ammonium
353 concentration in both single-stage and two-stage processes indicates that the capacity of the
354 CSTRs to fully degrade the particulate material at higher OLR and lower HRTs is reduced.
355 Ammonium is released during the degradation of organic matter and stand for the extent of
356 the hydrolysis process, primarily protein compounds (Forgács et al., 2013; Rincón et al.,
357 2009). However, this capacity limit is reached much sooner in single-stage process than in
358 two stage process.
359

360 4 Conclusion

361

362 The effect of OLR and HRT in single-stage and two-stage process using OFMSW and FPW
363 was evaluated. The single-stage process could at best handle an OLR of 3 gVS/l/d and a HRT
364 of 7 days. A more stable operation could be achieved at higher OLR and lower HRT in the
365 two-stage process. The two-stage process could handle an OLR of 8 gVS/l/d for FPW and up
366 to 12 gVS/l/d for OFMSW, while the HRT could also be decreased to 3 days. Using the two-
367 stage process, a higher theoretical yield and a lower reactor volume could be achieved
368 compared to the single-stage process.
369
370 Nomenclature
371
372 FPW-Food processing waste
373 OFMSW- organic fraction of municipal solid waste
374 OLR-Organic loading rate
375 HRT-Hydraulic retention time
376 VFA-volatile fatty acids
377 VS reduction-volatile solids reduction
378 TS-total solids

9
379 VS-Volatile solids
380 HPLC-High performance liquid chromatography
381 GC- Gas chromatography
382 CSTR- Continuous stirred tank reactor
383 UASB Upflow anaerobic sludge blanket
384
385

386 References
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388 imbalance in anaerobic digestors. Applied Microbiology and Biotechnology 43, 559-565.
389 Angelidaki, I., Ellegaard, L., Ahring, B.K., 1999. A comprehensive model of anaerobic
390 bioconversion of complex substrates to biogas. Biotechnology and Bioengineering 63, 363-
391 372.
392 Aslanzadeh, S., Rajendran, K., Jeihanipour, A., Taherzadeh, M.J., 2013. The Effect of
393 Effluent Recirculation in a Semi-Continuous Two-Stage Anaerobic Digestion System.
394 Energies 6, 2966-2981.
395 Cecchi, F., Pavan, P., Mata Alvarez, J., Bassetti, A., Cozzolino, C., 1991. Anaerobic digestion
396 of municipal solid waste: thermophilic vs. mesophilic performance at high solids. Waste
397 management & research 9, 305-315.
398 Cho, J.K., Park, S.C., Chang, H.N., 1995. Biochemical methane potential and solid state
399 anaerobic digestion of Korean food wastes. Bioresource Technology 52, 245-253.
400 Climenhaga, M., Banks, C., 2008. Uncoupling of liquid and solid retention times in anaerobic
401 digestion of catering wastes.
402 De La Rubia, M.A., Raposo, F., Rincón, B., Borja, R., 2009. Evaluation of the hydrolytic–
403 acidogenic step of a two-stage mesophilic anaerobic digestion process of sunflower oil cake.
404 Bioresource Technology 100, 4133-4138.
405 Demirer, G.N., Chen, S., 2005. Two-phase anaerobic digestion of unscreened dairy manure.
406 Process Biochemistry 40, 3542-3549.
407 Dinopoulou, G., Rudd, T., Lester, J.N., 1988. Anaerobic acidogenesis of a complex
408 wastewater: I. The influence of operational parameters on reactor performance.
409 Biotechnology and Bioengineering 31, 958-968.
410 Forgács, G., 2012. Biogas production from citrus wastes and chicken feather: pretreatment
411 and co-digestion, Chalmers University of Technology, Sweden.
412 Forgács, G., Lundin, M., Taherzadeh, M., Sárvári Horváth, I., 2013. Pretreatment of Chicken
413 Feather Waste for Improved Biogas Production. Applied Biochemistry and Biotechnology
414 169, 2016-2028.
415 Forgács, G., Pourbafrani, M., Niklasson, C., Taherzadeh, M.J., Hováth, I.S., 2012. Methane
416 production from citrus wastes: process development and cost estimation. Journal of Chemical
417 Technology & Biotechnology 87, 250-255.
418 Forster-Carneiro, T., Pérez, M., Romero, L.I., 2008. Anaerobic digestion of municipal solid
419 wastes: Dry thermophilic performance. Bioresource Technology 99, 8180-8184.
420 Heo, N.H., Park, S.C., Kang, H., 2004. Effects of mixture ratio and hydraulic retention time
421 on single-stage anaerobic co-digestion of food waste and waste activated sludge. Journal of
422 Environmental Science and Health, Part A 39, 1739-1756.

