TECHNIQUES TO ENHANCE METHANE PRODUCTION FROM TERRESTRIAL (SUGARBEET) AND ALGAL ( NANNOCHLOROPSIS OCULATA ) BIOMASS By SAMRIDDHI BUXY A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL F ULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2014
2014 Samriddhi Buxy
4 ACKNOWLEDGMENTS At the outset, I wish to expres s my sincere gratitude to my chair Dr. Pratap Pullammanappallil for his continual guidance and encouragement during the course of my research at University of Florida. I would also like to sincerely thank my committee members Dr. Jennifer Curtis, Dr. Edwar d Phlips, Dr. Bruce Welt and Dr. Ben Koopman for their time and diligent efforts to provide guidance for my research. I would like to thank the following individuals for their kind assistance in my research Dr. Patrick Dube, Dr. Abhay Koppar, Yang Shuncha ng, Wen Ji, Dr. Gayathri Ram Mohan and Cesar Moreira. I would like to highlight, Dr. Mandu Inyang, Dr. Zhouli Tian, Sneha Jain and Henna Tangri have been a constant source of motivation and support during the entire tenure and I am indebted to them for kee ping me in great spirits. I am grateful to my parents who have been always supportive of my education. Last but certainly not the least, my husband Saurabh Sinha has always been encouraging and enthusiastic towards my research, which was a great motivati on and support
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ................... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 12 Background ................................ ................................ ................................ ............. 12 Motiv ation ................................ ................................ ................................ ............... 14 Challenges ................................ ................................ ................................ .............. 17 Objectives ................................ ................................ ................................ ............... 19 2 EFFECT OF SIZE REDUCTION AND DIFFERENT TYPES OF STORAGE ON BIOMETHANE POTENTIAL OF SUGARBEETS ................................ .................... 20 Summary ................................ ................................ ................................ ................ 20 Introduction ................................ ................................ ................................ ............. 21 Materials and Methods ................................ ................................ ............................ 23 Feedstock ................................ ................................ ................................ ......... 23 Anaerobic digester ................................ ................................ ........................... 23 Experiments ................................ ................................ ................................ ..... 24 Analysis ................................ ................................ ................................ ............ 24 Results ................................ ................................ ................................ .................... 26 Discuss ion ................................ ................................ ................................ .............. 29 Effect of size reduction on biomethane potential of sugarbeets ....................... 29 Effect of different types of storage on biomethane potent ial of sugarbeets ...... 32 Conclusions ................................ ................................ ................................ ............ 34 3 PILOT SCALE, TWO STAGE, THERMOPHILIC BATCH ANAEROBIC DIGESTION OF SUGAR BEETS ................................ ................................ ............ 41 Summary ................................ ................................ ................................ ................ 41 Background ................................ ................................ ................................ ............. 41 Methods ................................ ................................ ................................ .................. 43 Two stage pilot scale anaerobic digester system design and operation ........... 43 Experimental Protocols ................................ ................................ ..................... 44 Analysis ................................ ................................ ................................ ............ 46 Results and Discussion ................................ ................................ ........................... 47
6 Methane yield and its rate of production ................................ ........................... 47 Biogas quality ................................ ................................ ................................ ... 49 Mixed liquor characteristics ................................ ................................ .............. 49 Digestion residue characteristics ................................ ................................ ...... 51 Nutrient requirements ................................ ................................ ....................... 51 Comparison of present work to previous work in literature ............................... 53 Conclusions ................................ ................................ ................................ ............ 55 4 ENZYMATIC SACCHARIFICATION OF DILUTE ACID PRETREATED SALINE MICROALGAE, NANNOCHLOROPSIS OCULATA ................................ ................ 62 Summary ................................ ................................ ................................ ................ 62 Background ................................ ................................ ................................ ............. 62 Materials and Methods ................................ ................................ ............................ 64 Feedstock ................................ ................................ ................................ ......... 64 Pretreatment ................................ ................................ ................................ ..... 65 Experimental setup for enzyme saccharification ................................ .............. 65 Ash Free Dry Matter Analysis (AFDM) ................................ ............................. 67 Results ................................ ................................ ................................ .................... 67 Ash Free Dry Matter (AFDM) of N.oculata ................................ ........................ 67 Control runs for saccharification of N.oculata ................................ ................... 67 Dilute acid hydrolysis ................................ ................................ ........................ 68 Discussion ................................ ................................ ................................ .............. 70 Conclusion ................................ ................................ ................................ .............. 74 5 PRETREATMENT OF NANNOCHLOROPSIS OCULATA FOR IMPROVED BIOGASIFICATION ................................ ................................ ................................ 78 Summary ................................ ................................ ................................ ................ 78 Background ................................ ................................ ................................ ............. 79 Materials and methods ................................ ................................ ............................ 81 Algae growth and characteristics ................................ ................................ ...... 81 Anaerobic digestion setup ................................ ................................ ................ 82 Loading ................................ ................................ ................................ ............. 82 Pretreatment procedures description ................................ ............................... 83 Results and Discussion ................................ ................................ ........................... 85 Conclusions ................................ ................................ ................................ ............ 90 6 CONCLUSIONS ................................ ................................ ................................ ..... 99 LIST OF REFERENCES ................................ ................................ ............................. 101 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 112
7 LIST OF TABLES Table page 2 1 Total solids and Volatile solids content of sugarbeet stored at different conditions ................................ ................................ ................................ ........... 35 2 2 Gompertz parameters of sugarbeet digestion runs ................................ ............. 35 2 3 Gompertz parameters of two fraction sugarbeet digestion of frozen beet with and without size reduction ................................ ................................ .................. 35 3 1 Comparison to results obtained by Lehtomaki and Bjrnsson (2006) ................. 56 4 1 Comparison of carbohydrate saccharification from different algal biomass ........ 75 4 2 Sugar released (in g sugars/ kg AFDM) at different loadings of enzyme during saccharification ................................ ................................ ........................ 76 4 3 Sugar released (g sugars/ kg AFDM) at nominal EII loading from 2% phosphoric acid pretreate d samples at 160oC for various durations ................. 77 4 4 Concentration of sugars and other by products from treatment with nominal EII loading ................................ ................................ ................................ .......... 77 5 1 Summary of anaerobic digestion of microalgae incorporating different pretreatments ................................ ................................ ................................ ..... 95 5 2 Rate of methane production from digestionof pretreated N.oculata .................... 98
8 LIST OF FIGURES Figure page 2 1 Photograph of sugarbeets taken out of airtight storage after 4 months and 20 days ................................ ................................ ................................ .................... 36 2 2 Schematic diagram of 30 L anaerobic digester ................................ .................. 37 2 3 Cumulative methane yield of sugarbeets stored at different conditions .............. 37 2 4 Percentage ultimate methane yield of sugarbeets at different storage conditions ................................ ................................ ................................ ........... 38 2 5 Cumulative methane yield comparing whole and shredded beet digestion ........ 38 2 6 Percentage ultimate yield of sugarbeets at different sizes ................................ .. 39 2 7 Soluble COD released during digestion of sugarbeets stored at different conditions ................................ ................................ ................................ ........... 39 2 8 Soluble COD release from whole and shredded sugarbeet digestion ................ 40 3 1 Schematic diagram of the pilot scale two stage anaerobic digester ................... 57 3 2 Biogas production and quality A: Cumulative methane yield at STP, B: Volumetric methane productivity, C: Solids digester biogas methane and H2S content and D: Anaerobic filter biogas methane and H2S content ..................... 58 3 3 Solids digester and anaerobic filter mixed liquor characteristics. A: pH, B : soluble COD and C: total organic acids expressed in mg COD/L. ...................... 59 3 4 Organic acid composition in the solids digester and anaerobic filter mixed liquor. A: Lactic acid, B: Acetic aci d and C: Butyric acid. Other organic acids like propionic and valeric acids were not detected. Ethanol was also not detected. ................................ ................................ ................................ ............. 60 3 5 Methane production in each digester expressed as a fraction of total methane that can be produced in that digester. ................................ .................. 61 5 1 Schematic diagram of bioreactor assembly ................................ ........................ 91 5 2 Photoreactor assembly ................................ ................................ ....................... 92 5 3 Methane yield of N.oculata after different pretreatment ................................ ...... 92 5 4 Soluble chemic al oxygen demand from digestion of pretreated N.oculata ......... 93
9 5 5 Hierarchal order from highest methane production to lowest from different pretreatments ................................ ................................ ................................ ..... 94
10 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy TECHNIQUES TO ENHANCE METHANE PRODUCTION FROM TERRESTRIAL (SUGARBE ET) AND ALGAL ( NANNOCHLOROPSIS OCULATA ) BIOMASS By Samriddhi Buxy August 2014 Chair: Pratap C. Pullammanappallil Cochair: Jennifer S. Curtis Major: Agricultural and Biological Engineering Biogasification (or anaerobic digestion) is a biochemical proces s that converts organic matter to biogas under anaerobic conditions. Biogas, a mixture of methane (50 70%) and carbon dioxide, can be used as Renewable Natural Gas (RNG) which is chemically identical to fossil natural gas after scrubbing carbon dioxide. Bi ogas is mainly produced as a byproduct from organic waste treatment. However, biogas generated from waste alone does not meet growing energy demands. Terrestrial biomass such as sugarbeet is cultivated once a year usually in excess of quantities regulated for sugar production. This potential availability and high amount of sugar in sugarbeet projects it as an ideal feedstock for anaerobic digestion. Howeve r, sugarbeets are not available in sufficient quantity to meet energy requirements hence aquatic biomas ses such as microalgae are also looked upon as a potential biomass resource for biogas production. Algae have higher growth rates and can be grown in saline or brackish water obviating the need for precious fresh water resources. This dissertation deals w ith the biogasification of sugar beets and the saline microalgae, Nannochloropsis oculata Studies were done to explore options for economical long term storage of sugarbeet for biogas production. Biomethane potential of beets stored under ambient
11 temper ature air tight storage conditions was compared to that of frozen and freshly harvested sugarbeets It was then verified that size reduction of sugarbeets did not considerably increase rate and yield of methane production. A two reactor pilot scale system (200/600 liter) was designed to efficiently biogasify whole sugarbeets without any size reduction. This approach overcame issues related to biomass compaction and rapid acidification Nannochloropsis oculata can be grown easily in brackish or seawater, has satisfactory growth rates, can tolerate a wide range of pH and temperature and is rich in carbohydrates. However, this feedstock was shown to be resistant to biogasification. Pretreatment techniques such as ultrasonication thermal hydrolysis and enzym e saccharification including novel technique of pho tocatalysis by titanium dioxide were assessed for biomethane potential of N. oculata This novel technique was assessed better than costly radiation pretreatment technology such as ultrasonication and was e qually good as best pretreatment technologies for microalgae such as thermal.
12 CHAPTER 1 INTRODUCTION Background Increasing demands for a comfortable lifestyle is causing the premature depletion of limited non renewable oil reserves around the world. In thi s present scenario it is predicted that developed countries like United States will need to import 2/3rd of oil to meet current demands. If this trend continues it is estimated that overall world resources would exhaust in 50 years ( House of Lords 2013). Due to progress achieved in research, development and commercialization, biofuels appears to be the most feasible option, among other alternative energies, to fulfill the growing energy demands. By 2035, biofuels aim to contribute to 20% of overall fuel de mands ( World Energy Outlook 2012). Most widely produced biofuels are ethanol, biodiesel and biogas. Other fuels that can be produced from organic feedstock are hydrogen, other alcohols, alkanes and Fischer Tropsch fuels. Biogas (a mixture of methane, 50 70%, and carbon dioxide) is produced from the process of anaerobic digestion. Biogasification (or anaerobic digestion) is a biochemical process that converts organic matter to biogas through the concerted and syntrophic action of microorganisms under anae robic conditions. The process is mediated by a mixed, undefined culture of microorganisms at near ambient conditions. Biogas could be looked upon as renewable fuel. Initial capital investment for biogas plant is low and it is relatively easy to operate. An aerobic digestion recovers maximum energy from biomass and mineralizes the organic nitrogen and phosphorous from the biomass. These nutrients can be collected as by products after digestion and used as fertilizers without any further processing.
