Pretreatment of cellulosic waste and high-rate biogas production. Solmaz Aslanzadeh

Transcription

1 Pretreatment of cellulosic waste and high-rate biogas production Solmaz Aslanzadeh

2 Copyright Solmaz Aslanzadeh School of Engineering University of Borås SE Borås (Sweden) Handle-ID ISBN (Printed) ISBN (pdf) ISSN X, Skrifter från Högskolan i Borås, nr. 47 Printed in Sweden by Ineko AB Borås 2014 ii

3 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, Nmethylmorpholine-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 semicontinuous 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

4 iv

5 List of Publications This thesis is mainly based on the results presented in the following articles: I. II. III. IV. V. Aslanzadeh S, Taherzadeh MJ and Sárvári Horváth I. (2011): Pretreatment of straw fraction of manure for improved biogas production. Bioresources 6: 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. 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: 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: 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. v

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

7 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 Biogas industry: current status and challenges The AD process and its complexities Factors influencing the AD process Bottlenecks of anaerobic digestion Organic loading rate Retention time Phase separation Chapter 3. Substrates for biogas production Substrate composition and its effect on AD Lignocellulosics-structural carbohydrates Textile waste-cellulose and synthetic fibers Starch-non structural carbohydrates Organic fraction of municipal solid waste Remarks on theoretical and experimental methods for determination of biogas potential Theoretical methods Experimental methods Chapter 4. Approaching the challenge of biomass recalcitrance Definition of substrate biodegradability Challenges with lignocellulosic recalcitrance Microbial strategy for lignocellulose recalcitrance: Cellulosome Goal of pretreatment Effect of pretreatment on biogas production Pretreatment technologies Physical pretreatment Physiochemical pretreatments Biological pretreatment Chemical pretreatments Chapter 5. High-rate anaerobic treatment systems Background and Status Upflow anaerobic sludge blanket reactor Biogranulation of microorganisms Factors influencing anaerobic granulation vii

8 Characteristics of anaerobic granules Two-stage process for high-rate methane production Batch process- single vs. two-stage Two-stage semi-continuous process Two-stage- open system vs. closed system Semi-continuous process- Single vs. two stage Concluding Remarks Future work Nomenclature Acknowledgments References viii

9 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

10 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 cellulosebased 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: 2 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).

11 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

12 4

13 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

14 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 % Cumulative (Kton/year) % 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 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

15 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 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

16 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

17 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 H 2. However, these oxidation reactions can only occur if the partial pressure of H 2 is low, which explains why the collaboration with the methanogens is very important, since they will continuously consume the H 2 to produce methane. Hence, during this symbiotic relationship interspecies 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 H 2, CO 2, and acetic acid. The methanogenic microorganisms can be divided into three main groups: (1) Acetoclastic methanogenesis Acetate CH 4 + CO 2 (2) Hydogenotrophic methanogenesis H 2 + CO 2 CH 4 (3) Methylotrophic methanogenesis Methanol CH 4 + H 2 O Complex organic matter 100% Biodegradable organic matter Proteins Carbohydrates Lipids Hydrolysis 21% 40% 5% 21% Acidogenesis Amino acids, Sugars Fatty Acids 46% 20% 0% 34% Intermediate products Propionate, Butyrate, etc. 20% 12% 8% 35% Acetogenesis 11% 23% Acetate H 2, CO 2 11% Methanogenesis 70% Rate-limiting step 30% Methane, CO 2 Figure 2. Schematic diagram of the anaerobic digestion process. Adapted from [31] 9

18 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 (onephase), 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 ( ) by feeding the system at an 10

19 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 (NH + 4 ) or ammonia (NH 3 ) 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 NH 3. 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

20 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 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] 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

21 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 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 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

22 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] 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 Solids Dissolved Solids Acid phase Liquification Acidification Organic Acids Acetate Methane Gas (Methane) Phase 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

23 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

24 16

25 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

26 Lignocellulosics-structural carbohydrates Lignocellulose is the most abundant renewable biomass worldwide [62] with an estimated annual production between 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 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 ,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

27 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 twostage semi-continuous high-rate biogas production was investigated in papers III and IV 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-1,4linked 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

28 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

29 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) Remarks on theoretical and experimental methods for determination of biogas potential Theoretical methods Biogas production from the organic substrates engages internal redox reactions that convert organic molecules into CH 4 and CO 2. 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 CH 4 and CO 2 is produced [27]: C 6 H 12 O 6 3 CH CO 2 (1) For proteins, the process can be described as follows: C 13 H 25 O 7 N 3 S + 6 H 2 O 6.5 CH CO NH 3 + H 2 S (2) The degradation of fats and vegetable oils (triglycerides) can be summarized by the following equation: C 12 H 24 O H 2 O 7.5 CH CO 2 (3) The ideal stoichiometry, for a two-phase digestion, considering the simple case of carbohydrate degradation, can theoretically be defined as follows: First stage: C 6 H 12 O H 2 O 4 H C 2 H 4 O 2 (acetic acid) + 2 CO 2 (4) 23

30 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 CH 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 Where: (9) 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 C6H10O Lipids/fats C57H104O 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 24 (10)

31 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] 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 semicontinuous 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 semi25

32 continuous process, only a CSTR with a retention time between 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

33 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 biologicallymediated reaction taking place in the anaerobic digestion process [83] 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

34 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

35 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 ratelimiting step in the overall AD process [90] 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

36 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] 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 ratelimited 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

37 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 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, ] 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) 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), CO 2 explosion, SO 2 explosion, steam pretreatment/autohydrolysis, hydrothermolysis, and wet oxidation [13]. These pretreatment methods have been successfully introduced prior to the biogas production. 31

38 Biological pretreatment Biological pretreatments make use of microorganisms (e.g., fungi) natural ability to degrade the lignin and hemicellulose, leaving the cellulose intact [ ]. 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, ]. 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 prehydrolyzed as well as acidified to some extent by the microorganisms present in the storage tank for 3 4 days. RM0 0,45 RM7 RM14 RM28 PM28 Volume m3 CH4/kg VS 0,4 0,35 0,3 0,25 0,2 0,15 0,1 0, 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

39 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, ]. 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

40 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, ]. 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 to (d-1) for the cattle and from to (d-1) for the horse manure (Figure 7). 34

41 (a) 1,0 ln [Gm/(Gm-G)] 0,8 0,6 0,4 0,2 Untreated 5h treatment 15h treatment 0, , Time(days) 1,2 (b) 1,0 ln[gm/(gm-g)] 0,8 0,6 0,4 untreated 5h treatment 15h treatment 0,2 0,0 0-0, 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

42 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 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

43 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 NMMOpretreatment of lignocelluloses [132, 137]. Volume Nml/gVS untreated 90, 3h, 85 % 120, 3h, 85% 120, 15h, 75% 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 CH 4 /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 carbonrich 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 NMMOpretreatment on pure cellulose. 37

44 38

45 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 fixedfilm 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 [ ]. 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

46 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 [ ]. 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 was estimated to be 2,266; however, this number declined to 610 between (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 LAGOON 5% Af 6% CSTR 7% HYBRID 3% FB 2% UASB 50% FB 2% HYBRID 2% CSTR 4% EGSB 22% Af 1% LAGOON 1% UASB 34% 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

47 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

48 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] 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

49 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 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 , 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

50 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 [161, 162] 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 2producing 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 hydrodynamic 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

51 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

52 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) 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 Methane (ml/gvs/day) Viscose/polyester 50 Cotton/polyester Days Figure 14. Cumulative methane production in a single stage batch process - Viscose/polyester and Cotton/polyester (paper III) 48