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423 Jash, T., Ghosh, D.N., 1996. Studies on the solubilization kinetics of solid organic residues
424 during anaerobic biomethanation. Energy 21, 725-730.
425 Jeihanipour, A., Aslanzadeh, S., Rajendran, K., Balasubramanian, G., Taherzadeh, M.J.,
426 2013. High-rate biogas production from waste textiles using a two-stage process. Renewable
427 Energy 52, 128-135.
428 Nordic Commity on Food Analysis, 1989. Fat. Determination according to SBR (Schmid-
429 Bondzynski-Ratslaff) in meat and meat products, NMKL method No 131, OSLO.
430 Revitt, M., Scholes, L., Donner, E., 2010. Priority pollutant behaviour in on-site treatment
431 systems for industrial wastewater. Urban Pollution Research Centre, Middlesex University,
432 UK.
433 Rincón, B., Borja, R., Martín, M.A., Martín, A., 2009. Evaluation of the methanogenic step of
434 a two-stage anaerobic digestion process of acidified olive mill solid residue from a previous
435 hydrolytic–acidogenic step. Waste Management 29, 2566-2573.
436 Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., 2005. Determination
437 of Ash in Biomass A national laboratory of the U.S. Department of Energy,Office of Energy
438 Efficiency & Renewable Energy, U.S.
439 Swedish Standard Institute, 1976. "Water quality-Determination of of ammonia nitrogen
440 content" SIS 28134, Artikel No STD-883,, Stochholm, sweden.
441 Swedish Standard Institute, 1984. Warer quality-Determination of kejldahl nitrogen-Method
442 after mineralization with selinium, ISO 5663,, Stockholm, Sweden.
443 Weiland, P., 2010. Biogas production: current state and perspectives. Applied Microbiology
444 and Biotechnology 85, 849-860.
445 Verrier, D., Roy, F., Albagnac, G., 1987. Two-phase methanization of solid vegetable wastes.
446 Biological wastes 22, 163-177.
447 Zhang, R., El-Mashad, H.M., Hartman, K., Wang, F., Liu, G., Choate, C., Gamble, P., 2007.
448 Characterization of food waste as feedstock for anaerobic digestion. Bioresource Technology
449 98, 929-935.
450
451
452
453
454
455
456
457
458
459
460
461
462

11
463 Table 1. Characterization of substrates OFMSW and FWP
464
Analyses FPW OFMSW
Total solid (TS) Wt% 7.95 13.1
Volatile solid (VS) Wt % 7.8 12.9
Carbohydrate Wt % 2.6 7.1
Fat Wt % 1.8 2.1
Protein Wt % 2.4 2.6
Kjeldahl nitrogen Wt % 0.38 0.41
Ammonium nitrogen Wt % 0.00056 0.00045
Volatile fatty acid (mg/l) 2133 -
465
466
467
468
469
470
471
472
473
474
475
476
477
478

12
479
480 Table 2. Operation condition in each step with connected organic loading rate, (OLR) and
481 hydraulic retention time (HRT) and the average chemical oxygen demand (COD), volatile
482 solid (VS) reduction and theoretical yield in the single-stage process.

OLR Operation VS Theorethical

Substrat Step HRT days Alkalinity
(gVS/l/d COD (mg/l) reduction methane
e s (days) (mg/l)
) (%) yields1 (%)

1 2.0 10.0 4000±1946 3188±136 95±2.1 81

30
FPW 2 3.0 7.0 3067±850 2866±369 94±2.5 84
21
3 4.0 5.0 5000±707 1695±350 97±1.4 2
15
1 2.0 10.0 3700±1212 3130±343 93±1.5 67
30
OFMSW
2 3.0 7.0 3433±321 2354±205 94±2.7 48
21
1
483 The theoretical methane yield is calculated based on fat, protein and carbohydrate content of the substrates. The
484 VFA concentration in FPW is not taken into account.