13 Biogas c an be used on site with very little clean up to produce heat and/or removing carbon dioxide and other contaminants to produce pure methane. RNG can be conveniently transported t hrough natural gas pipelines and utilized in lieu of natural gas. Life cycle analysis of biogas vs. ethanol from cellulosic biomass shows that biogas is a relatively cleaner fuel and contributes less to global warming. Biogasification reduces the amount of methane (23 times as potent a greenhouse gas (GHG) as carbon dioxide) emissions from untreated wastes. Studies claim higher yield of biogas can be achieved per hectare than ethanol for same area of land ( Braun et al., 2009). Also, heat of combustion of me thane is 55.7 MJ/kg, which is higher than ethanol (29.7 MJ/kg). Anaerobic digestion has been traditionally used for waste management. There are several commercial installations world wide producing biogas from variety of industrial wastewaters, municipal solid waste and wastewater, and manure slurries. Biomass used for biofuel production is broadly classified into three generations. First generation biomass includes crops that yield simple sugars or starch, and oil. Examples are sugarcane, sugarbeet, cor n, grains, oil seeds etc. Fuel can be produced from these feedstocks by using simple unit operations. Biofuel production from first generation biomass is unsustainable because of competition with food, and diversion of agricultural land for fuel produ ction. This led to the advent of second generation biomass consisting mainly of lignocellulosic biomass (essentially non edible biomass) such as agricultural, forestry and urban residues like corn stover, straw, municipal green waste, waste vegetable oil, and short rotation woody crops and herbaceous grasses grown on marginal lands like willow, oak, switch grass, and miscanthus. These have
14 relatively complex structure and needs additional unit operations like pretreatment and hydrolysis to release sugars f or biofuel production, but in long term have more cost reduction potential and higher net energy production than fuel generated from first generation biomass. The drawback associated with second generation biomass is that, high infrastructure is required a t initial setup of production. Owing to dependency on limited land for cultivation of first and second generation biomass, current research focuses on using algae based feedstocks for fuel production. Algae are categorized as third generation feedstock. Th e focus of the research addressed in this thesis is the production of biogas from sugar beet, a first generation biomass feedstock, and algae, a third generation biomass feedstock. Motivation Though not sustainable, first generation biomass is used as fe edstocks in well established industrial scale biofuel production facilities all over the world ( Lee et al., 2013). It dominates biofuel policies and is seen as a viable option to meet a certain percentage of energy demands in the current scenario. Sugar be et cultivation holds a significant value in the European agricultural market, occupying 1.4% of agricultural area of the continent with fresh and dry matter yield of 55 88 t/ha and 13 20 t/ha respectively ( Gain Dairy Feeds 2011). In Europe alone, there ar e 70 active sugar companies that use sugar beets for sugar production. Due to the policy of restricting sugar production by European government to control prices, a part of the harvested sugar beets goes unutilized which could be a raw material for biofue l production. European Union supports biofuels and has several schemes to stimulate the production of biofuels. These include tax incentives and mandating a percentage of biofuels in the
15 fuel needs. The European agricultural policy also encourage the produ ction of energy crop via new aid packages for energy crops and through schemes which allow growth of crops on land set aside for many non food uses. Europe also offers financial incentive of EUR 45 per hectare to farmers who produce energy crops. There are two possibilities for biofuel production from sugar beets; ethanol fermentation of the sugar syrup obtained from the beet or biogasifaction of the whole sugar beet or waste generated from sugarbeet processing. At present, sugarbeets are used as a feedsto ck for ethanol production. About 6.37 x 10 9 liters were produced annually in 2008 with additional production capacity of 2.17 x 10 9 ( Jung et al., 2010). Sugar beets can also be used as a feedstock for biogas production by anaerobic digestion. An advantage of this approach is that not only its sugar content but also the rest of the sugar beet (that is juice and pulp) can be digested to produce biogas. Additionally for biogas production, sugar beet need not be stored under controlled cold temperature to pre vent loss of sugar. Recently, a study showed that about 500 billion m 3 of methane per year in the form of biogas can be produced from organic wastes and energy crops in Europe to completely meet the current demand of natural gas (Kryvoruchko et al., 2009) In such a scenario, sugar beets could become an important feedstock for biogasification. Third generation biomass, mainly micro algae, are looked upon as a potential biomass resource for biofuels production. According to the International Energy Agency (IEA), by the year 2020, the source for a quarter of biofuels produced will be algae. Algae are autotrophic microorganisms utilizing a non fossil carbon source for growth by fixing carbon dioxide from atmosphere. Use of algae eliminates the need for terres trial
16 energy crops and hence will not impact availability of land for food and feed. Algae have higher growth rates, about 7 to 31 times greater than other terrestrial crops. Several species of algae can be grown in saline or brackish water obviating the n eed for precious fresh water resources. Recent developments in photo bioreactor designs have enabled algae to be grown 10 times more in quantity in compact areas, with high composition of lipids and carbohydrates. Algae can be broadly classified into macr o and microalgae. Macroalgae include seaweed, kelp, sea lettuce all found in marine water. Compared to microalgae, macroalgae usually have slower growth rates and higher nutrient demands for growth. Therefore microalgae is a preferred biomass for fuel prod uction and are rich in lipids and carbohydrates. Algae biomass can be directly combusted for energy or thermochemically gasified to produce syngas. Algae biomass can also be biologically gasified as well. For biogasification, algal slurries can be processe d without dewatering and sterile conditions need not be maintained for operating the fermenter. The process will also mineralize organic nitrogen and phosphorous, and these nutrients can be recycled for algae growth. The Department of Defense (DoD) is one of the largest consumers of energy (both in the form of liquid fuel and electricity) within the United States. The military requires vast amounts of electrical power to maintain a stateside presence, but with the current conflicts in the Middle East, and with operations being conducted all over the world, it also requires a substantial amount of mobile and deployable power systems. As the demand for power continues to increase, the DoD will face increasing costs in both capital and human resources, as lar ger convoys are necessary to provide
17 fuels, increasing costs to ship and protect these assets. Over the past eight years the US Air Force Research Laboratories have spent large research efforts into identifying microalgae species that can be grown for on s ite bioenergy generation. Nannochloropsis oculata (N. oculata ) was chosen as the appropriate microalgae species because it can be grown easily in brackish or seawater. It has satisfactory growth rate and can tolerate a wide range of pH (7 10) and temperatur e (17 27 C). Challenges This thesis aims to address some of the important engineering issues related to the biogasification of sugarbeets and the microalgae, Nannochloropsis oculata The outcomes from this research could be implemented for other terre strial biomass and microalgae species as well. Sugarbeets are harvested only once in a year around September. Due to the long cold winters in areas of North America where sugar beets are grown, beets can be stockpiled frozen in the fields for a longer time enabling sugar factories to be operated for over nine months in a year. However, in Europe, sugar factories run for only three months after harvest. After that, the harvested sugar beets cannot be used for sugar production, as there is a significant loss of sugars during storage and it does not remain fit for sugar production. Unlike ethanol or sugar production, if sugarbeets are to be biogasified then some loss of sugars can be tolerated. However, the beets cannot be left in the fields after harves t. So there is a need to come up with an efficient and inexpensive storage option for large amounts of unused beets or for beets grown for energy production.
18 Well established commercial units have been developed for anaerobic digestion of wastewater and slurries. To anaerobically digest sugarbeets, a readily implementable option would be size reduction of beets followed by mixing with water to form slurry and then pumping the slurry into a well mixed digester. This approach has drawbacks as it consumes water to prepare slurry, and poor methane productivity (i.e. L per L reactor volume per day) due to low concentration of substrate in the slurry which means reactor sizes are large ( Frost et al., 2011). Alternate approach would be to feed chopped sugarbeet s directly into digester. Previous studies on anaerobic digestion of sugarbeet tailings showed problems due to rapid acidification and compaction of biomass within digester ( Polematidis 2007). Rapid acidification occurred because of the high sugar conten t in sugarbeets which caused pH to drop as low as 3.0. This completely inhibited methanogenesis. Moreover, the biomass compacted within the digester which made it difficult to remove and dilute the acidified contents. Therefore, there is a need to develo p and validate an appropriate reactor design that can efficiently biogasify sugarbeets. Various species of algae differ in morphological structure, encapsulation behavior and cell wall rigidity which hinder release of carbohydrates ( Rooke et al., 2008). Large amounts of carbohydrates are embedded within the microalgal cell. Generally biomethane potential ( i.e. yield of methane from anaerobic digestion expressed on a unit ash free dry weight (afdw) basis of feedstock) of algae is very low. The high content of complex carbohydrates entrapped in the cell wall of the microalgae makes it essential to incorporate a pre treatment stage to release these complex carbohydrates into simple sugars or make them bioavailable during the fermentation process.
19 Pretreatme nt techniques that have been used to improve biogasification of algae include thermal treatment (cooking) and ultrasonication ( Fernandez et al., 2012). Pretreatment has shown to increase biomethane potential by 14 88%, however, there is room for further i mprovement. During the past few years a large number of pretreatment methods have been developed for lignocellulosic biomass, including alkali treatment, thermal, enzymatic, dilute acid hydrolysis, and others. Many methods have been shown to result in hig h sugar yields, above 90% of the theoretical yield for lignocellulosic biomasses such as woods, grasses, corn stover, and others. Such techniques could also potentially be applied to improve algae biogasification. Objectives The overall objectives of thi s research were to address challenges associated with the biogasification of (1) sugar containing biomass feedstocks and (2) microalgae feedstocks to improve the yield and kinetics of the process. Specific objectives of the study were to: 1. explore optio ns for economical long term storage of sugarbeet for biogas production. 2. study the effect of size reduction on biomethane potential of sugarbeets. 3. develop and test a novel process design for efficiently biogasifying sugarbeets. 4. adapt pretreatment approach es based on those developed for lignocellulosic biomass to efficiently saccharify N. oculata 5. develop an alternate, novel, low cost pretreatment method to improve biogasification of N. oculata 6. compare the efficiency of the novel pretreatment method to exi sting pretreatment approaches.
20 CHAPTER 2 EFFECT OF SIZE REDUCTION AND DIFFERENT TYPES OF STORAGE ON BIOMETHANE POTENTIAL OF SUGARBEETS Summary Long term storage of sugarbeets can result in loss of surface sugars and dry matter (DM) with time. Freezing room temperature storage, ensiling are different methods which have been tried previously for storing sugarbeets. Biomethane potential (BMP) of sugarbeets was studied in 30 L batch digesters. BMP of frozen, freshly harvested (fresh sugarbeets) and airt ight stored sugarbeets were studied. Freshly harvested sugarbeets stored over a period of 4 months and 20 days in an airtight container (bucket) is referred to as airtight stored beets in this dissertation. It was observed that there was loss of 15% dry m atter with airtight storage. The maximum rate of methane production for airtight stored sugarbeets was 12.4 L CH4 STP/kg/d, which is highest amongst the three types of storage studied. But, the methane yield from frozen sugarbeets was highest at 423.4 L C H4 STP/kg VS, whereas from fresh sugarbeets it was 220.2 LCH4 STP/kg VS and from airtight stored sugarbeets it was 360.8 LCH4 STP/kg VS. Based on weight of beets immediately after harvest airtight storage yielded 73.82 LCH4 STP/kg compared to 57.26 LCH4 ST P/kg for fresh beets. Therefore, airtight storage is a cheaper and feasible way of storing sugarbeets and results in the expedited biogas production with a higher yield to that of fresh sugarbeets. It was also observed that size reduction increases the r ate of production of biogas but not biomethane yield. Average methane yield is observed as 388 LCH4 STP/kg Volatile Solids (VS).
21 Introduction Sugar beet contains 18% sugar by weight and has high rate of degradation if it is not properly stored. Researcher s have developed storage technologies like freezing or cold storage to preserve the sugarbeets longer. These are expensive and demands labor and maintenance ( NDSU, biofuels digest 2012). These techniques could be economically feasible if applied for sugar production from sugarbeets. However, it may not be economical if sugarbeets are used for fuel production. Inexpensive storage like ensilage ( et al., 1999), storage in plastic containers (drums) with anti fungal solutions (Wagner et al., 2010), an d room temperature storage ( Burba et al., 1975), which are usually used to preserve sugarbeets for animal fodder, may be applicable for biogasification as well. Effects of ensiling of crops like corn, sugarbeet pulp, sugar cane etc. on animal feed nutriti on have been studied ( Cummins et al., 2007; Silvestre et al. 1976; Leupp et al., 2006). During ensiling of sugarcane, napier grass, maize stover, corn, and fodder beet the loss of DM over time was 8.2%, 15.2% 4.2%, 8.1%, 15% respectively ( Snijders et al. 2 011; Silvestre et al. 1976; Wagner et al. 2010; Toenjes et al.,1970; et al., 1999). Other studies have suggested storing sugarbeets in sealed chambers at ambient temperature as cheaper and convenient ( Burba et al., 1975). But closed chamber storage 7% to 17% of the initial fresh matter ( Kenter et al.,2006). Storage in plastic container (which was not hermetically sealed), the DM loss was 35% ( Wagner et al., 2010). The los s of sugars during storage using these techniques would also lower yield of ethanol if sugarbeets were used as feedstock for biofuel. Since anaerobic digesters contain microbial populations that can degrade a wide range of organic matter including sugars,
22 carboxylic acids, alcohols, proteins and fats, sugar loss due to partial fermentation of sugar during storage may not significantly impact biogas yields. Therefore, these approaches to store feedstock so as to operate a digester continuously throughout t he year may be feasible. In addition to storage another physical characteristic of feedstock that can affect anaerobic digestion is particle size. Particle size could affect the biomethane yield as well as rate of methane production. The effect of si ze reduction has been studied for food waste, municipal solid waste, tomato, reed canary grass, organic solid waste, high lignin material like paper and cardboard, and lignocellulosic biomass ( Hendriks et al., 2009 ; Edelmann et al., 2000; Palmowski et al., 2000; Hills et al., 1984; Izumi et al., 2010; Pommier et al., 2010 ). For highly degradable substrates, size reduction does not affect overall biogas yield but there is a 5 25% increase in the rate of methane production. The increased methane output if any comes at a cost as the operating and capital cost of process increases due to the additional size reduction unit. Size reduction may not always appear beneficial in terms of investments. It depends mostly on the type of substrate being digested. For a hi ghly degradable substrate, faster kinetics of degradation may compensate for increased size reduction costs. This chapter presents results from a study conducted to determine the effects of storage and size reduction on biomethane potential of sugar sugar beets Without size reduction, the effect of storing sugarbeets in airtight containers for several months on its biomethane potential and rate was compared to that from fresh sugarbeets (immediately after harvest) and frozen sugarbeets To study the ef fect of size reduction, biomethane potential and rate of methane production from anaerobic digestion of frozen
23 sugarbeets shredded to 3 cm size was compared to whole frozen sugarbeets The performance of anaerobic digestion was evaluated through an assess ment of methane yield, methane production rate, and residual organic matter remaining after digestion. Materials and Methods Feedstock Pails of frozen whole sugarbeets were shipped overnight from American Crystal Sugar, Moorehead, MN in coolers. Upon re ceipt, the coolers were stored in cold storage maintained at 20C. Later another pail of fresh sugarbeets were shipped in coolers overnight and these were stored at 4C. Immediately upon receipt of fresh sugarbeets, 14 kg were placed in a 5 gallon bucke t and closed airtight. The bucket was kept at room temperature 23C 2C for 4 months and 20 days (20 weeks). Henceforth, the For size reduction experiments, frozen sugarbeets were thawed and manually chopped using a kitchen knife to approximately 3 cm length. Anaerobic digester The digester was constructed by modifying 30 L Pyrex glass carboy bottle. As shown in Figure 2 Each carboy bottle was thermally cut at its base and a flanged lip was curled, resulting in an inverted carboy bottle with a cross sectional opening. The neck of the bottle was adjusted by thermally fusing a glass flange to increase overall length. The bottom of the digester was attached to a glass cup with outlet for liquid withdrawal and port for recirculation of liquid. were fabricated on top of lid to be used as outlet po rts for biogas and recirculation of mixed liquor. A rubber gasket is placed between the digester top and the lid to avoid