53 100 Methane (ml/gvs/day) Pretreated Jeans 100 Methane (ml/gvs/day) Days Share of reactor in methane production Share of reactor in methane production (%) (%) Untreated Jeans 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) Total VFAin CSTR (g/l) 1, ,2 4 0, ,4 Total VFA in UASB (g/l) Untreated Jeans Pretreated Jeans 7 1, ,2 4 0, ,4 1 0 Total VFA in UASB (g/l) Total VFA in CSTR (g/l) Days Figure 19. Total VFA concentration in ( ) the UASB and ( ) CSTR for the untreated jeans and the pretreated jeans (paper III) 53

54 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. Cotton Starch 450 CH 4 volume (ml/gvs/day) Share of methane production (%) Share of methane production (%) CH 4 volume (ml/gvs/day) 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) 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

55 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]. Cotton Starch Share of methane production (%) CH 4 volume (ml/gvs/day) Share of methane production (%) CH 4 volume (ml/gvs/day) 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

56 This also further confirms that the more degradable the substrate, the higher OLR and a lower HRT can be applied ,2 0 0 C-UASB Closed system D-UASB -Open system 1 Total VFA (g/l) 1 0,8 0,8 0,6 0,6 0,4 0,4 0,2 0,2 0 Total VFA (g/l) Total VFA (g/l) B-CSTR-Open system Total VFA (g/l) A-CSTR-Closed system Days Days Figure 22. Total VFA concentration. - Starch - Cotton in closed and open system 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

57 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 Volume CH4 m3/kg VS 0,7 0,6 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 58

59 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

60 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

61 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 twophase 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

62 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 63

64 Nomenclature AD CSTR UASB OLR HRT LCFAs SRT TS VS VFA COD OFMSW FPW NMMO ECP Anaerobic digestion Continuous stirred tank reactor Upflow anaerobic sludge blanket Organic loading rate Hydraulic retention time Long-chain fatty acids Solid retention time Total solids Volatile solids Volatile fatty acids Chemical oxygen demand Organic fraction of municipal solid waste Food processing waste N-methylmorpholine-N-oxide Extracellular polymeric substances 64

65 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

66 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 67

68 References [1] Korres NE. Storage and distribution of biomethane. In: N. E. Korres, P. O'Kiely, J. Benzie, J. Wes editors. Bioenergy production by anaerobic digestion: Using agricultural biomass and organic wastes Canada: Taylor & Francis p [2] Noike T, Endo G, Chang J-E, Yaguchi J-I, Matsumoto J-I. Characteristics of carbohydrate degradation and the rate-limiting step in anaerobic digestion. Biotechnology and Bioengineering 1985;27: [3] Jeihanipour A, Niklasson C, Taherzadeh MJ. Enhancement of solubilization rate of cellulose in anaerobic digestion and its drawbacks. Process Biochemistry 2011;46: [4] Nayono SE. Anaerobic digestion of organic solid waste for energy production vol. 46: KIT Scientific Publishing; p [5] Schnürer A, Jarvis A. Microbiological handbook for biogas plants. Swedish waste management, Swedish gas center; p [6] Rajendran K, Aslanzadeh S, Taherzadeh MJ. Household Biogas Digesters A Review. Energies 2012;5: [7] Bal AS, Dhagat NN. Upflow Anaerobic Sludge Blanket Reactor- A Review. Indian Journal of Environmental Health 2001;43:1-82. [8] Skiadas IV, Gavala HN, Schmidt JE, Ahring BK. Anaerobic Granular Sludge and Biofilm Reactors. Biomethanation II vol. 82: Springer Berlin Heidelberg; p [9] Van Lier JB, Tilche A, Ahring BK, Macarie H, Moletta R, Dohanyos M, Hulshoff Pol LW, Lens P, Verstraete W. New perspectives in anaerobic digestion. Water Science and Technology 2001;43:1 18. [10] Abbasi T, Abbasi SA. Formation and impact of granules in fostering clean energy production and wastewater treatment in upflow anaerobic sludge blanket (UASB) reactors. Renewable and Sustainable Energy Reviews 2012;16: [11] Lettinga G, van Velsen AFM, Hobma SW, de Zeeuw W, Klapwijk A. Use of the upflow sludge blanket (USB) reactor concept for biological wastewater treatment, especially for anaerobic treatment. Biotechnology and Bioengineering 1980;22: [12] Jeihanipour A. Waste textiles bioprocessing to ethanol and biogas. Sweden: Chalmers University of Technology; Doctoral thesis; [13] Forgács G. Biogas production from citrus wastes and chicken feather: pretreatment and co-digestion. Sweden: Chalmers University of Technology; Doctoral thesis; [14] De Baere L, Mattheeuws B. Anaerobic Digestion of the Organic Fraction of Municipal Solid Waste in Europe ; Status, Experience and Prospects. Waste Management 2012; 3: [15] Rapport J, Zhang R, Jenkins BM, Williams RB. Current Anaerobic Digestion Technologies Used for Treatment of Municipal Organic Solid Waste. California Environmental Protection Agency; [16] De Bere L. Anaerobic digestion of solid waste: state-of-the-art. Water Science and Technology 2000;41: [17] Pohland FG, Ghosh S. Developments in Anaerobic Stabilization of Organic Wastes - The Two-Phase Concept. Environmental Letters 1971;1: [18] Vandevivere P, De Baere L, Verstraete W. Types of anaerobic digester for solid wastes. In: J. Mata- Alvarez editor. Biomethanization of the organic fraction of municipal solid wastes; p

69 [19] US EPA. Biosolids Technology Fact Sheet- Multi-Stage Anaerobic Digestion. US Environmental Protection Agency 2006;Washington DC: EPA 832-F [20] De Baere L, Mattheeuws B. State-of-the-art 2008-Anaerobic digestion of solid waste Belgium: [21] European Bioplastics. anaerobic digestion. Germany: [22] De Baere L, Mattheeuws B. Anaerobic digestion of MSW in Europe; 2010 Updates and trends. 25th Annual Biocycle West Coast Conference p. 29. [23] van Foreest F. Perspectives for Biogas in Europe. Oxford Institute for Energy Studies [24] Teghammar A. Biogas Production from Lignocelluloses: Pretreatment, Substrate Characterization, Codigestion and Economic Evaluation. Sweden: Chalmers University of Technology; Doctoral thesis; [25] Ziemioski K, Frąc M. Methane fermentation process as anaerobic digestion of biomass: Transformations, stages and microorganisms. African Journal of Biotechnology 2012;11: [26] Naik SN, Goud VV, Rout PK, Dalai AK. Production of first and second generation biofuels: a comprehensive review. Renewable and Sustainable Energy Reviews 2010;14: [27] Deublein D, Steinhauser A. Biogas from waste and renewable resources: An introduction. Weinheim: Wiley-VCH; p [28] Schink B. Energetics of syntrophic cooperation in methanogenic degradation. Microbiology and Molecular Biology Reviews 1997;61: [29] Gerardi MH. The microbiology of anaerobic digesters. USA: Wiley p [30] Chandra R, Takeuchi H, Hasegawa T. Methane production from lignocellulosic agricultural crop wastes: A review in context to second generation of biofuel production. Renewable and Sustainable Energy Reviews 2012;16: [31] van Haandel AC, van der Lubbe J. Handbook of Biological Wastewater Treatment: Design and Optimisation of Activated Sludge Systems. Netherlands: IWA Publishing; p [32] Mittal KM. Biogas Systems: Principles and Applications. New Age International Limited Publishers; p [33] Yadvika, Santosh, Sreekrishnan TR, Kohli S, Rana V. Enhancement of biogas production from solid substrates using different techniques a review. Bioresource Technology 2004;95:1-10. [34] Dague RR. Application of digestion theory to digester control. Journal of Water Pollution Control Federation 1968: [35] Siegert I, Banks C. The effect of volatile fatty acid additions on the anaerobic digestion of cellulose and glucose in batch reactors. Process Biochemistry 2005;40: [36] Stafford DA. The effects of mixing and volatile fatty acid concentrations on anaerobic digester performance. Biomass 1982;2: [37] Wang Q, Kuninobu M, Ogawa HI, Kato Y. Degradation of volatile fatty acids in highly efficient anaerobic digestion. Biomass and Bioenergy 1999;16: [38] Hobson PN, Bousfield S, Summers R. Methane production from agricultural and domestic wastes. Springer; p [39] Chen Y, Cheng JJ, Creamer KS. Inhibition of anaerobic digestion process: A review. Bioresource Technology 2008;99: [40] Braun R, Drosg B, Bochmann G, Weiß S, Kirchmayr R. Recent developments in bio-energy recovery through fermentation. In: B. D. R. Braun, G. Bochmann, S. Weiß, R. Kirchmayr editor. Microbes at work: From wastes to resources: Springer; p [41] Lee S, Shah YT. Biofuels and Bioenergy: Processes and Technologies. CRC Press; [42] Martin-Ryals AD. Evaluating the potential for improving anaerobic digestion of cellulosic waste via routine bioaugmentation and alkaline pretreatmentuniversity of Illinois; [43] Zuo Z, Wu S, Zhang W, Dong R. Effects of organic loading rate and effluent recirculation on the performance of two-stage anaerobic digestion of vegetable waste. Bioresource Technology 2013;146: [44] Sharma A, Unni BG, Singh HD. A novel fed-batch digestion system for biomethanation of plant biomasses. Journal of Bioscience and Bioengineering 1999;87:

70 [45] Mata-Alvarez J, Macé S, Llabrés P. Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives. Bioresource Technology 2000;74:3-16. [46] Bouallagui H, Touhami Y, Ben Cheikh R, Hamdi M. Bioreactor performance in anaerobic digestion of fruit and vegetable wastes. Process Biochemistry 2005;40: [47] Appels L, Baeyens J, Degrève J, Dewil R. Principles and potential of the anaerobic digestion of wasteactivated sludge. Progress in Energy and Combustion Science 2008;34: [48] Demirer GN, Chen S. Two-phase anaerobic digestion of unscreened dairy manure. Process Biochemistry 2005;40: [49] Turovskiy IS, Mathai PK. Wastewater sludge processing. USA: John Wiley & Sons, Inc.; p [50] Pavlostathis SG, Giraldo Gomez E. Kinetics of anaerobic treatment: A critical review. Critical Reviews in Environmental Control 1991;21: [51] Demirel B, Yenigün O. Two-phase anaerobic digestion processes: a review. Journal of Chemical Technology and Biotechnology 2002;77: [52] Ghosh S, Pohland FG. Kinetics of substrate assimilation and product formation in anaerobic digestion. Journal of Water Pollution Control Federation 1974: [53] Massey ML, Pohland FG. Phase separation of anaerobic stabilization by kinetic controls. Journal of Water Pollution Control Federation 1978: [54] Cohen A, Zoetemeyer RJ, Van Deursen A, Van Andel JG. Anaerobic digestion of glucose with separated acid production and methane formation. Water Research 1979;13: [55] Pohland FG, Mancy KH. Use of ph and pe measurements during methane biosynthesis. Biotechnology and Bioengineering 1969;11: [56] Fernandes IAP. Application of Porous Membranes for Biomass Retention in a Two-phase Anaerobic Process. University of Newcastle upon Tyne; [57] Ince O. Performance of a two-phase anaerobic digestion system when treating dairy wastewater. Water Research 1998;32: [58] Zoetemeyer RJ. Acidogenesis of Soluble Carbohydrate-Containing Wastewaters. Universiteit van Amsterdam; p [59] Yen H-W, Brune DE. Anaerobic co-digestion of algal sludge and waste paper to produce methane. Bioresource Technology 2007;98: [60] Pagés Díaz J, Pereda Reyes I, Lundin M, Sárvári Horváth I. Co-digestion of different waste mixtures from agro-industrial activities: Kinetic evaluation and synergetic effects. Bioresource Technology 2011;102: [61] Ahring B, Angelidaki I, Macario EC, Gavala HN, Hofman-Bang J, Macario AJL, Elferink SJWHO, Raskin L, Stams AJM, Westermann P, Zheng D. Perspectives for Anaerobic Digestion. Biomethanation I vol. 81: Springer Berlin Heidelberg; p [62] Sanchez OJ, Cardona CA. Trends in biotechnological production of fuel ethanol from different feedstocks. Bioresource Technology 2008;99: [63] Claassen PAM, van Lier JB, Lopez Contreras AM, van Niel EWJ, Sijtsma L, Stams AJM, de Vries SS, Weusthuis RA. Utilisation of biomass for the supply of energy carriers. Applied Microbiology and Biotechnology 1999;52: [64] Taherzadeh MJ, Karimi K. Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. International Journal of Molecular Sciences 2008;9: [65] U. S. Environmental Protection Agency and Combined Heat and Power Partnership. Biomass Combined Heat and Power, Catalog of Technologies [66] Gopalakrishnan K, van Leeuwen J, Brown RC, Takara D, Khanal S. Biomass Pretreatment for Biofuel Production. Sustainable Bioenergy and Bioproducts: Springer London; p [67] Harmsen P, Huijgen W, Bermudez L, Bakker R. Literature review of physical and chemical pretreatment processes for lignocellulosic biomass. Wageningen UR, Food & Biobased Research; [68] Eriksson KEL, Babel W, Blanch HW, Cooney CL, Enfors SO, Fiechter A, Klibanov AM, Mattiasson B, Primrose SB, Rehm HJ, Rogers PL, Sahm H, Schügerl K, Tsao GT, Venkat K, Villadsen J, Stockar U, Wandrey C, Kuhad R, Singh A, Eriksson K-E. Microorganisms and enzymes involved in the degradation of plant fiber cell walls. Biotechnology in the Pulp and Paper Industry vol. 57: Springer Berlin Heidelberg; p