485
486

13
Table 3. Operation condition in each step with connected organic loading rate, (OLR) and hydraulic retention time (HRT) and the average
chemical oxygen demand (COD), volatile solid (VS) reduction and theoretical yield in the single-stage process

HRT Operation Theorethical

HRT in days COD
Step OLR in COD (mg/l) Alkalinity (mg/l) VS reduction (%) methane
CSTR Efficiency (%)
Substrate UASB yield(%)1
(gVS/l/d) (day) (day) CSTR UASB CSTR UASB CSTR
1 2.0 10.0 7.50 30 5667±1150 2367±231 56.9±10.8 10468±245 11714±614 95.2±1.2 82
2 3.0 7.0 5.25 21 4167±1154 2400±264 42.3±7.48 11906±404 12506±579 86.2±8.2 74
3 4.0 5.0 3.75 15 3000±400 1833±152 38.6±3.50 12516±292 11779±293 92.5±5.0 92
4 5.0 4.0 3.00 12 3050±212 1700±565 44.8±14.7 12131±584 12160±153 89.4±2.3 87
FPW 5 6.0 3.0 2.25 9 3700±283 1700±252 53.6±11.2 10465±116 11598±392 95.7±1,9 93
6 8.0 3.0 2.25 9 3100±141 1350±212 56.5±4.86 10442±311 11243±169 86.2±0.7 86
7 10.0 3.0 2.25 9 6800±1052 3200±135 53.7±1.13 10487±265 11130±109 79.1±2.1 72
8 12.0 3.0 2.25 9 11250±1768 3133±115 72.0±5.66 9506±81 11210±135 61.3±3.8 62
9 14.0 3.0 2.25 9 15000±2854 3900±657 74.0±8.00 9068±57 12116±162 54.7±5.4 57
1 2.0 10.0 7.50 30 4700±2253 2900±265 31.4±21.4 9625±1488 11652±315 94.2±1.2 72
2 3.0 7.0 5.25 21 3500±964 2733±702 21.3±6.1 9716±377 10942±364 95.1±0.5 77
3 4.0 5.0 3.75 15 2433±305 1733±306 27.4±20.2 10293±1410 10073±696 86.5±3.6 66
4 5.0 4.0 3.00 12 1950±919 1800±435 26.3±6.4 90421±201 8649±808 85.4±4.7 70
OFMSW 5 6.0 3.0 2.25 9 2900±141 1700±215 41.3±2.8 7429±538 8175±442 87.8±2.1 60
6 8.0 3.0 2.25 9 3550±1343 1500±138 57.7±8.0 7709±1046 8558±290 89.5±0.7 67
7 10.0 3.0 2.25 9 7000±1587 3300±647 47.6±7.4 7429±2342 7258±134 87.8±1.8 78
8 12.0 3.0 2.25 9 8700±1997 3267±451 64.6±13.7 6858±1249 7398±543 82.2±0.2 71
9 14.0 3.0 2.25 9 15000±3051 2500±583 83.0±9.5 4445±123 6248±354 78.1±7.3 40
1
The theoretical methane yield is calculated based on fat, protein and carbohydrate content of the substrates. The VFA concentration in FPW is not taken into account.

14
 Two-stage process

Biogas measurement
Single-stage process
Biogas Recirculation

Feed
Outlet Feed Hydrolysis

Methanogenesis

CSTR (55°C) CSTR (55 °C) UASB (34°C )

Sedimentation tank
Pump

Figure 1.Schematic sketch of the single-stage and two-stage process

15
Figure 2. Methane production in A- single-stage process B- two-stage process FPW, C-two
stage process (OFMSW)

16
Figure 3. Total and individual VFA concentration in single-stage process. A- CSTR –FPW,B-
CSTR-OFMSW

17
 20 7 Acetic acid
A OFMSW total VFA C
6 Butyric acid
FPW total VFA
15 Propionic acid
5
Valeric acid
4
10
3

VFA (g/L)
VFA (g/L)
2
5
1

0 0
0 50 100 150 200 250 0 50 100 150 200 250
Days Days
1,8 7
B D
1,5 6

5
1,2
4
0,9
3

VFA (g/L)
VFA (g/L)
0,6
2
0,3 1

0 0
0 50 100 150 200 250 0 50 100 150 200 250
Days Days

Figure 4. Total and individual volatile fatty acid concentration in two-stage process for FPW and OFMSW A-total VFA in CSTR, B-total VFA in
UASB, C-individual VFA in CSTR-FPW, D- individual VFA in CSTR-OFMSW

18
Figure 5. Ammonium concentration in A-single-stage and B- FPW-two-stage, C- OFMSW-
two-stage during the entire experimental period at each organic loading rate (OLR) and
Hydraulic retention time (HRT).

19