24 any gas leakages. Sugarbeets are loaded from top of digester before sealing the lid. A recirculation pump is provided to circulate the leachate in the digester from bottom to top. The gas production from the digesters was measured by using positive displacement gas meters. Digester was heated by wrapping heating tape around the Pyrex bottle. Temperature was monitored by inserting a therm ocouple inside digester and at surface as shown in Figure 2 1. Temperature was controlled and set at 60C at surface and at 55C inside digester by controller CR10X. Peristaltic pump was used for recirculation of mixed liquor of digester from bottom to top Further details of reactor setup can be found in a previous study ( Polematidis 2007). Experiments Frozen, fresh and airtight stored sugarbeets were digested in three batch anaerobic digester as described above. Digestion of each type of sugarbeet was do ne by loading 4.68 kg by weight in 28 L of active inoculum (of mixed microbial biomass), which is equivalent to a loading of 40 g VS/L of frozen sugarbeets, 38.9 g VS/L of fresh sugarbeets and 33.5 g VS/L of airtight stored sugarbeets. Buffer (baking soda) was added at 10 g/L concentration to maintain an average pH in range of 7 8. Digestion runs for frozen, fresh and airtight stored sugarbeets were triplicated. Mixed liquor during digestion was collected for various analysis described below. Analysis All forms of sugarbeet feedstock were analyzed for total solids (TS) or dry matter (DM) and volatile solids (VS). Mixed liquor samples collected from digester were analyzed for soluble chemical oxygen demand (sCOD) and pH. Volumetric gas production from diges ter was measured using a positive displacement gas measuring
25 device. Biogas was analyzed for methane and carbon dioxide content. Mixed liquor and biogas samples were analyzed daily. ( Greenbe rg et al.,1992) DM were determined by drying a known amount of sample for 24 hours at 104C and then weighing the dry sample to get the DM content of the sample. It was ensured that drying for 24 hours produced a constant dry weight. Then dried sample w as burned at 550C in muffle furnace for 2 hours. The ash produced was weighed and volatile content was determined by difference. The biogas from the anaerobic digester was quantified in terms of methane and carbon dioxide content using a gas chromatograph (Fisher Gas Partitioner). The sCOD was measured by using HACH COD measuring kit. The mixed liquor from digester was centrifuged at 300 rpm for 10 minutes and then filtered with a 0.45 micron pore size Whatman filter paper. The sample was appropriately dil uted and 2 ml of it was pipetted out in COD measuring vial (HACH, 2 150 ppm range). The vial contents were digested for 2 hours, allowed cool before reading on a HACH DR/890 colorimeter. Gompertz curve fit ( Koppar et al. 2007 ) was performed on the average cumulative methane data from experiments using whole frozen sugarbeet, shredded frozen sugarbeet, airtight stored sugarbeet and fresh sugarbeet. Gompertz equation modified for cumulative methane production is as follows: Fitting experimental data to Equation 1 allowed determination of digestion performance. Gompertz parameters P, R m and in Eq uation 1 are estimated cumulative methane potential (LCH4 STP/kg VS), maximum rate of methane production (LCH4
26 STP/kg VS/d) and duration of lag phase (d) for methane initiation respectively. Gompertz parameters from fits to average of data are summarized i n Table 2 2. Results A photograph of the sugarbeets immediately after being taken out from storage is shown in Figure 2 2. As can be seen, sugarbeets were found intact, non bruised and no microbial/fungal growth on the surface. There was no leachate (free liquid) accumulation in the container. The sugarbeets after being taken out of airtight storage weighed 12.25 kg, which was 87.5% of initial weight loaded into the bucket. This corresponded to a weight loss of 12.5% during airtight storage. The results for DM content (or total solids, TS) and VS of the sugarbeets stored under different conditions are summarized in Table 2 1. The DM and VS content of fresh sugarbeets were (24.80.54) %, (942.31) % respectively. The DM and VS content of frozen sugarbeets were 25.960.94% and 95.96% respectively. The DM content and VS content decreased to 220.65% and 912.31% respectively after airtight storage. Accumulated methane yield at STP per kg VS of sugarbeets loaded into digester is plotted against digestion dur ation (days) for frozen, airtight and fresh whole sugarbeets in Figure 2 3. From Figure 2 3, on a VS basis, frozen sugarbeets produced the highest yield of 423.4 LCH4 STP/kg VS. The yield from airtight stored sugarbeets and fresh sugarbeets were 360.7 LCH4 STP/kg VS and 220.2 LCH4 STP/kg VS respectively. Similar trend was observed for yield when normalized per kg weight of the sugarbeets immediately weighed after harvesting. It was not possible to estimate cumulative methane potential per kg feed as harvest ed for frozen sugarbeets as the original weight of the sugarbeet prior to freezing was not available. Airtight stored beet
27 digestion and fresh beet digestion resulted values of 73.82 LCH4 STP/kg and 57.26 LCH4 STP/kg of sugarbeets as harvested respective ly. Gompertz curve fit on the experimental data from anaerobic digestion of fresh, frozen and airtight stored sugarbeets gives an estimated methane yield potential of 236.98 LCH4 STP/kg VS, 456.87 LCH4 STP/kg VS and 374.12 LCH4 STP/kg VS respectively, when normalized per kilogram VS loaded into digester. Time required to degrade 95% of fresh beet was around 26 days. The 95% degradation time for frozen sugarbeets was 23.5 days and for airtight stored sugarbeets was 13 days (Figure 2 4). Methane production i nitiation in fresh sugarbeet digestion was at day 3.6 and for frozen and airtight stored sugarbeets, initiation was at day 0.25 and day 2.25 respectively. Maximum value of methane production normalized per liter of inoculum per period for fresh sugarbeets was 1.57 L CH4 STP/L/d and for frozen and airtight stored sugarbeets were 2.96 L CH4 STP/L/d and 0.58 L CH4 STP/L/d respectively. These maximum values were observed at 9.58 d, 8.75 d and 7.06 d during digestion of fresh sugarbeets, frozen sugarbeets and a irtight stored sugarbeets respectively. The maximum rate of methane production expressed per kg of feed loaded was 4.85 L CH4 STP /kg/d for fresh beet, 9.16 L CH4 STP /kg/d for frozen sugarbeets and 12.4 L CH4 STP /kg/d for airtight stored sugarbeets. Abo ve results indicates rates of digestion of airtight stored sugarbeet was highest amongst fresh, frozen and airtight stored sugarbeets. The soluble chemical oxygen demand (sCOD) was normalized per kg VS loaded in Figure 2 7. Normalized soluble chemical oxy gen demand for fresh sugarbeets digestion was measured 635.2 g/kg VS at 1.042 day and for frozen sugarbeets and
28 airtight stored sugarbeets digestion, 1594 g/kg VS at 1.5 day and consistent at the value of 1163 g/kg VS from day 2.6 to 5.24 day. The end sCOD from digestion of frozen beet and fresh sugarbeet is 220 g/kg loading where as it was higher in case of digestion of airtight stored beet as 360.8 g/VS loaded. Accumulated methane yield at STP per kg VS of frozen sugarbeets loaded into digester is plotte d against digestion duration (days) for whole sugarbeets and shredded sugarbeets in Figure 2 5. As seen from Figure 2 5, methane production from sugarbeets appears to occur in two stages irrespective of size reduction. Cumulative methane curve appears to level off after an initial increase (methane from first fraction) and then increases again before leveling off once more (methane from second fraction). For whole sugarbeets, the first fraction yielded 196.8 L of methane/kg VS at 6.7 days and total ultima te yield was 423.42 LCH4 STP/kg VS at 24.7 days. For shredded sugarbeets, first fraction yielded 148 LCH4 STP/kg VS at 4.7 days and total ultimate yield was 379.8 LCH4 STP/kg VS after 17.9 days. A Gompertz curve fit very well to the experimental data and gave similar total ultimate yield. The Gompertz curve fit for the entire set of data points did not predict the methane from two fractions. Gompertz fit estimated methane yield from whole beet digestion as 456.9 LCH4 STP/kg VS and from shredded beet as 4 34. 9 LCH4 STP/ kg VS. Maximum rate of methane production (Rm) was also similar; 58.25 LCH4 STP/kg VS/d for whole beet and 58.81 LCH4 STP/kg VS/d for shredded beet. Initiation of methane production was 34% faster (indicated by a shorter lag time or lower ) using shredded sugarbeets than whole beet. A Gompertz curve fit was done to fit two fractions of methane yield in whole as well as shredded sugarbeets digestion. In whole sugarbeets digestion, first fraction
29 methane yield from Gompertz curve was 191 LCH 4 STP/kg VS and rate of methane production was 63 LCH4 STP/kg VS/d and second fraction yield was 460.9 LCH4 STP/kg VS and rate of methane production was 24.2 LCH4 STP/kg VS/d. In shredded sugarbeets digestion, first fraction methane yield from Gompertz cur ve was 159 LCH4 STP/kg VS and rate of methane production was 55.8 LCH4 STP/kg VS/d and second fraction yield is 421.5 LCH4 STP/kg VS and rate of methane production is 32.9 LCH4 STP/kg VS/d. Percentage of ultimate methane yield is plotted against duration o f digestion in Figure 2 6. Whole beet gives 95% of the total obtained yield in 20.3 days, whereas it takes only 14.7 days for the shredded sugarbeets to produce 95% of the ultimate yield. At the end of digestion when methane production ceases sCOD remain ing was around 220 g/kg VS for both whole and shredded sugarbeets. Soluble COD accumulates rapidly during shredded beet digestion peaking on day 1.8 at 1126 g/kg VS. Soluble COD accumulated to 1594 g/kg VS on day 5.7. As shown in Figure 2 8, the sCOD co ncentration in shredded beet digestion remained lower throughout the digestion when compared to the whole beet digestion. Less than 2% of errors are associated with all analytical measurements. They are not shown in Figure 2 8, as it is considerably low. Discussion Effect of size reduction on biomethane potential of sugarbeets The average methane yield of sugarbeet obtained from lab scale experiments is 388 LCH 4 /kg VS which is higher when compared to other terrestrial biomass such as Napier grass (340 LCH 4 STP/kg VS), poplar (320 LCH 4 STP/kg VS), willow (300 LCH 4
30 STP/ kg VS) etc. and hence a better substrate for anaerobic digestion than other terrestrial biomasses. The biomethane production from shredded sugarbeet digestion was observed to be less than tha t from whole sugarbeet digestion. Sugarbeet consists of sugar and non sugar DM. The sugar concentration in a sugarbeet is highest in the vascular zone ( central part of the sugarbeet) and has lower fresh and dry weight concentration of sugar ( Draycott 2006 ) Also, the lower part of the root contains highest concentration of sugar about 16 20% on wet weight and concentration decreases towards hypocotyl (15%) and lower (13%) and upper parts of the crown (7 9%). Crown is the part of sugarbeet which lies above the level of lowest leaf scar. It is possible while loading the shredded sugarbeets, parts of higher concentration of sugars were left behind. Therefore, the loading in whole sugarbeet digestion and shredded sugarbeet digestion may be equal in terms of wet weight, but not equivalent in terms of sugar content. Lower methane yield from shredded beet digestion can be accounted from the above reason. Sugarbeet consists of 52% sugars, 22% non digestible carbohydrates or dietary fibers and rest being proteins, m inerals and ash ( Asadi 2007 ), which make it a highly degradable substrate but not a rapid one. The surface sugars should degrade first in a digester followed by pulp (fibrous component). The two step degradation observed in whole sugarbeets and shredded s ugarbeet digestion (Figure 2 5) indicated that at first the surface sugars degrade followed by the remaining pulp. Shredding does not help in improving the initial rate of methane production as in both cases the readily soluble sugars degrade first.
31 Previ ous study has shown problems of rapid acidification and compaction of sugarbeet fibers in digester, which makes size reduction in sugarbeets an unnecessary step. However, size reduction studies were done to investigate any increase in methane production ra te compared to no size reduction (whole sugarbeets). Gompertz fit was done on whole sugarbeet and shredded sugarbeet digestion data and it was observed that rate of methane production during first fraction of sugar degradation is same in both digestion as shown in Table 2 3. Kinetics of pulp (fiber) degradation is 36% faster in shredded sugarbeet than whole sugarbeet as measured by rate of methane production. Degradation of 95% of shredded sugarbeets was achieved in 42% less time than degradation of 95% of whole sugarbeets. This shows shredding helps in reducing the degradation time of sugarbeets, but there is considerable amount of ene rgy required for size reduction. Due to faster kinetics of digestion of shredded sugarbeets, a digester can handle more num ber of digestions in a year and faster methane generation than whole sugarbeet. But additional operation of size reduction demands energy and could affect the net energy from the anaerobic digestion system. If the proposed lab scale system is allowed to ru n for one year, 18 cycles of whole sugarbeet (4.68 kg/cycle) can be digested per annum and 26 cycles of shredded sugarbeet (4.68 kg/cycle) can be digested per annum. This results in generation of 690 MJ and 942.7 MJ of energy per annum from batch digestion systems. Shredding of sugarbeets alone may demand 854 MJ of energy per annum and this makes net energy from shredded sugarbeet digestion much less than whole beet digestion ( Naimi 2006) despite of faster kinetics of
32 degradation. So it can be concluded th at size reduction does increase kinetics of methane production from sugarbeets but at expense of high energy requirement. Effect of different types of storage on biomethane potential of sugarbeets A decrease of 15% and 4.4% was observed in DM content and VS content respectively for airtight stored sugarbeets when compared to frozen and fresh sugarbeets. Similar observations were made in a previous study for sugarbeets stored in a closed chamber, with a loss of 7% to 17% of initial fresh matter ( Kenter et a l. 2006). The DM and VS content of the sugarbeets decreased after airtight storage. During storage period, sugarbeets loose DM due to continued respiration. Studies done previously on corn silage, grass silage, dried sugar beet pulp silage (natural, withou t any chemical addition) has shown a reduction in DM in the range from 60 70% (Toenjes et al.,1970; et al. 1999; Owens et al.1970; McEniry et al. 2007). There is also significant loss of moisture with the duration of storage which is responsible fo r reduction in total weight of stored sugarbeets. Airtight storage leads to partial fermentation of surface sugars and fibers in sugarbeets if stored for a shorter period of time. Mostly, dissacharide sucrose gets converted to ethanol and other acids durin g storage. This results in higher rate of methane production in airtight stored sugarbeets. Also, with time, the water in the beet evaporates leading the concentration of sucrose in sugarbeets to increase up to 5.0% of initial value ( Kenter et al.,2006). T he total nitrogen in the sugarbeets remains unchanged but it converts to amino Nitrogen (amino N) which is easier to breakdown than protein in the anaerobic digestion process. The concentration of amino N increases by a range of 57% to 153% (Kenter et al., 2006). Partial fermentation of sugarbeets often can be responsible for faster rate of degradation of sugarbeets and higher sucrose concentration for high rate of methane
33 production in digestion of airtight stored beet. Limited degradation of surface sugars could result in lower rate of methane production in initial 3 days. Cumulative methane yield was observed highest (423.42 LCH4 STP/kg VS) from digestion of frozen sugarbeets and lowest from fresh sugarbeets (220.22 LCH4 STP/kg VS). Cumulative methane yie ld from airtight storage was 85% of yield obtained from frozen sugarbeets. The rate of respiration decreases with lowering of temperature. This prevents the loss of sugar from the sugarbeet. Freezing increases membrane fragility and modifies the transtonop last electrical potential difference. Freezing also affects the vacuolar membrane and increases its permeability for sucrose ( Barbier et al., 1981). Hence it could facilitate higher sucrose release. This could be accounted for highest methane production fr om the frozen beet. Sugarbeets disinterred from ground is considered as fresh. Freshly harvested sugarbeets are resistant to bacterial attack (diseases) as a part of action against microbial attack to preserve sugars in sugarbeets (Asadi, 2007). Similar pr otective action of sugarbeets inside anaerobic digester could have prevented increased degradation of fresh sugarbeets and explains lower methane yield. Further, observation of a larger amount of fibrous material remaining at the end of digestion of fresh sugarbeets suggested only partial degradation of sugarbeets The sCOD at the end of digestion reached lower values at the end of digestion of frozen, fresh and airtight stored sugarbeets. This indicates sugars released were degraded completely. However, 50 60% less sCOD was released from fresh sugarbeets as compared to frozen and airtight stored sugarbeets. This could be due to bacterial resistivity of fresh sugarbeets as discussed above. 25% less sCOD released
34 from airtight stored sugarbeets than froze n sugarbeets can be explained from partial fermentation of surface sugars during airtight storage. Conclusions Biomethane potential of sugarbeets on an average is estimated to be 388 L STP/kg VS, which makes it a very good feedstock for methane production. Airtight storage is a cheaper alternative of storing sugarbeets and results in only 17% less cumulative methane yield than frozen sugarbeets. In addition, 95% of total methane production during digestion of airtight stored sugarbeets was attained in half the amount of time than obtained during digestion of frozen sugarbeets. Airtight storage method is similar to plastic tube silos used to store large amounts of sugarbeets in fields. This method of storage can be used in places facing short winters where i t is difficult to store sugarbeets piles in the open by freezing. Fresh sugarbeets are not good choice as feedstock for biomethane production. Size reduction of sugarbeets has no effect on ultimate methane yield. Not surprisingly there is an improvement in rate of methane production after size reduction of sugarbeets but this would be at a cost from additional unit operation and increased energy consumption. Whole sugarbeets digestion of frozen sugarbeets was further studied in two stage digestion systems i n pilot scale in Chapter 3 for improving the rate of methane production.