71 [69] Kumar P, Barrett DM, Delwiche MJ, Stroeve P. Methods for Pretreatment of Lignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production. Industrial & Engineering Chemistry Research 2009;48: [70] Mohnen D, Bar-Peled M, Somerville C. Cell Wall Polysaccharide Synthesis. In: M. E. Himmel editor. Biomass recalcitrance :Deconstructing the plant cell wall for bioenergy. USA: Blackwell Publishing Ltd.; p [71] Van Dyk JS, Pletschke BI. A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes Factors affecting enzymes, conversion and synergy. Biotechnology Advances 2012;30: [72] Pérez J, Munoz-Dorado J, de la Rubia T, Martinez J. Biodegradation and biological treatments of cellulose, hemicellulose and lignin: an overview. International Microbiology 2002;5: [73] Peng P, Bian J, Sun R-C. Extractives. Cereal Straw as a Resource for Sustainable Biomaterials and Biofuels. Amsterdam: Elsevier; p [74] Kostamo A, Holmbom B, Kukkonen JVK. Fate of wood extractives in wastewater treatment plants at kraft pulp mills and mechanical pulp mills. Water Research 2004;38: [75] Leiviskä T, Rämö J, Nurmesniemi H, Pöykiö R, Kuokkanen T. Size fractionation of wood extractives, lignin and trace elements in pulp and paper mill wastewater before and after biological treatment. Water Research 2009;43: [76] Rubin EM. Genomics of cellulosic biofuels. Nature 2008;454: [77] Rosa Estela Q-C, Jorge Luis F-M. Hydrolysis of Biomass Mediated by Cellulases for the Production of Sugars p [78] Quiroz-Castañeda RE, Folch-Mallol JL. Hydrolysis of biomass mediated by cellulases for the production of sugars. In: A. K. Chandel,S. Silvério da Silva editors. Sustainable degradation of lignocellulosic biomass - Techniques, applications and commercialization InTech; p [79] Food and Agriculture Organization Of The United Nations and International Cotton Advisory Committee. World apparel fibre consumption survey July [80] Palm. David. Improved waste management of textiles IVL Swedish Environmental Research Institute Ltd, vol. IVL Report B1976 Stockholm Sweden [81] Jeihanipour A, Taherzadeh MJ. Ethanol production from cotton-based waste textiles. Bioresource Technology 2009;100: [82] Kaplan DL. Biopolymers from renewable resources. Springer; [83] Labatut RA. Anaerobic biodegradability of complex substrates: performance and stability at mesophilic and thermophilic conditions.usa: Cornell University; Doctoral thesis; [84] Nallathambi Gunaseelan V. Anaerobic digestion of biomass for methane production: A review. Biomass and Bioenergy 1997;13: [85] Bouallagui H, Torrijos M, Godon JJ, Moletta R, Ben Cheikh R, Touhami Y, Delgenes JP, Hamdi M. Twophases anaerobic digestion of fruit and vegetable wastes: bioreactors performance. Biochemical Engineering Journal 2004;21: [86] Schnürer A, Nordberg Å. Ammonia, a selective agent for methane production by syntrophic acetate oxidation at mesophilic temperature. Water Science and Technology 2008;57: [87] Malik A, Grohmann E, Alves M, Albanna M. Anaerobic Digestion of the Organic Fraction of Municipal Solid Waste. Management of Microbial Resources in the Environment: Springer Netherlands; p [88] Hoornweg D, Bhada-Tata P. What a Waste: A Global Review of Solid Waste Management. Urban development series Washington, DC: World Bank; [89] Rajendran K, Björk H, Taherzadeh MJ. Borås, a zero waste city in Sweden. Journal of Development and Management 2013;1:3-8. [90] Fdez.-Güelfo LA, Álvarez-Gallego C, Sales Márquez D, Romero García LI. Biological pretreatment applied to industrial organic fraction of municipal solid wastes (OFMSW): Effect on anaerobic digestion. Chemical Engineering Journal 2011;172: [91] Cirne DG, Paloumet X, Björnsson L, Alves MM, Mattiasson B. Anaerobic digestion of lipid-rich waste Effects of lipid concentration. Renewable Energy 2007;32:

72 [92] Sousa DZ, Pereira MA, Stams AJM, Alves MM, Smidt H. Microbial communities involved in anaerobic degradation of unsaturated or saturated long-chain fatty acids. Applied and Environmental Microbiology 2007;73: [93] Angelidaki I, Ahring BK. Effects of free long-chain fatty acids on thermophilic anaerobic digestion. Applied Microbiology and Biotechnology 1992;37: [94] Woolley P, Petersen SB. Lipases: their structure, biochemistry and application. Cambridge University Press Cambridge; [95] Sprott GD, Patel GB. Ammonia toxicity in pure cultures of methanogenic bacteria. Systematic and Applied Microbiology 1986;7: [96] Hashimoto AG. Ammonia inhibition of methanogenesis from cattle wastes. Agricultural Wastes 1986;17: [97] Breure AM, Mooijman KA, Andel JG. Protein degradation in anaerobic digestion: influence of volatile fatty acids and carbohydrates on hydrolysis and acidogenic fermentation of gelatin. Applied Microbiology and Biotechnology 1986;24: [98] Krich K, Augenstein D, Batmale J, Benemann J, Rutledge B, Salour D. Biomethane from Dairy Waste: A Sourcebook for the Production and Use of Renewable Natural Gas in California; p [99] Møller HB, Sommer SG, Ahring BK. Methane productivity of manure, straw and solid fractions of manure. Biomass and Bioenergy 2004;26: [100] Bidlack J, Malone M, Benson R. Molecular structure and component integration of secondary cell walls in plants. Proceedings of the Oklahoma Academy of Science 1992;72: [101] Himmel ME, Picataggio SK. Our Challenge is to Acquire Deeper Understanding of Biomass Recalcitrance and Conversion. In: M. E. Himmel editor. Biomass recalcitrance :Deconstructing the plant cell wall for bioenergy. USA: Blackwell Publishing Ltd.; p [102] Colberg PJ. Anaerobic microbial degradation of cellulose, lignin, oligolignols, and monoaromatic lignin derivatives. In: A. J. B. Zehnder editor. Biology of Anaerobic Microorganisms. USA: Wiley; p [103] Hendriks ATWM, Zeeman G. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresource Technology 2009;100: [104] Van Soest PJ. Nutritional Ecology of the Ruminant ; Ruminant Metabolism, Nutritional Strategies, the Cellulolytic Fermentation and the Chemistry of Forages and Plant Fibers. O & B Books; p [105] Angelidaki I, Ahring BK. Methods for increasing the biogas potential from the recalcitrant organic matter contained in manure. Water Science and Technology 2000;41: [106] Keys JE, Van Soest PJ, Young EP. Comparative Study of the Digestibility of Forage Cellulose and Hemicellulose in Ruminants and Nonruminants. Journal of Animal Science 1969;29: [107] Zeikus JG. Chemical and fuel production by anaerobic bacteria. Annual Reviews in Microbiology 1980;34: [108] Hu Z-H, Liu S-Y, Yue Z-B, Yan L-F, Yang M-T, Yu H-Q. Microscale analysis of in vitro anaerobic degradation of lignocellulosic wastes by rumen microorganisms. Environmental Science & Technology 2007;42: [109] Bayer EA, Chanzy H, Lamed R, Shoham Y. Cellulose, cellulases and cellulosomes. Current Opinion in Structural Biology 1998;8: [110] Taherzadeh MJ, Jeihanipour A. Recalcitrance of Lignocellulosic Biomass to Anaerobic Digestion. In: A. Mudhoo editor. Biogas Production:Pretreatment methods in anaerobic digestion. USA: Wiley p [111] Fan LT, Lee Y-H, Beardmore DH. Mechanism of the enzymatic hydrolysis of cellulose: Effects of major structural features of cellulose on enzymatic hydrolysis. Biotechnology and Bioengineering 1980;22: [112] Bayer EA, Henrissat B, Lamed R. The cellulosome: A natural bacterial strategy to combat biomass recalcitrance. In: M. E. Himmel editor. Biomass Recalcitrance: Deconstructing the Plant Cell Wall for Bioenergy. USA: Blackwell Publishing Ltd.; p [113] Bayer EA, Belaich J-P, Shoham Y, Lamed R. The cellulosomes: multienzyme machines for degradation of plant cell wall polysaccharides. In: M. E. Himmel editor. Biomass recalcitrance :Deconstructing the plant cell wall for bioenergy. USA: Blackwell Publishing Ltd.; p