35 Table 2 1. Total solids and Volatile solids content of sugarbeet stored at different conditions Feedstock condition Total Solids (TS) % Volatile Solids (VS) (% Total Solids) F rozen sugarbeets 25.96094 95.692.31 Airtight stored sugarbeets 220.65 912.31 Fresh sugarbeets 24.80.54 942.31 Table 2 2. Gompertz parameters of sugarbeet digestion runs Run feedstock Feedstock condition Experimental Methane yield (L STP/kg VS) Gompertz parameter P (L STP /kg VS) R m (L STP/kg VS/day) (day) Frozen Whole beet 423.42 456.87 58.25 0.24 Frozen Shredded 379.86 434.89 58.81 0.007 Fresh Whole beet 220.22 236.98 15.53 3.57 Airtight storage Whole beet 360.77 374.12 51.87 2.24 Table 2 3. Gompertz parameters of two fraction sugarbeet digestion of frozen beet with and without size reduction Run feedsto ck Feedstock condition Experim ental Methan e yield (L STP/kg VS) Gompertz parameter Fraction I Gompertz parameter Frac tion II P (L STP /kg VS) R m (L STP/kg VS/day ) (day) P (L STP /kg VS) R m (L STP/kg VS/day ) (day) Frozen Whole beet 423.42 191 63 0.3 460.9 24.2 0 Frozen Shredded 379.86 159 55.8 0 421 32.9 1.67
36 Figure 2 1. Photograph of sugarbe ets taken out of airtight storage after 4 months and 20 days
37 Figure 2 2. Schematic diagram of 30 L anaerobic digester Figure 2 3. Cumulative methane yield of sugarbeets stored at different conditions 0 50 100 150 200 250 300 350 400 450 500 0 5 10 15 20 25 30 Cumulative Methane Yield (L STP/kg VS) Time Elapsed (days) Frozen (Whole beet) Airtight storage (Whole beet) Fresh (Whole beet)
38 Figure 2 4. Percentage ultimate methane yield of sugarbeets at different storage conditions Figure 2 5. Cumulative methane yield comparing whole and shredded beet digestion 0 10 20 30 40 50 60 70 80 90 100 0 5 10 15 20 25 30 Percentage of ultimate methane yield Time Elapsed (days) Frozen (Whole beet) Airtight stored beet (Whole beet) Fresh beet (Whole beet) 0 50 100 150 200 250 300 350 400 450 500 0 5 10 15 20 25 30 Cumulative Methane Yield (L@STP/kg VS) Time Elapsed (days) Whole beet (frozen) Shredded beet (frozen)
39 Figure 2 6. Percentage ultimate yield of sugarbeets at different sizes Figure 2 7. Soluble COD released during digestion o f sugarbeets stored at different conditions 0 10 20 30 40 50 60 70 80 90 100 0 5 10 15 20 25 30 Percentage of ultimate methane yield Time Elpased (days) Whole beet (frozen) Shredded beet (frozen) 0 200 400 600 800 1000 1200 1400 1600 1800 0 5 10 15 20 25 30 35 Soluble Chemical Oxygen Demand (g/kg VS) Time Elapsed (days) Frozen (Whole beet) Airtight storage (Whole beet) Fresh (Whole beet)
40 Figure 2 8. Soluble COD release from whole and shredded sugarbeet digestion 0 200 400 600 800 1000 1200 1400 1600 1800 0 5 10 15 20 25 30 Soluble Chemical Oxygen Demand (g/kg VS) Time Elapsed (days) Whole beet (frozen) Shredded beet (frozen)
41 CHAPTER 3 PILOT SCALE, TWO STAGE, THERMOPHILIC BATCH ANAEROBIC DIGESTION OF SUGAR BEETS Summary Sugar beets without the tops and without size reduction were anaerobically digested in a batch two stage thermophilic pilot scale digestion system. The two stage system consisted of a solids digester (SD; 150 L working volume) into which the sugar beets were loaded and an anaerobic filte r (600 L working volume). The methane yield from the system was 305 L at STP/kg VS loaded and was obtained within 17 days. This is equivalent to 52 L of methane at STP/ kg sugar beet as received from the stockpiles. 60% of the methane was produced in the AF and the rest in the SD. The methane yield from this pilot system was 80% of that obtained in laboratory scale trials. 90% of the total methane produced in the anaerobic filter was collected within 10 days. A 10 day residence time would yield a total o f 256 L at STP/kg VS or 84% of total yield from pilot scale system. The digester required addition of nitrogen and phosphorous nutrients. The biogas may not require hydrogen sulfide clean up before utilization. Background This chapter presents results fro m pilot scale two stage batch digestion of solely sugar beets obtained from stockpiles on the fields. These studies were conducted in a two stage system. Previous studies on anaerobic digestion of a rapidly fermentable feedstock, like sugarbeet tailings, which contains a large fraction of soluble organic matter, have shown that to successfully digest this material at appreciable decomposition rates, it is necessary to wash the soluble components away and digest it separately ( Liu et al., 2008) or carry ou t the digestion in a two stage system ( Polematidis 2007). The two stage system consisted of two separate vessels. The
42 solid feedstock is loaded into one vessel (called the solids digester) and filled with inoculum. The second vessel is a conventional w astewater anaerobic digester like for example a stationary fixed film reactor. The liquid from the solids digesters is exchanged with the liquid in the anaerobic filter. This process removes the solubilized and fermented organic matter from the solids dig ester and inoculates it with buffered mixed liquor from the anaerobic filter. Initially, methane is produced only from the anaerobic filter as the solubilized organic matter is biogasified in this vessel. Eventually through continued inoculation and remova l of fermented soluble, methane production is established in the solids digester also ( Polematidis 2007). As sugar beets contain large fraction of sugars which was expected to solubilize and rapidly ferment, a two stage system was employed. Anaerobic digestion can be co nducted at mesophilic (32 38 C) or ther mophilic temperatures (52 58 C) ( Sung and Santha 2003). Thermophilic operation facilitates higher degradation rates, which in turn makes the system robust for a wide range of org anic loading rates (OLR) and guarantees better pathogen (primarily plant pathogen in this case) destruction in the digester effluent ( Han et al., 1997; Veeken and Hamelers 1999; Sung and Santha 2003; Hegde and Pullammanappallil 2007; Koppar and Pullamma nappallil 2009; Rubio Loza and Noyola 2010; Riau et al., 2010; Kim et al., 2011) Hence, thermophilic digestion was preferred for the present study. The sugarbeets were digested as the whole beet without any size reduction. Laboratory scale digestion s tudies shown in Chapter 2 had indicated that the rate of decomposition did not significantly improve by chopping the beets down to 2 cm size. The performance of the pilot scale system with regards to biogas production rate,
43 composition and quality, and ch aracteristics of effluent and digested residue are presented in this chapter. Methods Two stage pilot scale anaerobic digester system design and operation Figure 3 1 shows a schematic diagram of the two stage system. A 160 L (working volume 150 L) stainle ss steel cylindrical vessel with a conical bottom was used biogas outflow and sampling, insertion of temperature probes, and attachment of recirculation and sequencing line with two knife gate valves. Knife gate valves were used to create a chamber in the chute. The solids digester was equipped with a diamond back conical bottom to be able to easily remove the digested resi (AF). The total volume was 678 L (working volume 600 L). A pressure gauge, gas outflow and sampling ports were provid ports were used for the peristaltic pump transfer lines. The AF contained bundles of plastic meshes (mesh size 3 cm x 3 cm) tethered to the bottom plate. Each bundle was tethered with cords of different lengths so when filled with liquid each floated to different height. These bundles acted as a packing material providing surface for biofilm development and to retain m icrobial. The digesters were heated by hot water flowing through copper tubing wound around the vessels. Two household electric water heaters provided hot water to each digester. The SD was insulated with Hull Epoxy 2 Part Foam insulation and AF
44 with R30 fiberglass insulation. Hayward SP1580 centrifugal pumps were used to recirculate the liquid in SD and AF. The recirculation pumps were turned for 3 minutes (at 240 liters per minute) every 45 minutes to pump liquid from top to bottom thereby mixing thoro ughly without creating any foam. A Cole Parmer (Model No. 7553 30) two head peristaltic pump with variable flow control (20 400 L/day) was used to transfer a specific volume of liquid between digesters (called the sequencing operation). Masterflex Neopr ene food grade flexible tubing was used for sequencing. Positive displacement, U tube gas meters were used to monitor biogas production in both digesters ( Koppar and Pullammanappallil, 2009 ). Thermocouples were inserted in both digesters and connected to a Campbell Scientific CR 10X Datalogger to monitor and record temperature. In the SD thermocouples were placed in cylindrical and conical sections in order to ensure that temperature was uniform temperature throughout the digester. Experimental Protocols F rozen whole sugar beets were shipped from American Crystal Sugar Company (Moorehead, Minnesota, USA) stockpiles and kept in cold storage upon receipt as described in Chapter 2. Each sugar beet weighed on average 310 g. Beets did not include any top and le aves. Sugar beets were loaded as received into the SD. The pilot scale system was operated for over six months with sugar beets as feedstock before the experiment discussed here was carried out. Prior to sugar beets the system was fed citrus pulp for over a year. During initial operations with sugar beets, the system was first used as a single stage with only the SD being active. About 1 kg (~ 3 beets) of whole sugar beet was added daily for over a month. It was found that the loading could not be increas ed beyond 1 kg as it would acidify the digester. The AF then was brought
45 into the process and sequencing was initiated. Initially 50 L/d of liquid was exchanged between the digesters. The mass of sugar beet loaded was ramped up. Just prior to this expe riment the system was successfully operated with a 60 kg batch addition of sugar beets. For the experiment discussed here, 71 kg of sugar beets were loaded manually into the SD as follows. The top valve (KGV 1) was opened first keeping KGV 2 closed (Figu re 3 1). The chute was filled with sugar beets and digester mixed liquor from previous run to expel air. KGV 1 was closed and then KGV 2 opened to drop the beets and mixed liquor into digester. This was repeated until 71 kg of sugar beet as well as 75 L of digester mixed liquor was loaded. This created a 10 L headspace. The liquid level in the digester was such that any floating debris was above the intake port of the recirculation pump 1 so as to avoid clogging. Sequencing flow rate was set to 100 Lite rs/day to yield a 6 day HRT in the AF. Samples were taken from the sampling ports on both digesters. pH was measured immediately and the samples were then stored in a 4 C refrigerated chamber for further analysis. Throughout the digestion process preca utions were taken to prevent gas and liquid leaks by maintaining a close look at all major ports, attachments, valve connections. Once the experiment was completed the SD was emptied using the bottom knife gate valves KGV 3 and 4. Keeping KGV 4 closed KGV 3 was opened to allow digester contents to fill the chute. KGV 3 was closed and KGV 4 opened to empty the chute. The procedure was repeated. The drained contents were sieved using a 1 mm screen mesh.
4 6 Analysis Total solids (TS) were determined by gravim etric method after drying for a few days in an oven at 105oC. Volatile solids (VS) were determined by burning the dried solids in a muffle furnace drying at 550 C for 2 hours. Details of TS and VS analysis are in Chapter 2. Biogas composition was measured using a LANDTEC GEM 2000 Gas Analyzer and Extraction Monitor (Colton, California, USA). The analyzer measured the percentage (by volume) concentrations of methane, carbon dioxide and oxygen in the biogas. Additionally the analyzer measured carbon monoxide and hydrogen sulfide in parts per million (ppm). pH was measured using a digital pH probe, OAKON Waterproof Big Display pH Tester 30. The probe was calibrated once a week using HACH Standard Buffer Solutions pH 4,7,10. Liquid samples were centrifuged in a Fisher Marathon micro H centrifuge for 10 minutes at the speed of 350 rpm. The supernatant was filtered using Whatman filter paper (0.20 m). The filtrate was used for soluble chemical oxygen demand (sCOD), organic acids, ethanol, ammonia N and phospha te P analysis. sCOD analysis was carried out using HACH kit. Two ml of filtrate was pipetted out in COD vials (range: 2 150 ppm, HACH) containing the reagents and heated in COD reactor (HACH) for 2 h. The COD of the reacted and cooled sample was measured by using colorimeter (HACH DR/890). Organic acids (acetic, formic, butyric, valeric, lactic and succinic acids) and ethanol was analyzed using a HPLC HP1090 (Agilent Technologies, CA) equipped with a HPX 87H column (Bio Rad laboratories, CA). Photometri c and refractive index detectors were used in series for estimation of organic acids and ethanol present in the sample. Th e operating temperature was 45 C, and the carrier liquid was1 M sulfuric
47 acid at a flow rate of 0.4 ml/min. Chromatograms were process ed using chemstation software. The filtrate was acidified with HCl at a volume ratio of 1:0.05, filled in a HPLC sampling vial and sealed with a rubber top. The vials were loaded into an automatic sampler. The volume of sample injected was 10 L. HACH Nitrogen, Ammonia Salicylate, Method 8155 (range of 0.01 to 0.50 mg/L NH 3 N) was used to determine NH3 N in the mixed liquor from the digester. Filtrate was taken in a sample cell and mixed with one ammonium salicylate powder pillow allowing 3 minute of reaction time, followed by addition of ammonia cyanurate powder pillow allowing 15 minute of reaction time. Sample cell was read on a colorimeter ( HACH DR/890). Ascorbic acid method was utilized to determine phosphate concentration. 50 ml of diluted filtrate was mixed with 8 ml of combined reagent and allowed to react for 10 minutes. Absorbance of the reacted mixture was measured at 880 nm in (Thermo Scientific EV60 spect rophotometer). From a calibration curve, corresponding phosphate concentration was determined. The phosphate concentration was reported as PO4 P. Results and Discussion Methane yield and its rate of production Whole sugar beets without any size reduction were successfully digested in a two stage batch system at thermophilic temperature. Cumulative methane yield from the individual reactors (SD and AF) and from the combined system expressed as L at STP per kg VS of sugar beet loaded in SD is shown in figur e 3 2A. Methane productivity per liter of reactor volume per day from both individual reactors and for the combined system is shown in figure 3 2B. The concentration of methane in biogas from both digesters is shown in figure 3 2 C and D.
48 After loading the SD, it took five days before significant methane content (~15%) to be detected in the biogas (figure 3 2C). The methane content of biogas fluctuated between 0.4 and 2 % for the first four days. During this period the biogas was primarily carbon dioxid e (> 90%). Once methanogenesis was established in the SD, biogas methane content continued to increase reaching more than 50% by day 9. After day 10, the methane content fluctuated between 51 and 57.6% until the end of digestion. The methane production rate peaked by day 10 to 1.9 L at STP/L/day and then dropped as substrate was consumed (Figure 3 2B). Methane was produced in the AF from the solubilized material that was fed to it from SD by the sequencing operation. Methane production was initiated in AF as soon as sequencing was started. However, it quickly dropped before increasing again. The biogas methane content in AF was around 80% initially dropped to 27% before increasing to 60% within two days of the drop. This temporary drop was due t o an overloading of AF with mixed liquor containing high concentrations of solubilized and acidified organic matter from SD. But AF recovered quickly and methane production as well as biogas methane content increased. Biogas methane content in AF increase d to 60% by day 3 and was maintained around 80% after day 4 (figure 3 2D). Methane production rate reached a peak value of 0.9 L at STP/L/day by day 6 and then dropped as concentration of organic matter in the mixed liquor from SD decreased (figure 3 2B). It should be noted that by this time methanogenesis was also established in the SD. Methane productivity from the combined system peaked at its highest value of 0.76 L/L/kg VS corresponding to peak value from SD before decreasing. The total methane yie ld from the system was 305 L at STP / kg VS of sugar beets loaded (figure 3 2A).