73 [114] Veeken A, Hamelers B. Effect of temperature on hydrolysis rates of selected biowaste components. Bioresource Technology 1999;69: [115] McMillan James D. Pretreatment of Lignocellulosic Biomass. Enzymatic Conversion of Biomass for Fuels Production vol. 566: American Chemical Society; p [116] Wyman C. Handbook on Bioethanol: Production and Utilization. Taylor & Francis; [117] Zheng Y, Pan Z, Zhang R. Overview of biomass pretreatment for cellulosic ethanol production. International Journal of Agricultural and Biological Engineering 2009;2: [118] Brodeur G, Yau E, Badal K, Collier J, Ramachandran KB, Ramakrishnan S. Chemical and Physicochemical Pretreatment of Lignocellulosic Biomass: A Review. Enzyme Research :1-17 [119] Kumar R, Wyman CE. Effects of cellulase and xylanase enzymes on the deconstruction of solids from pretreatment of poplar by leading technologies. Biotechnology Progress 2009;25: [120] Sánchez C. Lignocellulosic residues: biodegradation and bioconversion by fungi. Biotechnology Advances 2009;27: [121] Shi J, Chinn MS, Sharma-Shivappa RR. Microbial pretreatment of cotton stalks by solid state cultivation of Phanerochaete chrysosporium. Bioresource Technology 2008;99: [122] Sun Y, Cheng J. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technology 2002;83:1-11. [123] Johnson DK, Elander RT. Pretreatments for Enhanced Digestibility of Feedstocks. In: M. E. Himmel editor. Biomass recalcitrance :Deconstructing the plant cell wall for bioenergy. USA: Blackwell Publishing Ltd.; p [124] Cheng Y-S, Zheng Y, Yu C, Dooley T, Jenkins B, VanderGheynst J. Evaluation of High Solids Alkaline Pretreatment of Rice Straw. Applied Biochemistry and Biotechnology 2010;162: [125] Tormo J, Lamed R, Chirino AJ, Morag E, Bayer EA, Shoham Y, Steitz TA. Crystal structure of a bacterial family-iii cellulose-binding domain: a general mechanism for attachment to cellulose. The EMBO Journal 1996;15:5739. [126] Liu R-G, Shen Y-Y, Shao H-L, Wu C-X, Hu X-C. An Analysis of Lyocell Fiber Formation as a Melt spinning Process. Cellulose 2001;8: [127] Woodings CR. The development of advanced cellulosic fibres. International Journal of Biological Macromolecules 1995;17: [128] Eckelt J, Eich T, Röder T, Rüf H, Sixta H, Wolf BA. Phase diagram of the ternary system NMMO/water/cellulose. Cellulose 2009;16: [129] Cuissinat C, Navard P. Swelling and Dissolution of Cellulose Part 1: Free Floating Cotton and Wood Fibres in N Methylmorpholine N oxide Water Mixtures. Macromolecular Symposia vol. 244: Wiley Online Library; p [130] Lindman B, Karlström G, Stigsson L. On the mechanism of dissolution of cellulose. Journal of Molecular Liquids 2010;156: [131] Jeihanipour A, Karimi K, Taherzadeh MJ. Enhancement of ethanol and biogas production from high crystalline cellulose by different modes of NMO pretreatment. Biotechnology and Bioengineering 2010;105: [132] Kabir MM, del Pilar Castillo M, Taherzadeh MJ, Horváth IS. Effect of the N-Methylmorpholine-N-Oxide (NMMO) Pretreatment on Anaerobic Digestion of Forest Residues. Bioresources 2013;8: [133] Shafiei M, Karimi K, Taherzadeh MJ. Pretreatment of spruce and oak by N-methylmorpholine-N-oxide (NMMO) for efficient conversion of their cellulose to ethanol. Bioresource Technology 2010;101: [134] Lennartsson P. Zygomycetes and cellulose residuals: hydrolysis, cultivation and applications.sweden: Chalmers University of Technology; Doctoral thesis; [135] Zhao H, Kwak JH, Wang Y, Franz JA, White JM, Holladay JE. Interactions between cellulose and N- methylmorpholine-n-oxide. Carbohydrate Polymers 2007;67: A comparative kinetic evaluation of the anaerobic digestion of untreated molasses and molasses previously fermented with Penicillium decumbens in batch reactors. Biochemical Engineering Journal 2004;18:

74 [137] Shafiei M, Karimi K, Taherzadeh MJ. Techno-economical study of ethanol and biogas from spruce wood by NMMO-pretreatment and rapid fermentation and digestion. Bioresource Technology 2011;102: [138] Teghammar A, Forgács G, Sárvári Horváth I, Taherzadeh MJ. Techno-economic study of NMMO pretreatment and biogas production from forest residues. Applied Energy 2014;116: [139] Lier JBv. Anaerobic industrial wastewater treatment; perspectives for closing water and resource cycles. Proceedings of ACHEMA 2006, 28th International Exhibition Conference on Chemical Technology, Environmental Protection and Biotechnology. Frankfurt, Germany; [140] Lens P, Verstraete W. New perspectives in anaerobic digestion. Biological Treatment Processes 2001;43:1. [141] Nordberg Å, Jarvis Å, Stenberg B, Mathisen B, Svensson BH. Anaerobic digestion of alfalfa silage with recirculation of process liquid. Bioresource Technology 2007;98: [142] Pol LH, Lettinga G. New technologies for anaerobic wastewater treatment. Water Science and Technology 1986;18: [143] Youngsukkasem S, Rakshit SK, Taherzadeh MJ. Biogas production by encapsulated methaneproducing bacteria. Bioresources 2011;7: [144] Talebnia F. Ethanol Production from Cellulosic Biomass by Encapsulated Saccharomyces cerevisiae.sweden: Chalmers University of Technology; Doctoral thesis; [145] Barber WP, Stuckey DC. The use of the anaerobic baffled reactor (ABR) for wastewater treatment: a review. Water Research 1999;33: [146] Chynoweth DP, Owens JM, Legrand R. Renewable methane from anaerobic digestion of biomass. Renewable Energy 2001;22:1-8. [147] Van Lier J, Van der Zee F, Tan N, Rebac S, Kleerebezem R. Advances in high rate anaerobic treatment: staging of reactor systems. Water Science and Technology 2001;44: [148] Lettinga G. Anaerobic digestion and wastewater treatment systems. Antonie van Leeuwenhoek 1995;67:3-28. [149] Hulshoff Pol L. The phenomenon of granulation of anaerobic sludgelandbouwuniversiteit te Wageningen; [150] Henze M. Biological wastewater treatment: principles, modelling and design. IWA publishing; [151] Winslow C-EA, Phelps EB. Investigations on the Purification of Boston Sewage. The Journal of Infectious Diseases 1911;8: [152] Lettinga G. Sustainable integrated biological wastewater treatment. Water Science and Technology 1996;33: [153] Rajeshwari KV, Balakrishnan M, Kansal A, Lata K, Kishore VVN. State-of-the-art of anaerobic digestion technology for industrial wastewater treatment. Renewable and Sustainable Energy Reviews 2000;4: [154] Seghezzo L, Zeeman G, van Lier JB, Hamelers HVM, Lettinga G. A review: The anaerobic treatment of sewage in UASB and EGSB reactors. Bioresource Technology 1998;65: [155] Schmidt JE, Ahring BK. Granular sludge formation in upflow anaerobic sludge blanket (UASB) reactors. Biotechnology and Bioengineering 1996;49: [156] Chan YJ, Chong MF, Law CL, Hassell DG. A review on anaerobic aerobic treatment of industrial and municipal wastewater. Chemical Engineering Journal 2009;155:1-18. [157] Hulshoff Pol LW, de Castro Lopes SI, Lettinga G, Lens PNL. Anaerobic sludge granulation. Water Research 2004;38: [158] Tay J, Xu H, Teo K. Molecular Mechanism of Granulation. I: H+ Translocation-Dehydration Theory. Journal of Environmental Engineering 2000;126: [159] Schmidt JEE, Ahring BK. Extracellular polymers in granular sludge from different upflow anaerobic sludge blanket (UASB) reactors. Applied Microbiology and Biotechnology 1994;42: [160] Zhou W, Imai T, Ukita M, Li F, Yuasa A. Effect of loading rate on the granulation process and granular activity in a bench scale UASB reactor. Bioresource Technology 2007;98: [161] Liu Y, Xu H-L, Show K-Y, Tay J-H. Anaerobic granulation technology for wastewater treatment. World Journal of Microbiology and Biotechnology 2002;18: [162] Gabriel B. Wastewater Microbiology. Wiley;