49 About 60% (182 L/kg VS) of this yield was produced in the AF and the rest 40% (123 L/ kg VS) in the SD. Biogas quality Figures 3 2 C and D also show the composition of hydrog en sulfide in the biogas from SD and AF respectively. Hydrogen sulfide was detected only in low concentrations in both digesters. In the SD, 145 ppm was detected one day after start of digestion and dropped below 10 ppm for the next 2 days increasing to 392 ppm on day 4. After that no H 2 S was detected until day 13. On day 14 there was another peak in H 2 S content at 59 ppm followed by a drop and increase to 147 ppm by the end of the run. In the AF, H 2 S was detected in the biogas only during the first 7 da ys of operation. During this period H 2 S content fluctuated between 1 and 50 ppm. There were two distinct peaks in H 2 S content. The first peak at 45 ppm occurred one day after startup and the second peak of 50 ppm occurred five days after startup. The biogas may not require H 2 S clean up before utilization. It is likely that biogas produced in the SD during the first five days will not be used since the methane content is very low. It is only during this period that the biogas from SD contained H 2 S. When H 2 S was detected in biogas from AF, it ranged between 1 and 50 ppm. This concentration is sufficiently low to require any clean up. Mixed liquor characteristics The characteristics of the mixed liquor in both digesters are shown in figures 3 3 and 3 4. The pH, soluble COD and organic acid and alcohol concentration was measured in the mixed liquor samples. The pH in the AF before the start of the run was 8.5. The pH decreased as soon as the sequencing process was initiated. It dropped to as low as 6.66 by day 3 before increasing again (figure 3 3A). The drop in pH was due
50 to an accumulation of organic acids in the AF (figure 3 4). High concentrations of acetic and butyric acids were detected in AF until day 5. Acetic acid concentration peak ed at 4000 mg COD/L and butyric acid concentration was between 1000 and 2000 mg COD/L during this period. About 360 mg COD/L of lactic acid was measured on day 1 after which it was not detected until day 10 when about 800 mg COD/L was measured. As the aci d levels decreased the pH increased and was maintained above 7.5 after day 5. The pH in the SD showed more dramatic changes, dropping to 4.96 within 2 days. It continued to increase after this and was maintained above 7.0 after day 7. The low pH in SD w as due to high levels of organic acids. For two days after start of experiment lactic acid levels reached 30,000 mg COD/L. From day 3 onwards lactic acid was not detected. Acetic acid concentration remained above 2,000 mg COD/L for five days peaking at 4,000 mg COD/L on day 3. Butyric acid levels were also above 1,300 mg COD/L for the first 7 days remaining above 8,000 mg COD/L from day 3 to day 5. Other organic acids like propionic acid and valeric acid were not detected. Ethanol was also not detec ted in the samples. About 75 mg COD/L of formic acid was measured on day 2, and 150 and 115 mg COD/L of succinic acid was measured on days 2 and 3 in the solids digester. These were not detected on other days in SD and were also not detected at any time in AF. The concentration of total organic acids expressed in mg COD/L is shown in figure 3 3C. In the SD, the organic acid concentration peaks to 35,000 mg COD/L by day 2 before decreasing. In the AF, organic acid concentration peaks to around 5,000 mg CO D/L and drops to 200 mg COD/L by day 6. By day 10 the organic acid concentrations in both digesters were
51 below 300 mg COD/L. The soluble COD in the AF was between 4000 and 7000 mg/L for the first 7 days of operation and then it decreased quickly to below 1000 mg /L by day 10 (figure 3 3B). The soluble COD in SD was high initially but decreased below 1,000 mg/L after day 12 (data not shown). Digestion residue characteristics The contents of the solids digester were drained and sieved at the end o f digestion. This was done for the experiment prior to the one discussed here as well as for this experiment. Upon draining one half of the liquid volume, the solids residue that ter contents were drained. Towards the end of drain operation some more solids collected on the ere separately dried. layer ranged between 14 and 18%. The sludge sediment was 55% of the dry weight of residual solids and the floating layer was 45% of dry weight of residual solids. The total dry residual solids (> 1mm) was 13% of the dry matter loaded. Nutrient requirements Ammonia and ortho phosphate concentrations in the mixed liquor of both solids digester and anaerobic filter were monitored every two to thr ee days through the entire period of digester operation not only for the experiment reported here but also during prior operation. When ammonia and phosphate concentrations dropped below 50 mg/L, additional nitrogen and phosphorus were supplemented in the form of urea (46 0 0) and superphosphate (0 46 0) respectively. The total urea added up to and including this experiment was tallied and was divided by the total mass of sugar beets digested after
52 subtracting the residual ammonia remaining in the digeste rs at the end of this experiment. It was found that an additional 3 g of nitrogen was required per kg of sugar beet digested (or equal to 15.5 g of additional nitrogen required per kg sugar beet VS digested). Based on methane production this requirement is equal to 51 mg/ L of methane. This requirement was verified theoretically with bacterial nitrogen composition as follows. Assume that nitrogen content in bacterial cells is about 11% of its dry weight ( Doran 2006). If 1 kg VS is digested typically 1 0% of the digested organic matter is diverted to anabolic cell synthesis reactions in an anaerobic digester. This means that 100 g VS ends up as cell biomass for kg VS digested. Nitrogen required to produce 100 g of cell biomass is approximately 11 g. In other words 11 g of nitrogen is required per kg VS digested. The value obtained here is 15.5 g which is close to the theoretical calculation. Note that it is assumed that sugar beet does not contribute any nitrogen for cell growth. About 11.2 g of phos phorus (or 53.3 g of superphosphate) was added per kg VS of sugar beets loaded. This was equivalent to 2.2 g of phosphorus per kg of sugar beets digested. It was not possible to estimate phosphorus requirements like that done above for nitrogen due to s low dissolution of superphosphate in the digester mixed liquor. This was inferred upon observing undissolved pellets in the dried residual solids. So the superphosphate added would have been far in excess of requirements. It was observed that ammonia an d phosphorus requirements were greater towards the end of the digestion process. This observation regarding nutrient supplementation was contradictory to that made by Lehtomaki and Bjornsson (2006). Their two stage digester did not require any nutrient
53 su pplements possibly because beet tops along with beets was used as feedstock for digestion. Comparison of present work to previous work in literature Table 3 1 compares the results from the present study with that obtained by Lehtomaki and Bjornsson, 2006 Other studies used ensiled beets or beets were co digested with other biomass feedstocks ( Parawira et al., 2004; Lehtomaki et al., 2007; Seppala et al., 2008; Kryvoruchko et al., 2009 ; Kacprzak et al., 2010 ) and therefore could not be compared to the pr esent study. Both these studies used a two stage batch system for digestion of sugar beets. Henceforth, we refer the Lehtomaki and Bjornsson study (2006) as L&B study in the present chapter. The sugar beets were loaded in one vessel and the liquid from th is vessel recirculated back after pumping it through an anaerobic filter. Differences between the studies were the temperature of digestion (thermophilic vs mesophilic) and the particle size of the feedstock (whole vs shredded). In addition, ensiled beet tops were also mixed with beets in a ratio of 1:3 (wet weight) by L&B study. The pilot scale rector used by L&B study was a much larger scale than the one used in the present study. The average yield obtained from size reduction experiments as mentione d in Chapter 2, was 388 L at STP/kg VS after on average 26 days of digestion. The methane yield from the pilot scale system was 305 L at STP/kg VS which was about 80% of yield from laboratory scale digestion and was obtained in 17 days. The methane yield obtained by L&B study was higher both in laboratory scale digesters and pilot scale system. It should be noted that L&B study used a mixture of beets and beet tops as feedstock. Better digestibility of beet tops may contribute to higher yield. Their pilo t scale system produced 85% of the yield from laboratory scale system. The
54 methane yield from pilot scale system when corrected for temperature of measurement, assu ming biogas was measured at 22 C, was 354 L/VS and 85% of this yield, i.e. 301 L/kg VS was p roduced in 30 days. Mesophilic digestion took about twice as long to produce the same yield as a thermophilic digester. In the L&B study pilot scale system a major fraction, about 92% of methane was produced in the anaerobic filter over the first 30 days. After 30 days of digestion, the rest of the methane was mostly produced in the hydrolysis reactor. In other words it took about 30 days for methanognesis to become established in the hydrolysis reactor. In contrast, in our study, methanogenesis was est ablished within 6 days in the thermophilic system using whole beets by which time the biogas methane content reached 30% in solids digester (figure 3 2C). So, a higher fraction about 40% of the total methane yield was produced in the solids digester and t he rest 60% in the anaerobic filter. In figure 3 5, methane produced in each vessel of two stage system is plotted as a fraction of total methane that was produced in that vessel. About 90% of the total methane produced in the anaerobic filter was produc ed in 10 days (point a) and 90% was produced in 12 days from the solids digester (point b). Within a 10 day retention time, the solids digester produced 75% of the total methane that was produced in the vessel (point c). A two stage batch system operated at a 10 day residence time would produce a total of 256 L/kg VS (equal to 44 L/kg sugar beet) or 84% of total yield from pilot scale system. This design should scale up readily as there are no moving parts within both vessels and does not require any pret reatment (for example size reduction). Size reduction can incur significant operating costs. Whole sugar beets can be loaded at a bulk density of 500 kg/m3 in the solids digester. To sustain continuous
55 biogas production at a predetermined flow rate and met hane content, one SD vessel then the system will require 10 vessels to serve as SDs. Each SD could be sequenced with the same AF which will be sized to receive mixed liqu or from all SDs containing solids at various stages of digestion. So the entire anaerobic digestion facility will consist of ten solids digesters and one anaerobic filter. The system would be able to process a significant amount of sugar beets due to the r elatively short retention time required. Sugar beet growers and production facilities with an excess supply would greatly benefit from such systems by offsetting their energy usage. Conclusions The methane yield from the whole sugar beet after 17 days of a naerobic digestion in the two stage system was 305 L at STP/kg VS (which is equivalent to 52 L at STP/kg sugar beet as received from the stockpiles). About 40% of the methane was produced in the solids digester and 60% in the anaerobic filter. Only about 1 3 % of dry matter of sugar beets loaded remained as residue > 1 mm in size in the digester. The solids residue remaining in the digester partitioned into a Floating mat layer and a sludge sediment layer with about 45% dry matter > 1mm in the mat and 55% i n the sludge sediment. Methane composition of biogas in the anaerobic filter was about 80%. In the solids digester it took about 10 days for methane content to reach 60%. Hydrogen sulfide was not detected most of the digestion period. In the solids digest er when hydrogen sulfide was detected methane content was below 16%. In the anaerobic filter H 2 S content fluctuated between 1 and 50 ppm during the first 7 days of operation. H 2 S removal may not be required prior to using the biogas. The digester require d supplementation of both nitrogen and phosphorus. About 3 g of nitrogen supplement was required per kg of sugar beets digested. Additionally 2.2 g of phosphorus (in the form of superphosphate) per kg of sugar beets digested was also added.
56 Compared to l iterature it appeared that the rate of biogasification is about twice as fast at thermophilic temperature as that at mesophilic temperature. Table 3 1. Comparison to results obtained by Lehtomaki and Bjrnsson (2006) Parameter This study Lehtomaki and Bjrnsson (2006) L&B study* Feedstock Sugar beet Sugar beets and tops (3:1) Particle size Whole beet (no size reduction) Shredded Digestion Temperature Thermophilic (55 o C) Mesophilic (37 o C) Digester configuration Two stage (solids digester + anaerob ic filter) Two stage pilot scale (hydrolysis reactor + anaerobic filter) Mode of operation Batch Batch Methane yield (from laboratory digesters) 380 L at STP/kg VS in 26 days **450 L/kg VS in 90 days Methane yield (from pilot scale digester) 305 L at ST P/kg VS **383 L/kg VS in 55 days Residence time to obtain 85% of methane yield from pilot scale digester 11 days 30 days Methane recovery in each stage of combined system 40% (solids digester) 60% (anaerobic filter) 17% (hydrolysis reactor) 83% (anaerobi c filter) Nutrient limitation Yes No Lehtomaki and Bjrnsson 2006, referred as L&B study in the text. *The condition at which the gas volume was measured was not reported. The condition at which the gas volume was measured was not reported.
57 Figure 3 1. Schematic diagram of the pilot scale two stage anaerobic digester
58 Figure 3 2. Biogas production and quality A: Cumulative methane yield at STP, B: Volumetric methane productivity, C: Solids dige ster biogas methane and H2S content and D: Anaerobic filter biogas methane and H2S content
59 Figure 3 3. Solids digester and anaerobic filter mixed liquor characteristics. A: pH, B: soluble COD and C: total organic acids expressed in mg COD/L.
60 Figure 3 4. Organic acid composition in the solids digester and anaerobic filter mixed liquor. A: Lactic acid, B: Acetic acid and C: Butyric acid. Other organic acids like propionic and valeric acids were not detected. Ethanol was also not detected.
61 Figure 3 5. Methane production in each digester expressed as a fraction of total methane that can be produced in that digester.