75 [163] Lepistö R, Rintala J. Extreme thermophilic (70 C), VFA-fed UASB reactor: performance, temperature response, load potential and comparison with 35 and 55 C UASB reactors. Water Research 1999;33: [164] Fang HHP, Lau IWC. Startup of thermophilic (55 C) UASB reactors using different mesophilic seed sludges. Water Science and Technology 1996;34: [165] Teo K-C, Xu H-L, Tay J-H. Molecular mechanism of granulation. II: Proton translocating activity. Journal of Environmental Engineering 2000;126: [166] Guiot SR, Pauss A, Costerton JW. A structured model of the anaerobic granule consortium. Water Science and Technology 1992;25:1-10. [167] MacLeod FA, Guiot SR, Costerton JW. Layered structure of bacterial aggregates produced in an upflow anaerobic sludge bed and filter reactor. Applied and Environmental Microbiology 1990;56: [168] Kosaric N, Blaszczyk R, Orphan L, Valladarfs J. The characteristics of granules from upflow anaerobic sludge blanket reactors. Water Research 1990;24: [169] Jian C, Shi-yi L. Study on mechanism of anaerobic sludge granulation in UASB reactors. Water Science and Technology 1993;28: [170] Pereboom J, Vereijken T. Methanogenic granule development in full scale internal circulation reactors. Water Science and Technology 1994;30:9-21. [171] Jeihanipour A, Karimi K, Niklasson C, Taherzadeh MJ. A novel process for ethanol or biogas production from cellulose in blended-fibers waste textiles. Waste Management 2010;30: [172] Alimahmoodi M, Mulligan CN. Anaerobic bioconversion of carbon dioxide to biogas in an upflow anaerobic sludge blanket reactor. Journal of the Air & Waste Management Association 2008;58: [173] Eastman JA, Ferguson JF. Solubilization of particulate organic carbon during the acid phase of anaerobic digestion. Journal of Water Pollution Control Federation 1981: [174] Wu M-c, Sun K-W, Zhang Y. Influence of temperature fluctuation on thermophilic anaerobic digestion of municipal organic solid waste. Journal of Zhejiang University SCIENCE B 2006;7: [175] Climent M, Ferrer I, Baeza MdM, Artola A, Vázquez F, Font X. Effects of thermal and mechanical pretreatments of secondary sludge on biogas production under thermophilic conditions. Chemical Engineering Journal 2007;133: [176] Mata-Alvarez J. Biomethanization of the organic fraction of municipal solid wastes. UK: IWA publishing; p

76

77 I

78

79 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 and Nm 3 CH 4 /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, k 0, was increased from to (d -1 ) for the cattle and from to (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, , Borås, Sweden *Corresponding author: Tel: , Fax: , 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 prestorage 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). Aslanzadeh et al. (2011). Pretreatment for better biogas BioResources 6(4),

80 PEER-REVIEWED ARTICLE bioresources.com Straw, when used as bedding material in proper ratios or after appropriate pretreatment, 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 Aslanzadeh et al. (2011). Pretreatment for better biogas BioResources 6(4),

81 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%) Volatile Solids(wt%) Protein(wt%) Kjeldahl Nitrogen(wt%) Ammonium Nitrogen(wt%) Fat Content(wt%) Carbohydrates(wt%) 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-m 3 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. Aslanzadeh et al. (2011). Pretreatment for better biogas BioResources 6(4),

82 PEER-REVIEWED ARTICLE bioresources.com 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 NMMOpretreated 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% N 2 and 20% CO 2. 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. Aslanzadeh et al. (2011). Pretreatment for better biogas BioResources 6(4),

83 PEER-REVIEWED ARTICLE bioresources.com Kinetic Modeling The kinetics of the degradation process were evaluated using the following firstorder kinetic model (Jiménez et al. 2004): or G = G m [1 exp( k 0 t)] (1) ln[g m / (G m -G)] =k 0 t (2) where G (ml) is the volume of methane accumulated after a period of time t (days), G m (ml) is the maximum accumulated gas volume at an infinite digestion time, k 0 (day -1 ) is the specific rate constant, and t (days) is the digestion time. Plotting the calculated data of ln[g m / (G m G)] vs. time, t, gives a straight line with a slope equal to k 0 with intercept of zero. The value of G m 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 (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 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 hydrochloric 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 H 2 SO 4 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),

84 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 Nm 3 CH 4 /kgvs, which increased to Nm 3 CH 4 /kgvs after 5 h pretreatment and further to Nm 3 CH 4 /kgvs after the 15 h pretreatment (Table 2). The same pattern was observed for the horse manure. The specific methane production increased to and Nm 3 CH 4 /kgvs after 5 h, respective, 15 h pretreatments, while the methane yield of the untreated horse manure was Nm 3 CH 4 /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 m 3 CH 4 /kgvs and m 3 CH 4 /kgvs, respectively. These results are in accordance with the theoretical methane yield for dairy cattle manure of m 3 CH 4 /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. Aslanzadeh et al. (2011). Pretreatment for better biogas BioResources 6(4),

85 PEER-REVIEWED ARTICLE bioresources.com 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 (k 0 ) were obtained during batch digestion of manure mixed with untreated vs. pretreated straw. Sample Total Lignin (% of TS) Total Carbohydrates (%) LOI a Specific Methane Yield(Nm 3 /kgvs) b Horse manure untreated ± h-treatment ± h-treatment ± Specific rate constant k 0 (day -1 ) Cattle manure untreated ± h-treatment ± h-treatment ± 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 (k 0 ) for treated vs. untreated horse and cattle manures, respectively. Aslanzadeh et al. (2011). Pretreatment for better biogas BioResources 6(4),

86 PEER-REVIEWED ARTICLE bioresources.com As shown in Fig. 3, k 0 increased with the pretreatment time for both the cattle and the horse manure samples (Table 2). After 15 h pretreatment, k 0 increased from (untreated) to d -1 in the case of horse manure, while similar treatment conditions resulted in an increase from (untreated) to d -1 for cattle manure. The ph was around 7 at the end of each digestion setup. (a) 1,0 0,8 ln [Gm/(Gm-G)] 0,6 0,4 0,2 0,0 Untreated 5h treatment 15h treatment ,2 Time(days) (b) 1,2 1,0 0,8 ln[gm/(gm-g)] 0,6 0,4 0,2 0,0-0, Time(days) untreated 5h treatment 15h treatment Fig. 3. Values of ln [Gm/ (Gm 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 (wt %) to (wt %) and to (wt %) after 5 and 15 h Aslanzadeh et al. (2011). Pretreatment for better biogas BioResources 6(4),

87 PEER-REVIEWED ARTICLE bioresources.com 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 OH stretching vibrations (Denise S. Ruzene 2007) 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) Carbonylgroup (C=O) (MacKay, O'Malley et al. 1997) conjugated to aromatic ring Aromatic skeletal vibrations (Buta, Zadrazil et al. 1989) 1595 Aromatic ring with C=O (MacKay, et al. 1997) stretching 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) 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 Aslanzadeh et al. (2011). Pretreatment for better biogas BioResources 6(4),

88 PEER-REVIEWED ARTICLE bioresources.com crystallinity index decreases as the time of the pretreatment increases 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). Aslanzadeh et al. (2011). Pretreatment for better biogas BioResources 6(4),

89 PEER-REVIEWED ARTICLE bioresources.com An additional effect of the pretreatment was the elimination of waxes, which can be observed from the reduced CH 2 - 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, k 0, 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). REFERENCES CITED Adney, W. S., McMillan, J. D., Mielenz, J., Klasson, K. T., Ruzene, D. S., Silva, D. P., Vicente, A. A., Gonçalves, A. R., and Teixeira, J. A. (2008). "An alternative application to the Portuguese agro-industrial residue: Wheat straw," Biotechnology for Fuels and Chemicals, Humana Press, Aslanzadeh et al. (2011). Pretreatment for better biogas BioResources 6(4),

90 PEER-REVIEWED ARTICLE bioresources.com Amon, T., Amon, B., Kryvoruchko, V., Zollitsch, W., Mayer, K., and Gruber, L. (2007). "Biogas production from maize and dairy cattle manure. Influence of biomass composition on the methane yield," Agriculture, Ecosystems & Environment 118(1-4), Budiyono, I., Widiasa, N., Johari, S., and Sunarso (2010). "The kinetic of biogas production rate from cattle manure in batch mode," Internationa Journal of Chemical and Biomolecular Engineering 3(1), Comino, E., Rosso, M., and Riggio, V. (2009). "Development of a pilot scale anaerobic digester for biogas production from cow manure and whey mix," Bioresource Technology 100(21), Davidsson, Å. (2007). "Increase of biogas production at wastewater treatment plants, Addition of urban organic waste and pre-treatment of sludge," Ph.D. Thesis, Department of Chemical Engineering, Lunds University, Sweden Nordic Committee on Food Analysis (1989). "Fat, determination according to SBR (Schmid-Bondzynski- Ratslaff) in meat and meat products," NMKL method no. 131, NMKL, Oslo. Hansen, T. L., Schmidt, J. E., Angelidaki, I., Marca, E., Jansen, J. l. C., Mosbæk, H., and Christensen, T. H. (2004). "Method for determination of methane potentials of solid organic waste," Waste Management 24(4), Hashimoto, A. G. (1983). "Conversion of straw-manure mixtures to methane at mesophilic and thermophilic temperatures," Biotechnology and Bioengineering 25(1), Hassan Dar, G., and Tandon, S. M. (1987). "Biogas production from pretreated wheat straw, lantana residue, apple and peach leaf litter with cattle dung," Biological Wastes 21(2), He, J., Cui, S., and Wang, S.-y. (2008). "Preparation and crystalline analysis of highgrade bamboo dissolving pulp for cellulose acetate," Journal of Applied Polymer Science 107(2), Jeihanipour, A., Karimi, K., and Taherzadeh, M. J. (2009). "Enhancement of ethanol and biogas production from high-crystalline cellulose by different modes of NMO pretreatment," Biotechnology and Bioengineering 105(3), Jiménez, A. M., Borja, R., and Martín, A. (2004). "A comparative kinetic evaluation of the anaerobic digestion of untreated molasses and molasses previously fermented with Penicillium decumbens in batch reactors," Biochemical Engineering Journal, 18(2), Kristensen, J., Thygesen, L., Felby, C., Jorgensen, H., and Elder, T. (2008). "Cell-wall structural changes in wheat straw pretreated for bioethanol production," Biotechnology for Biofuels 1(1), 5. Lawther, J. M., Sun, R., and Banks, W. B. (1996). "Fractional characterization of wheat straw lignin components by alkaline nitrobenzene oxidation and FT-IR spectroscopy," Journal of Agricultural and Food Chemistry 44(5), Lennartsson, P. R., Niklasson, C., and Taherzadeh, M. J. (2011). "A pilot study on lignocelluloses to ethanol and fish feed using NMMO pretreatment and cultivation with zygomycetes in an air-lift reactor," Bioresource Technology 102(6), Aslanzadeh et al. (2011). Pretreatment for better biogas BioResources 6(4),

91 PEER-REVIEWED ARTICLE bioresources.com MacKay, J. J., O'Malley, D. M., Presnell, T., Booker, F. L., Campbell, M. M., Whetten, R. W., and Sederoff, R. R. (1997). "Inheritance, gene expression, and lignin characterization in a mutant pine deficient in cinnamyl alcohol dehydrogenase," Proceedings of the National Academy of Sciences of the United States of America 94(15), Møller, H. B., Sommer, S. G., and Ahring, B. K. (2004). "Methane productivity of manure, straw and solid fractions of manure," Biomass and Bioenergy 26(5), Nelson, M. L., and O'Connor, R. T. (1964). "Relation of certain infrared bands to cellulose crystallinity and crystal latticed type. Part I. Spectra of lattice types I, II, III and of amorphous cellulose," Journal of Applied Polymer Science 8(3), Neves, L. C. M. d., Converti, A., and Penna, T. C. V. (2009). "Biogas production: New Trends for alternative energy sources in rural and urban zones," Chemical Engineering & Technology 32(8), Shafiei, M., Karimi, K., and Taherzadeh, M. J. (2009). "Pretreatment of spruce and oak by N-methylmorpholine-N-oxide (NMMO) for efficient conversion of their cellulose to ethanol," Bioresource Technology 101(13), Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., and Templeton, D. (2005). "Determination of ash in biomass," National Renewable Energy Laboratory. Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., and Crocker, D. (2008). "Determination of structural carbohydrate and lignin in biomass." National Renewable Energy Laboratory. Taherzadeh, M., and Karimi, K. (2008). "Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: A review," International Journal of Molecular Sciences 9(9), Swedish Standard Institute (1976). "Water quality- Determination of ammonia nitrogen content of water," SIS 28134, Article No STD-883, Stockholm, Sweden. Swedish Standard Institute (1984). "Water quality - Determination of Kjeldahl nitrogen - Method after mineralization with selenium," ISO 5663,Stockholm, Sweden. Zhao, H., Baker, G. A., and Cowins, J. V. (2009). "Fast enzymatic saccharification of switchgrass after pretreatment with ionic liquids," Biotechnology Progress 26(1), Article submitted: July 15, 2011; Peer review completed: September 24, 2011; Revised version received and accepted: October 27, 2011; Published: November 1, Aslanzadeh et al. (2011). Pretreatment for better biogas BioResources 6(4),

92

93 II

94

95 Biogas production from N-Methylmorpholine-Noxide (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: * Solmaz.Aslanzadeh@hb.se Keywords; Biogas, Anaerobic digestion, Forest residues, Batch experiment, Continuous experiment 1

96 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

97 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 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

98 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

99 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 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

100 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 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

101 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% N 2 and 20% CO 2. 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

102 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 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

103 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

104 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

105 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 CH 4 /gvs added (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 CH 4 /gvs added, and 100 NmL CH 4 /gvs added, 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 NmLCH 4 /g VS added, and methane production rate of 2.75 Nml/g VS/day. The highest methane production rate, i.e., 4.27 NmL CH 4 /gvs/day, was observed after pretreatment with 75% 11

106 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 in reactor 1 and between 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

107 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

108 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, NMMOtreatment enables a doubling of the digestibility of forest residues. Nonetheless, there is still twothirds 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

109 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

110 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 dewatering have a potential value as a fuel for combustion. 16