62 CHAPTER 4 ENZYMATIC SACCHARIFICATION OF DILUTE ACID PRETREATED SALINE MICROALGAE, NANNOCHLOROPSIS OCULATA Summary Present chap ter evaluates saccharification potential of marine microalgae N. oculata by employing preexisting conventional techniques of hydrolysis and subsequent saccharification often used for lignocellulosic biomass. N.oculata was first hydrolyzed using dilute acid such as, 5% (v/v) sulfuric acid, and 5% (v/v) and 2% (v/v) phosphoric acid at 160 C before subjecting to enzymatic saccharification by two commercial cellulases, EI and EII. Neither dilute acid hydrolysis nor enzymatic saccharification alone released any sugars. However, hydrolysates after acid hydrolysis were readily saccharified on addition of enzymes EI or EII. The extent of saccharification ranged between 8 and 100% in all experiments. Sulfuric acid hydrolysis produced furfurals whereas no side produ cts were detected after phosphoric acid hydrolysis. Maximum sugar yield using EI was 345 g sugars/kg AFDM within 4 hours whereas EII yielded 360 g sugars/kg AFDM within 12 hours. Twice of the nominal enzyme loading facilitated 35% more sugar release and ha lf the nominal enzyme loading yielded 64% less sugars. It was concluded that conventional dilute acid hydrolysis followed by enzymatic saccharification using commercially available could be efficient for saccharification of marine microalgae. Ongoing work focusses on optimization of phosphoric acid loading and duration of thermal hydrolysis for 100% saccharification. Background Challenges related to microalgae anaerobic digestion was discussed in Chapter 1. There are only a very limited number of studies on optimization of enzyme
63 saccharifica tion of saline microalgae in the literature. Even though in the present study, N.oculata was saccharified at optimum conditions to study enzymatic saccharification effects on biogas production, but since a literature r eview shows this pretreatment technique is commonly used for ethanol production, comparisons are done with studies used for ethanol production. A considerable amount of work has been done on the simultaneous saccharification and fermentation (SSF) of macr oalgae or aquatic biomass like spirogyra, sea lettuce, invasive algal feedstock such as Gracilaria salicornia, etc. for ethanol production ( Eshaq et al.,2010; Wang et al.,2011). Previous studies shows potential for promising yields of ethanol from macroalg ae owing to its high composition of carbohydrates ( Eshaq et al.,2010). Compared to macroalgae, microalgae usually have higher growth rates and lower nutrient demands for growth ( Hein et al., 1995). Therefore microalgae could be a preferred biomass for fuel production and they can also be cultivated to be rich in lipids and carbohydrates ( Smith et al., 2010). Utilization of algae for ethanol requires saccharification of the carbohydrate content followed by fermentation. With terrestrial biomass saccharifiato n is accomplished by pretreatment processes like acid hydrolysis, steam explosion or heat treatment followed by enzymatic hydrolysis ( Kumar et al.,2009). There are numerous studies using terrestri al biomass. However, there are only a handful studies deali ng with enzymatic saccharification of microalgae as listed in Table 4 1. All studies were done on fresh water microalgae species. These studies utilized commercial and bacterial enzymes for saccharification. From commercial enzyme saccharification literat ure, only one study demonstrates acid hydrolysis pre treatment (carried out at 121 C with dilute sulfuric acid) before enzymatic
64 saccharification of mixed undefined microalgae culture ( Sander et al., 2006). Rest of the studies involving both commercial and bacterial enzymes are subjected to direct enzymatic hydrolysis without any heat pretreatment and uses sulfuric acid to bring down the pH as shown in Table 4 1. In the present study, the saccharification potential of a defined culture of marine microalgae N oculata was studied. Saccharification was carried out by using acid hydrolysis pretreatment before enzyme hydrolysis. Acid hydrolysis using different concentrations of phosphoric acid was compared with sulfuric acid. Phosphoric acid was tested because i t is milder than sulphuric acid on materials used for construction of off the shelf process equipment and produces less inhibitors for fermentation ( Lenihan et al.,2011). Commercial cellulase developed for lignocellulosic biomass hydrolysis was tested for saccharification of N.oculata and its carbohydrate conversion was studied with different enzyme loading rates. The objectives of this study were to determine an optimal saccharification procedure for N.oculata using commercially available enzymes and to co mpare it with studies done with other algal biomass previously. Materials and Methods Feedstock N.oculata was grown in an open raceway pond at 25C, with 1% CO2 and 99% air supplied at Tyndall Air Force Base, Florida. N.oculata was grown for 2 3 weeks to f inal concentration of 600 800 mg/L, and then harvested in a 30 gallon batch by adding base and concentrating algae to 3.15% volatile solids ( Buxy et al.,2013). N oculata was dewatered to a thick slurry of 8% solids. This was accomplished as follows: pot assium hydroxide was added to increase pH to 10.8 so as to settle the biomass overnight followed by filtration of settled sludge using a cheese cloth. A batch of 1 gallon of
65 N. oculata was shipped overnight in coolers to Bioprocess Engineering Research Lab oratory at University of Florida. On receiving the shipment, N. oculata was stored in a chamber at a temperature of 5 C. The batch was well mixed prior to withdrawing samples for saccharification experiments. pH of the feedstock was 10.8. The salt content of the algae slurry was 3.5% and no salt removal was done prior to pretreatment or saccharification experiments. Pretreatment pH of each batch of N.oculata for dilute hydrolysis was brought down to 5 by adding dilute acids. 5% (v/v) sulfuric acid pretrea tment (referred as 5% H2SO4) is addition of 5% (v/v) sulfuric acid solution in N.oculata till pH reaches 5. Similarly 5% (v/v) phosphoric acid pretreatment (referred as 5% H3PO4) and 2% (v/v) phosphoric acid pretreatment (referred as 2% H3PO4) are addition of 5% (v/v) and 2% (v/v) phosphoric acid solution in N.oculata respectively. A Mathis dye beaker apparatus, type number BFA24 manufactured by LAbOMAT, Oberhasli, Zurich, was used for acid pretreatment at 160 C and 5 bar pressure. For acid treatment dilut e sulfuric acid or phosphoric acid was added to the sample, this decreased pH of sample to 5. 2% phosphoric acid added samples were hydrolyzed for 30, 60 and 90 minutes. 5% acid addition pretreatment was carried out for 90 minutes. Control pretreatments without acid addition was also carried out. Experimental setup for enzyme saccharification Enzyme saccharification experiments were conducted at 50C in 500 ml glass flasks using different loadings of enzymes EI and EII. Two commercial cellulases, Accell erase Trio from Genencor (EI) and Cellic CTech2 from Novozymes (EII), were used for saccharification experiments. The nominal loading of EI and EII for optimum
66 hydrolysis was 0.25 ml/g AFDM and 0.05 ml/g AFDM respectively as recommended by supplier. As enzyme activity at saline condition was not tested before (Matsumoto et al.,2003), it was verified that cellulase activity of both EI and EII was not affected by high salt content in separate experiments using cellulose powder as substrate. It was ensured that 4 g (dry weight) of algae was used in each experiment. Experiments were done using nominal, half the nominal and twice the nominal dosage of EI and EII. These flasks will be referred to as reaction flasks henceforth. The pH was all reaction mixtures was 5. For thermal pretreated samples without acid addition pH was adjusted to 5 before enzyme reaction. The following treatments were used as controls. For control run I, a batch of algae without pretreatment or enzyme addition was allowed to sit in th e reaction flasks at 50C and pH 5 for 12 hours. Control run II was done with only enzyme additions without acid pretreatment. For control run III algae samples were pretreated at 160C without acid addition followed by enzyme treatment. Two replications o f all control runs and each experiment were performed. All glass wares were autoclaved at 120C for 30 minutes to minimize any contamination. The reaction flasks are then kept in an incubator shaker set at 50 C and 350 rpm. pH was maintained in range of 4 .8 to 5 during enzyme saccharification without addition of acid or base. Samples were withdrawn at 4 hours and 12 hours, centrifuged for 1 min at 14000 rpm and supernatant was filtered with 0.2 m filter paper and instantly diluted 10 times for 3,5 Dinitro salicylic acid (DNS) analysis. DNS method was used for measuring reducing sugars. A calibration curve was made using 0.4, 0.8, 1.2, 1.6 and 2.0 mg/ml glucose concentration saline (3.5%) solutions. 1 ml of diluted samples were baked with 2 ml of DNS reage nt at 100 C for 10
67 minutes. After cooling the baked sample to room temperature and mixing 2 ml of deionized water, optical density was measured in spectrometer at 580 nm. It was verified that DNS method was not hindered by the presence of salt. Glucose, su crose, cellobiose, xylose, galactose, arabinose, mannose, fructose, furfural and hydroxymethyl furfural (HMF) concentrations in reaction mixtures was also measured using High Performance Liquid Ch romatography (Agilent1200HPLC). Carbohydrate content in micr oalgae samples were measured in terms of grams per dry weight. The extent of conversion of carbohydrate in microalgae was reported as grams sugar released per kilogram AFDM of algae. Ash Free Dry Matter Analysis (AFDM) The algae paste was analyzed for dry matter, volatile solids and ash content. Dry matter and ash analysis was done by conventional standard method of drying the solids ( Greenberg et al.,1992). Total Solids (TS) were determined after drying the wet sample overnight at 105C. The dried sample w as burned at 550C in a muffle furnace for 2 h to determine the Volatile Solids (VS) content and the AFDM of algae. Results Ash Free Dry Matter (AFDM) of N.oculata Algae samples received contained 7.75% dry matter and 30.45% of this dry matter was ash fre e So AFDM of samples of algae paste was 2.4 %. Control runs for saccharification of N.oculata Control run I, II and III released 1.5 g sugars/ kg AFDM (0.6% carbohydrates conversion), 3.1 g sugars/ kg AFDM (1.22% carbohydrates conversion) and 11.4 g sug ars/ kg AFDM (4.6% carbohydrates conversion) respectively. The sugar released after acid hydrolysis gave 2.5 g sugars/ kg AFDM (1% carbohydrates conversion). This
68 is a low value and is ignored in final sugar results noted after enzymatic saccharification f rom hydrolysate. Also, the sugar content of enzyme EI and EII are measured as 0.36 and 0.29 g/ml respectively. The correction for sugars accounting from is done in the final results. Dilute acid hydrolysis Enzyme EI: As shown in Table 4 2, enzyme hydrolys is with EI after 5% H2SO4 pretreatment, released 88.6 g sugars /kg AFDM (i.e. equivalent to 35.95% carbohydrate conversion) in 4 hours and 29.5 g sugars/kg AFDM (12% carbohydrate conversion) in 4 hours for nominal and half the nominal loading of enzyme res pectively. The sugar release dropped to 50.3 sugars/kg AFDM (24% carbohydrate conversion) in 4 hours on doubling the enzyme loading. The drop in sugar concentration could be due to contamination in the reaction system. With 5% H3PO4 pretreatment, nominal and half the nominal enzyme loading yielded 99.0 g sugars/kg AFDM (40.24% carbohydrate conversion) in 4 hours and 52.1g sugars/kg AFDM (21.17% carbohydrate conversion) in 12 hours respectively. Double enzyme loading gave higher sugar release of 121.6 g sug ars/kg AFDM (49.4% carbohydrate conversion) in 12 hours. 2% H3PO4 pretreatment gave 241.0 g sugars/kg AFDM (97.9% carbohydrate conversion) in 12 hours and 89.3 g sugars/kg AFDM (36.3% carbohydrate conversion) in 12 hours with nominal and half the nominal enzyme loading respectively. Double enzyme loading released 248.0 g sugars/kg AFDM (100% carbohydrate conversion) in 12 hours. Enzyme II: As shown in Table 4 2, enzyme hydrolysis with EII after 5% H2SO4 pretreatment, released 112.9 g sugars/kg AFDM (45.9 % carbohydrate conversion) in 4 hours and 53.8 g sugars/kg AFDM (21.8% carbohydrate conversion) in 12 hours for nominal and half the nominal loading of enzyme respectively. The sugar release
69 increased to 140.7 g sugars/kg AFDM (57.1% carbohydrate conversi on) in 12 hours on doubling the enzyme loading. The saccharification reaction was done for 12 hours and maximum sugar release in the reaction setup was attained in 12 hours. With 5% H3PO4 pretreatment, nominal and half the nominal enzyme loading yielded 1 53.0 g sugars/kg AFDM (62.2% carbohydrate conversion) in 12 hours and 66.8 g sugars/kg AFDM (27.15% carbohydrate conversion) in 12 hours respectively. Double enzyme loading gave higher sugar release of 195.4 g sugars/kg AFDM (79.3% carbohydrate conversion) in 12 hours. 2% H3PO4 pretreatment released maximum attainable sugars in 4 hours only. Experiments gave 155.0 g sugars/kg AFDM (63% carbohydrate conversion) in 4 hours and 69.5 g sugars/kg AFDM (28.25% carbohydrate conversion) in 4 hours with nominal and half the nominal enzyme loading. Double enzyme loading released 224.0 g sugars/kg AFDM (91% carbohydrate conversion) in 4 hours. Phosphoric acid pretreatment gave higher sugar release than sulfuric acid pretreatment. 2% loading of phosphoric acid facilit ated higher release of sugars than 5% loading. The rate of sugar release was observed to be slower in phosphoric acid than sulfuric acid as maximum sugar release in reaction flask with phosphoric acid is recorded at 12 hours and with sulfuric acid at 4 hou rs. The difference between sugars released at 12 hours and at 4 hours varied from 15% to 89%. Though at 2% phosphoric acid EII gave maximum sugar release at 4 hours. Enzyme EII produced a higher rate of sugar release than EI, irrespective of loading and ty pe of acid. EII showed highest sugar yield among all runs with 2% phosphoric acid treatment. On increasing the enzyme loading, a change between 20 30% in sugar release was observed. Nominal loading
70 could be the optimal loading of commercial enzyme, as it w ould perform equally well for hydrolysis of microalgae. As shown in Table 4 3, 30 minutes, 60 minutes and 90 minutes of pretreatment with 2% H3PO4 at 160 C followed by a nominal loading of EII, yields 69 g sugars/kg AFDM (27.8% carbohydrate conversion), 23 8 g sugars/kg AFDM (95.96% carbohydrate conversion) and 242 g sugars/kg AFDM (97.6% carbohydrate conversion) respectively. This indicates that pretreatment time can be further reduced to 60 minutes to get almost equivalent amount of sugar yield as obtained from 90 minutes pretreatment process. Trace amount of HMF and cellobiose was produced during saccharification of algae treated with sulfuric acid as compared to undetected levels of these during phosphoric acid pretreatment. In Table 4 4, concentration o f different sugars and by products measured from HPLC analysis is reported and compared to reducing sugars measured from DNS analysis (it should be noted that sucrose is not a reducing sugar). Quantities of undetected sugars are not mentioned in Table 4 4 Discussion N.oculata is a unicellular, thick cell walled spherical microalgae. The total carbohydrate composition of N.oculata is 7.8% of dry matter, out of which 88% is polysaccharide. 68.2% of polysaccharide is glucose, rest being fucose, galactose, mannose, rhamnose, ribose and xylose. 35% of dry matter is protein and 18% is lipid ( Brown et al.,1991). Rest of the composition is amino acids, fatty acids, omega 3, unsaturated alcohols, ascorbic acid. The carbohydrate content can be as high as 26% if gr own outdoors ( Banerjee et al.,2011). C: N ratio in the N.oculata can be controlled by different growth conditions. N.oculata has very high productivity and cheaper to grow
71 under saline condition. Hence it is a promising feedstock for commercial biofuel pro duction. Algae have a simpler structure as compared to lignocellulosic biomass, but a thick cell wall is responsible for entrapping cellulose and other carbohydrates. The cell wall of chlorophytic phytoplankton (N. oculata) is composed of cellulose fibers distributed within a complex organic matrix ( Brown et al., 1991), which makes cellulose accessibility difficult for enzymes as seen from control run I. A pretreatment is required to break open the cell wall and cellulose becomes accessible to enzyme hydro lysis. Disruption in micro algal cell wall due to acid hydrolysis has been shown in previous studies ( Harun et al.,2011). Acid hydrolysis could yield 2.5 g sugars/ kg AFDM which equals only 1% of total carbohydrates conversion in N.oculata. As shown in res ults, N.oculata could not be saccharified with heat, acid or enzyme hydrolysis alone. In all published studies, algal biomass was subjected to a pretreatment in the form of dewatering, freezing, drying, and in some cases extraction. The carbohydrate conver sion varied from 7.5% to 95%. In the present study N.oculata was concentrated only by alkali treatment and was not exposed to any other additional treatment prior studies conducted here. Some algal biomass like Chlamydomonas reinhardtii (microalgae) and s pirogyra (algal biomass) can accumulate high starch content via photosynthesis and have simpler cell wall rich in cellulose ( Choi et al.,2010). They may not require any pretreatment prior to enzymatic hydrolysis. These species result in higher sugar yields and carbohydrate conversion of up to 100% but at the expense of fresh water utilization, high cost and longer growth rates. As shown in Table 4 1, enzyme hydrolysis of marine microalgae with 53% of carbohydrates could result only in
72 23.8% carbohydrate con version in biomass. Another study shows that after ultrasonication pretreatment, enzyme hydrolysis (from fungi derived enzyme) of fresh water species Chlorococcum humicola gives up to 68.2% of carbohydrate conversion ( Harun et al.,2009) Studies done with micro algal cell wall debris produced after lipid extraction show carbohydrate conversion as low as 7.5% after acid and enzyme hydrolysis ( Sande r et al.,2009). Compared to these published studies, N.oculata gave 100% carbohydrate conversion to sugars aft er dilute acid hydrolysis followed by saccharification using commercial cellulases. Most common pretreatment technique to improve cellulose digestibility before enzyme hydrolysis is dilute acid hydrolysis. Sulfuric acid is often used in hydrolysis of macro /micro algae, saline crops, starch, cellulosic and woody biomass as it is considered a stronger hydrolyzing agent than phosphoric acid ( Zheng et al.,2006). Sulfuric acid causes dehydration of monosaccharides and the side reactions results in formation of H MF, which could significantly inhibit biological reactions. On the other hand phosphoric acid pretreatment is mild, non corrosive on process vessels, non toxic, safe, economic and resulted in no furfural production in microalgae hydrolysis making it a pre ferable candidate for pretreatment. Cellobiose indicates the incomplete breakdown of cellulose to simple sugars. HMF is produced due to side reactions in sulfuric acid pretreatment ( Lenihan et al.,2011). This explains the lower sugar yield in sulfuric acid pretreatment than phosphoric acid. Phosphoric acid pretreatment studies have been conducted for corn stover biomass. It has achieved only 56 % of hydrolysis as compared to 75 % of hydrolysis from sulfuric acid ( Um et al.,2003). Present study compares the performance of dilute acid pretreatment for microalgae with sulfuric acid
73 with phosphoric acid and it indicates microalgae have different behavior for dilute phosphoric pretreatment as compared to lignocellulosic biomass. It was observed phosphoric acid t reatment gives equivalent saccharifciation yield from microalgae as sulfuric acid treatment in lignocellulosic biomass. But our results show that phosphoric acid treatment could give 27% more saccharification than sulfuric acid. Difference between sugar r eleased at 4 hour and at 12 hour is of only 15%. The short duration for optimum release of fermentable sugars offers the advantages of eliminating contamination, reducing inhibition effects, and making the process economically effective. 5% H3PO4 pretreate d algae has higher solids loading than 2% H3PO4 pretreated algae (more volume of reaction mixture than 5%). Higher substrate loading results in higher viscosity, which in turn can affect mixing and thus hinder efficiency of enzyme hydrolysis. This could ex plain higher saccharification resulting in microalgae treated with 2% H3PO4 than 5% H3PO4. Cellulases are being commercially produced for specifically breaking down plant cellulose to sugars in lignocellulosic biomass. Hydrolysis studies with commercial en zymes shows higher yields and faster kinetics of sugar release, easy to use and is apt for commercial applications ( C ybulska et al.,2014 ). There are only handful studies of enzymatic hydrolysis of fresh water algae (macro and micro) using cellulase but non e with saline microalgae. This study throws light on feasibility, optimization and possible scale up applications of enzymatic hydrolysis of a defined marine microalgae culture by a commercial enzyme, cellulase.
74 Conclusion Commercial enzymes having define d optimized working conditions and loading rates for cellulosic biomass were used for enzymatic hydrolysis of microalgae in present study. Enzymes hydrolysis experiments were conducted with nominal, half and double the nominal dosage to optimize the loadin g rates of cellulase for N.oculata. The results show that half the nominal enzyme loading performs poorer than nominal loading and double the enzyme loading gives only 34 % higher sugar release than nominal enzyme loading. Hence it was concluded that cellu lase performance on N.oculata is similar to that for lignocellulosic biomass. EI and EII are two commercial enzymes, which targets cellulose, hemicellulose and cellobiose for degradation. In the recommended dosage, EII enzymes have high concentration and s tability, higher conversion yields and more tolerant towards inhibitors. EII had 5 times less recommended loading than EI and works efficiently in the high solids concentration, which ensures higher sugar release per batch of biomass. 100% carbohydrate con version to sugars was observed when treated with 2% phosphoric acid at 160 C for 60 minutes and subsequently hydrolyzed by commercial enzyme EII at nominal loading without addition of any buffer in the reaction flasks.
75 Table 4 1. Comparison of carbohydra te saccharification from different algal biomass Pretreatme nt Carbohydrate Sugar released % carbohydrat e S.no. Algae Type Type Pretreatment time % dry matter (g/kg DM) conversion Reference 1 Spirogyra (macro) Fresh water Alkali 2 hours 64.0% 666 104 ( Eshaq et al.,2010) 2 N. oculata (micro) Salt water Phosphoric acid 1 hour 25.0% 248 100. 3 Nizammudini (macro) Fresh water Sulfuric acid 1 hour 41.5% 70.2 16.3 ( Yazdani et al.,2011) 4 Chlorella Vulgaris (micro) Salt water None 53.0% 126 23. 8 ( Morris et al.,208) 5 Dead micro algae (micro) Unknown Sulfuric acid 1 hour 1.00% 60.0 7.50 ( Sander et al.,2009) 6 7 Chlorococcum humicola (micro) Chlamydomonas reinhardtii (micro) Fresh water Freshwater Sulfuric acid None 0.5 hour 32.5% 60.0% 2 21 570 68.2 95.0 ( Harun et al.,2011) ( Nguyen et al., 2009) *present study
76 Table 4 2 Sugar released (in g sugars/ kg AFDM) at different loadings of enzyme during saccharification 5% H 2 SO 4 5% H 3 PO 4 2% H 3 PO 4 0% acid No pretreatment Enzyme loading Duration of enzymatic saccharification (hours) 4 12 4 12 4 12 4 12 4 12 0.5XEI 29.53 29.53 43.44 52.14 16.54 89.34 1XEI 88.63 88.63 99.04 99.04 82.04 241.04 7.20.5 7.20.5 3.10.5 3.10.5 2XEI 50.33 59.13 13.04 121.64 172.04 248.04 0.5XEII 45.13 53.83 15.24 66.84 69.54 69.54 1XEII 112.93 112.93 99.04 153.04 155.04 155.04 11.40.5 11.40.5 2.80.5 2.80.5 2XEII 111.23 140.73 167.24 195.44 224.04 224.04
77 Table 4 3. Sugar released (g sugars/ kg AFDM) at nominal EII loading from 2% phosphoric acid pretreated samples at 160oC for various durations Time of pretreatment (minutes) Duration of enzymatic saccharification (hours) 4 12 0 3.1 3.1 30 69 69 60 238 238 90 342 342 Table 4 4. Concentration of sugars and other by products from treatment with nominal EII loading Component 5% H 2 SO 4 5% H 3 PO 4 2% H 3 PO 4 g/L g/L g/L HMF 0.036 Cello biose 0.60 2 Glucose 1.59 1.86 2.1 DNS (reducing sugars) 1.75 1.9 2.12
78 CHAPTER 5 P RETREATMENT OF NANNOCHLOROPSIS OCULATA FOR IMPROVED BIOGASIFICATION Summary Anaerobic digestion of microalgae faces numerous challenges due to complex structure and thick cell wall of algae cells, the main challenge being hydrolysis Present study deals with comparing different pretreatment techniques such as thermal (160C for 90 minutes), acid catalyzed thermal (with 2% phosphoric acid at 160C for 90 minutes), ultrasonication (1500 W for 5 minutes) and a well established technique for ethanol production, enzymatic hydrolysis (with thermal pretreatment w/ and w/o acid catalysis) with photocatalysis (30 minutes exposure of microalgae smeared over titanium dioxide coated media at 350 nm ultraviolet radiation), which is a novel approach of pretreatment for anaerobic digestion of microalgae Nannochloropsis oculata ( N .oculata) Methane yield from untreated N. oculata (MYUO) was 0.22 LCH 4 /g VS. Highest methane yield of 2 times MYUO (close to t heoretical of 0.5 LCH 4 /g VS) was observed after enzyme hydrolysis (w/ thermal pretreatment both w/ and w/o acid addition). Acid catalyzed thermal treatment and ultrasonication gave 1.7 time MYUO and 1.2 time MYUO respectively. Photocatalysis treatment gave 1.8 times MYUO which is similar to acid catalyzed thermal treatment. Photocatalysis is a much cheaper, less energy intensive and easy to use technique when compared to ultrasonication or thermal treatment. Thermal treatment showed no increase in methane y ield. P value analysis was done to support the results.
79 Background Unlike defined microbial cultures used for production of biofuels like ethanol or butanol, the microbial consortia in an anaerobic digester is capable of secreting extracellular enzymes to hydrolyze and solubilize macromolecules like cellulose, hemicellulose, proteins and fats. This characteristic has enabled several terrestrial biomass feedstocks like sugarbeets, sugarbeet tailings, napier grass, sorghum and aquatic biomass like water hyac inth and giant kelp to be successfully digested without pretreatment. However, degradability of feedstocks containing high fraction of lignin (for example sugarcane bagasse, switchgrass, miscanthus and woody biomass like pine, eucalyptus) is poor in an an aerobic digester. The refractoriness of these feedstocks has been attributed to low moisture, crystalline nature of the cellulose, and complex association of the component carbohydrates within lignin ( Chynoweth and Jerger 1985). The anaerobic digestibili ty of microalgae species varies. Species with no cell wall or cell encapsulation like Chlorella vulgaris and Phaeodactylum tricornutum, has a higher rate of methane production. Dunaliella tertiolecta has very low methane yield of 0.018 L/kg VS due to inhi bition caused by higher salinity ( Lakaniemi et al.,2011). The cell wall of N.oculata is composed of cellulose fibers distributed within an organic matrix. This is expected to offer greater resistance to digestion ( Northcote et al., 1958). Different pretrea tment techniques like physical pretreatment (thermal, high pressure thermal), radiation pretreatment (ultrasonication, microwave), chemical pretreatment (alkali) have been tried on microalgae to enhance methane production during anaerobic digestion. A brie f summary of studies is listed in Table 5 1. All these studies were done on fresh water species. Thermal and ultrasonication were widely
80 studied pretreatment techniques for anaerobic digestion of microalgae. In thermal pretreatment, high temperatures of 15 0 200C are used adjoined by pressure in the range of 5 25 bar. The process produces partially solubilized slurry with disintegrated biological cells. The organic material in this form is much more available for anaerobic digestion an d good for anaerobic digestion. Ultrasonication employs sound energy to disintegrate cellular structure. When a standing wave field is propagated into a liquid at high intensities, alternative high pressure (compression) and low pressure (rarefaction) cycles are generated whic h causes cell disintegration. Most of these studies were batch experiments using serum bottles and shown successful positive yields of methane from pretreated algae, however, a drawback with the methods employed in some studies was that accumulated volume tric biogas was measured and then multiplied by the methane concentration in the biogas at the end of digestion to estimate methane produced in the experiment. This method assumes that methane content of biogas was constant throughout the experimental run which is not correct. In batch digestion methane content initially increases from 0% to over 50% over a period of time and is then maintained at this higher value. The biogas initially produced is primarily carbon dioxide. This approach would lead to es timating higher methane yield values than actually produced. A previous study also reports effect of thermal pretreatment on N.oculata ( Kinnunen et al.,2014), but in order to accurately estimate methane yield from this pretreatment, it was performed again in present study. Photocatalysis is an oxidation technique used in wastewater treatment and air purification, and is the mode of action by some cleaning reagents. Titanium Dioxide exhibits strong oxidizing photo activity when irradiated by ultraviolet (UV ) ray. When a
81 photocatalyst like titanium dioxide (TiO2) absorbs UV radiation from sunlight or illuminated light source (fluorescent lamps), it produces negative electron (e ) and positive hole (h+) pair. The positive hole of titanium dioxide breaks the wa ter molecule apart to form hydrogen gas and hydroxyl radical. The negative electron reacts with oxygen molecule to a form super oxide anion. This cycle continues when light is available. It works well at high pH and is a self cleansing agent (hydrophilic n ature). Also, TiO2 regenerates after the reaction and hence could save catalyst costs by reusing the catalyst. Effect of photocatalysis on microalgae has not been studied so far so this technique was tested on N.oculata as pretreatment in anaerobic dige stion, expecting it oxidizes cellular wall of N.oculata and breaks down complex organic components present. In present study, performance of photocatalysis as pretreatment is assessed and compared with other pretreatment techniques in terms of methane yiel d. Materials and methods Algae growth and characteristics Shipment of N.oculata received was same as described in Chapter 4. Details of algae growth can be found in a previous study ( Buxy et al.,2013). N. oculata was dewatered to thick slurry of 8% solid s by adding potassium hydroxide. The algae paste was analyzed for dry matter, volatile solids and ash content. The analysis was done by conventional standard method of drying the solids (Greenberg,1992). Dry matter (DM) were determined after drying the we t sample overnight at 105C. The dried sample was burned at 550C in a muffle furnace for 2 h to determine the Volatile Solids (VS) content of algae. Free ammonia nitrogen analysis on paste was also performed. Details of analytical procedures were describe d in Chapter 3
82 Anaerobic digestion setup A 5 L anaerobic digester was constructed by modifying a Pyrex glass jar. The digester was provided with ports for venting and sampling biogas, and for liquid withdrawal as shown in Figure 5 1. The biogas outlet fro m digester was attached at the bottom of a carbon dioxide scrubbing unit consisting of vertical glass tube of length 10 cm and inner diameter of 3.5 cm, filled with soda lime. Biogas passed through 0.085 L of soda lime that absorbs carbon dioxide releasing only methane from outlet located at top of the scrubber. The methane released from scrubbing unit was measured using a positive displacement gas meter. The digester contents were bulked with lava rocks in order to increase retention time of biomass and pr event compaction of media in case of TiO2 pretreated algae digestion. The digester was placed in a chamber maintained at 38 C. The pH of the feedstock is measured as 10.8. 2% dilute phosphoric acid is used to neutralize N.oculata to pH 7 before subjectin g to different pretreatments. Loading Anaerobic digesters were run as a fed batch system. N.oculata was codigested with sugar. 1 g VS was loaded with 1 g of sugar in each loading along with nutrient solution (Owens et al., 1993). Hydraulic retention time ( HRT) was 4 days. At each loading, 160 ml of inoculum was withdrawn from digester and100 ml of algae paste (equivalent to 1 g VS) is loaded with 60 ml of nutrient solution. Individual digesters for each different pretreated N.oculata were activated with 1 g of sugar loaded every 4 days till a consistent methane yield was obtained. HRT was based on observation when methane production is exhausted. N.oculata without any pretreatment (control run) and N.oculata with different pretreatment procedures described below were digested in this fed batch setup. 10 15
83 loadings of algae along with sugar are done for each run. Methane production from N.oculata was estimated by subtracting methane from sugar. The soluble chemical oxygen demand (sCOD) was measured at the e nd of 4 days in each run. Pretreatment procedures description Titanium dioxide photocatalytic pretreatment N.oculata paste amounting to 1 g VS, was smeared on titanium dioxide coated nonwoven media (TiO2 media). TiO2 media supplied by Innovative Material s Development Company (IMDC), Gainesville, FL was developed using proprietary technology and accounts for 3 mg TiO2/cm2 of media. N.oculata was treated at pH of 7 for optimum photocatalytic activity (RamMohan et al., 2014). Each batch of N.oculata smeared media was then kept in photoreactor assembly, exposing it to UV radiations (350 nm) separately for 30 and 60 minutes for photocatalytic reactions. Photoreactor assembly consists of the smeared media kept in tray below UV light (7 cm distance between UV li ght and media) under a black box as shown in Figure 5 2. After photocatalytic pretreatment, the media was manually cut into small pieces of size 1X1 cm and loaded in anaerobic digester assembly with a lava rocks as bulking agent to avoid compaction. Ultra sonication Ultrasonication of N.oculata was done in a VCX 1500 CT system from Sonics & Materials, Inc., CT, USA. System was used to generate power ultrasound and a standard titanium alloy probe (L=254mm, d=25mm, W=680 g) was used to apply the power ultras ound. The system was designed to provide 1500 W of maximal output power at 20 kHz and was operated at 90% of the amplitude level. N.oculata was treatment was done for 5 minutes. Temperature during treatment increased to only