111 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

112 REFERENCES 1. Núñez-Regueira, L., Rodrıíguez-Añón, J., Proupín, J. and Romero-García, A. (2003) Bioresour. Technol. 88, Kabir, M. M., del Pilar Castillo, M., Taherzadeh, M. J. and Sárvári Horváth, I. (2013) BioResources. 8, Swedish Waste Management. (2008:2) Den svenska biogaspotentialen från inhemska råvaror Swedish Waste Management, Sweden. 4. Skinner, I., Essen, H., Smokers, R. and Hill, N. (2010) Final report produced under the contract ENV.C.3/SER/2008/0053 between European Commission Directorate-General Environment and AEA Technology,. 5. Ferreira, S., Gil, N., Queiroz, J. A., Duarte, A. P. and Domingues, F. C. (2010) Bioresour. Technol. 101, Hahn-Hägerdal, B., Galbe, M., Gorwa-Grauslund, M. F., Lidén, G. and Zacchi, G. (2006) Trends Biotechnol. 24, Hendriks, A. T. W. M. and Zeeman, G. (2009) Bioresour. Technol. 100, Lennartsson, P. R., Niklasson, C. and Taherzadeh, M. J. (2011) Bioresour. Technol. 102, Shafiei, M., Karimi, K. and Taherzadeh, M. J. (2010) Bioresour. Technol. 101, Teghammar, A., Karimi, K., Sárvári Horváth, I. and Taherzadeh, M. J. (2012) Biomass Bioenergy. 36, Rosenau, T., Potthast, A., Sixta, H. and Kosma, P. (2001) Prog. Polym. Sci. 26, Aslanzadeh, S., Taherzadeh, M. J. and Sárvári Horváth, I. (2011) Bioresources. 6, Cuissinat, C. and Navard, P. (2006) Macromolecular Symposia. 244, Jeihanipour, A., Karimi, K. and Taherzadeh, M. J. (2010) Biotechnol. Bioeng. 105, Hansen, T. L., Schmidt, J. E., Angelidaki, I., Marca, E., Jansen, J. l. C., Mosbæk, H. and Christensen, T. H. (2004) Waste Manage. (Oxford). 24, 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, Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J. and Templeton, D. (2005) Determination of Ash in Biomass. Standard Biomass Analytical Procedures. National Renewable Energy Laboratory. 19. Jonsson, S. and Borén, H. (2002) J. Chromatogr. A. 963, Jeihanipour, A., Aslanzadeh, S., Rajendran, K., Balasubramanian, G. and Taherzadeh, M. J. (2013) Renewable Energy. 52, Schink, B. (1997) Microbiology and Molecular Biology Reviwes. 61,

113 22. Larsen, J., Østergaard Petersen, M., Thirup, L., Wen Li, H. and Krogh Iversen, F. (2008) Chemical Engineering & Technology. 31, Henriksson, G., del Pilar Castillo, M., Jakubowicz, I., Enocksson, H., Contreras, J. A. and Lundgren, P. (2010) Miljöeffekter av polymerer inom biogasbranschen-förstudie. Projektnummer WR Shafiei, M., Karimi, K. and Taherzadeh, M. J. (2011) Bioresour. Technol. 102, Murphy, J. D. and Power, N. (2009) Applied Energy. 86, Davidsson, Å. (2007) Ph.D., Lunds University, Lund. 27. Teghammar, A., Castillo, M. D. P., Ascue, J., Niklasson, C. and Sárvári Horváth, I. (2013) Energy Fuels. 27, Teghammar, A., Forgács, G., Sárvári Horváth, I. and Taherzadeh, M. J. (2014) Applied Energy. 116,

114 FIGURES Volume Nml/gVS untreated 90, 3h, 85 % 120, 3h, 85% 120, 15h, 75% 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

115 140 Volume (Nml/gVS) Time (Day) Figure 2. Specific gas production for untreated forest residues (...) and NMMO-treated forest residue (o ) 21

116 7,9 7,8 7,7 7,6 7,5 7,4 7,3 7,2 7, Organiic loading rate [g VS/L & day] ph A ,8 4 7,6 3 7,4 2 7, Organiic loading rate [g VS/L & day] ph B 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

117 TABLES. Table 1. Pretreatment condition, Methane production, Carbohydrates, lignin of untreated and NMMO- Pretreated forest residue Pretreatment conditions Temperature C NMMO (%) Treatment time (h) 120 C 120 C 90 C Untreated Total solids (%) Volatile Solids (%) Total lignin (wt%) Total carbohydrates (%) Methane production Initial reaction rate Yield (Nml/g VS/day)a (Nml/gVS added)b 4.27 ± ± ± ± ±16 109,5±20 87,68±17 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

118

119 III

120

121 Renewable Energy 52 (2013) 128e135 Contents lists available at SciVerse ScienceDirect Renewable Energy journal homepage: 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 , Iran b School of Engineering, University of Borås, Borås, Sweden article info abstract Article history: Received 3 July 2012 Accepted 30 October 2012 Available online 22 November 2012 Keywords: Rapid digestion Biogas NMMO pretreatment UASB NMMO Waste textiles The efficacy of a two-stage Continuously Stirred Tank Reactor (CSTR), modified as Stirred Batch Reactor (SBR), and Upflow Anaerobic Sludge Blanket Bed (UASB) process in producing biogas from waste textiles was investigated under batch and semi-continuous conditions. Single-stage and two-stage digestions 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 a two-stage process was comparable to the production in a single-stage SBR, and in less than two weeks, more than 80% of the theoretical yield of methane was acquired. However, for cotton/polyester, the twostage batch process was significantly superior to the single-stage; the maximum rate of methane production was increased to 80%, and the lag phase decreased from 15 days to 4 days. In the two-stage semi-continuous process, where the substrate consisted of jeans textiles, the effect of N-methylmorpholine-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 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. Interestingly, 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.: þ ; fax: þ address: Karthik.Rajendran@hb.se (K. Rajendran). production of different biofuels, such as biogas [2]. For instance, in 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 celluloseiswasted every year. Ayield of415 ml methane (at STP) perg cellulose implies that the amount of waste textiles produced in Sweden would suffice as substrate for producing more than 20 million Nm 3 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 /$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.

122 A. Jeihanipour et al. / Renewable Energy 52 (2013) 128e 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 environmentally 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 problems related to transport of both wastes and biogas. Such systems (so-called on-farm biogas plants), have in Germany been commercially installed in thousands [14], mainly using biomass from agriculture. 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 agricultural 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 pretreating 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 interconnection 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 regenerating it. Jeihanipour et al. [16] recently developed a process for separating the cellulosic part from waste textiles, using an environmental 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 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 used blue jeans textiles (100% cotton). Prior to the experiments, the first two textiles were cut into small pieces (approximately cm 2 ), 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-m 3 municipal solid waste digester, operating under thermophilic (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) 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 atmospheric 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 continuously, 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 Experimental setup 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) Reactor seeding and start up The UASB reactors were seeded with 1.28 L of granular anaerobic 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 dissolved methane. The CSTR and SBR were filled with 2.5 L of inoculum 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 macronutrients) were (in mg/l): NH 4 Cl (76.4), KH 2 PO 4 (5.18), MgSO 4 $7H 2 O (0.27), CaCl 2 $2H 2 O, (10.00), and trace nutrients, 1 ml/l [20] 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

123 130 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 arrangements 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 continuously 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) 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 separated, washed, and studied in a stereomicroscope. In the semicontinuous 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 experimental 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)

124 A. Jeihanipour et al. / Renewable Energy 52 (2013) 128e volumes of produced gas were recorded. The gas samples were analyzed directly by gas chromatography (GC), while the liquid samples were stored in the freezer at 20 C for later analyses Analytical methods Thecellulosecontentof thesubstrateswasdeterminedaccording to the method provided by the National Renewable Energy Laboratory in the USA [21]. The gas production was recorded by using the Automatic Methane Potential Testing System (AMPTS, Bioprocess control AB, Lund, Sweden), whose function is based on water displacement and buoyancy, with a measuring resolution of 13 ml. The instrument was equipped with a laptop computer and the volumesofproducedgasvs. timewererecorded foreachreactor. The composition of the biogas produced during anaerobic digestion was measured using a gas chromatograph (Auto System Perkin Elmer, Waltham, MA) equipped with a packed column (Perkin Elmer, OD, 80/100, mesh) and a thermal conductivity detector (Perkin Elmer) set to 200 C. The inject temperature was set to Methane (ml/gvs/day) Days Viscose/polyester Cotton/polyester Fig. 2. Rate of methane production from (A) viscose/polyester and (C) cotton/polyester in the single-stage batch process. Fig. 3. Stereomicroscopic pictures of viscose/polyester and cotton/polyester before and after single-stage and two-stage digestions.