84 40C. After ultrasonication, 1 g VS equivalent N.oculata was loaded every four days in anaerobic digester as described in loading section. Thermal and Acid catalyzed thermal hydrolysis Thermal hydrolysis (cooking) was carried out at 160 C for 90 minutes in Mathis equipment described in previous chapter. pH of N. oculata was lowered to 7 by 2% phosphoric acid before cooking. After cooking, pH was measured as 7 and 1 g VS equivalent N. oculata was loaded in anaerobic digester. For acid catalyzed hydrolysis, 2% phosphoric acid was used to further bring down the pH of N. oculata to 5 before heating at 160 C for 90 minutes. After acid catalyzed hydrolysis, pH of 1 g VS equivalent of N. oculata was adjusted to 7 before loading in anaerobic digester. Enzymatic saccharification Reaction setup a nd loading protocols for enzymatic saccharification of N. oculata is described in chapter 4 Experiments were performed in similar way. Three types of enzyme hydrolysis was carried out: 1. Enzymatic saccharification of untreated N. oculata pH of microalgae wa s adjusted to 5 by 2% phosphoric acid and 1 g VS equivalent amount was subjected to twice the nominal loading of Cellic T 2 enzyme for 4 hours at 50 C. 2. Enzymatic saccharification of a cid catalyzed thermally treated N. oculata pH of microalgae was adjust ed to 5 by 2% phosphoric acid and then 1 g VS equivalent microalgae was heated at 160 C for 90 minutes. pH of acid catalyzed thermal treated microalgae was measured as 5 after treatment and then subjected to twice the nominal loading of Cellic T 2 enzyme f or 4 hours at 50C. 3. Enzymatic saccharification of thermal treated (cooked) N. oculata pH of microalgae was adjusted to 7 by 2% phosphoric acid and then 1 g VS equivalent microalgae was heated at 160 C for 90 minutes. pH of acid catalyzed thermal treated m icroalgae was readjusted to 5 after treatment and then subjected to twice the nominal loading of Cellic T 2 enzyme for 4 hours at 50C. After saccharification experiments, pH of N.oculata was adjusted to pH of 7 and loaded in anaerobic digester as describe
85 Results and Discussion Moisture analysis showed 7.75% DM 30.45% VS. Free ammonia nitrogen of N.oculata was 0.029 g/L. After adjusting pH of N.oculata to 7, VS content of algae reduced to 1 g per 100 ml. Net methane yield from contro l run or MYUO was recorded as 0.22 LCH4/g VS which is less than half of theoretical methane yield of 0.5 LCH4/g VS. The cell wall of N.oculata is composed of cellulose fibers distributed within an organic matrix. This is expected to offer greater resistanc e to digestion ( Northcote et al., 1958). Digestion of 1 g VS pretreated N. oculata with ultrasonicator yielded 0.286 L methane at STP that is 26.8% increase in methane than untreated N. oculata This is consistent with previous studies, double than 14% incr ease in methane yield obtained by ultrasonic pretreatment of Chlorella and S cenedesmus biomass that used half the energy than present study. Ultrasonication employs sound energy to disintegrate cellular structure. When a standing wave field is propagated i nto a liquid at high intensities, alternative high pressure (compression) and low pressure (rarefaction) cycles are generated which causes cell disintegration Previous study indicates 100% increase in methane production after ultrasonication. Although inc reased methane can also be attributed to thermal effect induced by rise in temperature as high as 85 C during ultrasonication. The amount of energy invested (129 MJ/kg) is 286 times more than present study and is hypothetical for practical usage/ energy i nefficient. Ultrasonication is feasible technique but energy inefficient. Other pretreatment techniques are explored. Thermal hydrolysis in turn requires comparatively less energy input ( Fernandez et al.,2012 ) and most widely
86 studied pretreatment on microa lgae for anaerobic digestion as can be seen from Table 5 1. In high pressure thermal pretreatment (HPTH), high temperatures of 90 200C were used adjoined by pressure in the range of 1 25 bar so far. The process produces partially solubilized slurry with disintegrated biological cells. The organic material in this form provides larger fraction of degradable material for anaerobic digestion ( Phothilangka et al., 2008 ). Majority of thermal treatment studies are done with Chlorella and Scenedesmus. Though Sc enedesmus possesses tough cellular walls than Chlorella, it has potential to release higher methane than Chlorella after thermal pretreatment at high temperatures and long durations. This shows feasibility of thermal treatment for methane production. HPTH has capacity to increase 80% methane production on an average during digestion of Chlorella and Scenedesmus. Nannochloropsis species cell wall toughness is less than Scenedesmus but higher than Chlorella. HPTH of N. s alina yielded 185% higher methane as co mpared to 33.3% by N. oculata ( Schwe de et al., 2013 ; Marsolek et al., 2013 ). N. s alina has high composition of lipids as compared to N. oculata ( Mohammady et al.,2011) and high methane from N. salina after HPTH could be attributed to this fact. In Marsolek et al., 2013 N. oculata was freezed and thawed before HPTH which could be accounted as pretreatment before HPTH and increase errors in results. HPTH of N. oculata was repeated for present study in order to verify this limitation. N. oculata digestion was found to be consistent with Marsolek et al., 2013 study. Untreated N.oculata yielded 0.22 (LCH 4 STP/g VS), as compared to 0.24 LCH 4 STP/g
87 VS reported in Marsolek et al., 2013 HPTH N. oculata yielded 0.26 L CH4 STP/ gVS, which is 18% increase in methane from unt reated N. oculata This is slightly less than obtained in Marsolek et al., 2013 which utilized serum bottle experiments that mostly reports higher methane production than true methane production. HPTH catalyzed by acid requires less pretreatment time than non catalyzed HPTH to yield same increase in methane as seen in studies ( Cho et al., 2013 ; Mendez et al., 2014 ) in Table 5 1. Further effect of acid catalysis in HPTH was studied by catalyzing HPTH of N. oculata with phosphoric acid for same amount of time. Digestion of acid catalyzed HPTH (TAH) yielded 0.39 L CH4 STP/ gVS which is 77% more methane than untreated N. oculata as compared to only 18% from HPTH. This concludes HPTH is more effective pretreatment for methane production from N. oculata when catalyze d with acid. A well established enzyme hydrolysis pretreatment was explored for anaerobic digestion of N. oculata Commercial cellulase (Cellic T 2) was used for hydrolysis of untreated, TAH and HPTH N. oculata Methane yield after digestion of hydrolyzed N. oculata was adjusted by subtracting methane produced from enzymes. Enzyme hydrolysis of untreated N. oculata (EH) showed no significant increase in methane production. E nzymatic saccharification of thermal hydrolyzed (ETTH) and enzymatic saccharificaiton of acid catalyzed thermal hydrolyzed (ETAH) N. oculata yielded 0.5 1 L CH 4 STP and 0. 485 L CH 4 STP of methane at STP per g VS respectively as shown in Figure 5 3 ETTH N. oculata yielded 131% more methane than control and ETAH N. oculata showed 115.5% increase i n methane production than control.
88 Novel technique of pretreatment by photocatalytic pretreatment (TiO2 PT) by titanium dioxide of N. oculata yielded higher methane 0.408 L CH 4 STP (for 30 minutes treatment) that is 80.9% more than control. Methane yield af ter 60 minutes TiO 2 PT yielded 0.415 L CH 4 STP which is similar to 30 minutes TiO 2 PT and hence indicates 30 minutes of pretreatment exposure is suff icient for enhancing methane yield. Photocatalysis serves as oxidizing agent for carbohydrates, lipids and proteins in algae. As it has been shown in previous studies, the organic matter gets completely oxidized to carbon dioxide, hence the components of the algae also have the tendency to degrade completely ( Peller, J.R. et al., 2007). However, if the photocat alysis is done partially, it may restrict the degradation of algae complex organic matter to simpler organic matter and could facilitate higher rate of methane production. TiO 2 photocatalysis breaks open/distort the algae cell and facilitate the release of organelles and other components inside the cell for digestion purpose (Peller, J.R. et al., 2007) The rate and extent of photocatalysis needs to be optimized. Also, photocatalysis reduces all the pigments like chlorophyll and chloroplasts and inhibits th e growth of the algae, thus decreasing the resistivity of algae in the digester. It has also been shown that photocatalysis from TiO 2 reduces the toxicity (Peller, J.R. et al., 2007) and high ly calcitrant materials. P value test done on data set of methane production from photocatalytic pretreated N. oculata and ultrasonicatied N. oculata gives a value of P as 0.3836 which is >0.1. This supports null hypothesis. Since average methane yield from photocatalytic pretreatment (TiO2 PT) (0.408 L CH 4 STP/g VS) is hi gher than ultrasonicated N. oculata (0.286 L CH 4 STP/g VS), it can be concluded that photocatalytic treatment performed better in terms of biogasification than ultrasonicated N. oculata In a similar way, P value
89 comparing methane yield of photocatalytic pret reated N. oculata and enzyme treated thermal hydrolyzed N. oculata (ETTH) is 0.3254 (>0.1). Mean value of ETTH being greater (0.521 L CH 4 STP/g VS) than TiO2 PT (0.408) shows performance of ETTH better than TiO2 PT. P value comparing TiO2 PT and control is 0. 2367 (also >0.1), in this case TiO2 shows higher methane yield that means undoubtedly TiO2 PT performed better than control. The hierarchy is listed in Figure 5 5. Further, Gompertz fit (as described in chapter 2) was performed on methane yield of pretreat ed and control N. oculata to analyze rate of methane production. Summary of rate of methane production is presented in T able 5 2. Highest rate of methane production was observed in TiO2 PT digestion as 0.7 L CH 4 STP/d, followed by ETTH digestion as 0.26 L CH 4 STP/d. TiO2 PT is 7 times faster than control. ETTH yielded higher methane than TiO2 PT but methane production rate of TiO2 PT is 3 times faster than ETTH. Acid hydrolyzed, ultrasonicated N. oculata and ETAH N. oculata digestion shows methane production rat e ranging from 0.1 0.12 L CH 4 STP/d which is same as digestion of N. oculata without any pretreatment. TiO2 not only increases overall methane yield of N. oculata but is the fastest among other pretreatment techniques studied in present study. Ammonia was me asured at the end of each run and found to be in range of 0.06 0.12 g/L. The sCOD hiked after each loading of pretreated N. oculata and remain ed consist ent after 4 days of operation. In Figure 5 4, sCOD of photocatalytic pretreated microalgae digestion is c ompared with widely studied ultrasonicated pretreated microalgae digestion. In photocatalytic pretreated N. oculata maximum sCOD increase observed in loadings is 1.86 g/L and in ultrasonicated and control runs as 1.465 g/L and
90 1.044 g/L respectively. This i ndicate d sCOD remained cons istent throughout digestion run and showed no build up or decrease in sCOD as shown in F igure 5 4 The consistent level of sCOD could be non degradable sCOD accumulated at each addition of N. oculata Further, it could be specula ted it could be organelles, which are released at uniform rate from algal cell after its cell wall rupture and slow in digestion as compared to cell wall and could account for high levels of sCOD. TiO2 PT works on principle of electron hole pair diode syst em. On the other hand, radiation techno logy such as ultrasonication is energy and cost intensive. Studies done apart from ultrasonication such as microwave pretreatment referred in T able 5 1 shows decent increase of 50 70% in methane production b ut at the cost of high er energy requirements, which might make the pretreatment techniques unsustainable. Enzymatic hydrolysis on other hand utilizes costly commercial enzymes and demands prerequisite pretreatment before hydrolysis, hence adding to increased cost of operation. Conclusions Titanium dioxide is a cost effective and easy to use technology, which consumes less energy than its radiation counterparts. TiO2 PT N.oculata yields 0.408 LCH4 STP methane/g VS loaded, with maximum rate of methane production at 0. 72 LCH4 STP/d. TiO2 PT shows 80.9% increase in methane yield from untreated N.oculata. This novel pretreatment technique is 3 times faster in terms of methane production than well studied radiation technology of ultrasonication and 7 times faster than conv entional thermal and acid hydrolysis techniques. Another approach of pretreatment, enzymatic hydrolysis is studied on N.oculata with different variations of further pretreatment such as heating, acid hydrolysis on N.oculata before enzyme treatment. Enzyme hydrolysis is
91 commonly used for sugar production from lignocellulosic biomass, but in present study it was used for microalgae pretreatment to see potential of methane production. Enzyme hydrolysis shows 131% increase in methane production from untreated N .oculata and 27% higher methane production from TiO2 PT N.oculata. Owing to much higher rate of methane production and cost effectiveness, this novel technique of photocatalysis looks promising for future energy production ventures. Figure 5 1. Schemati c diagram of b ioreactor assembly
92 Figure 5 2. Photoreactor assembly Figure 5 3. Methane yield of N.oculata after different pretreatment 0 0.1 0.2 0.3 0.4 0.5 0.6 Methane yield (L STP/g VS) Methane yield Theoretical methane yield (L STP/g VS) Tray UV lamps
93 Figure 5 4. Soluble chemical oxygen demand from digestion of pretreated N.oculata 0 0.5 1 1.5 2 2.5 0 2 4 6 8 10 Soluble Chemical Oxygen demand (g/L) Loading number Control Photocatalysis Ultrasonication Control avg Photocatalytic avg Ultrasonication avg
94 Figure 5 5. Hiera rchal order from highest methane production to lowest from different pretreatments ETTH (0.501 LCH 4 STP/g VS) (0.26 LCH 4 STP/d) ETAH ( 0.48 LCH 4 STP/g VS) (0.21 LCH 4 STP/d) TiO 2 PT ( 0.41 LCH 4 STP/g VS) (0.72 LCH 4 STP/d) TAH (0.39 LCH 4 STP/g VS) (0.12 LCH 4 STP/d) Ultrasonication (0.28 LCH 4 STP/g VS) (0.11 LCH 4 STP/d) HPTH (0.26 LCH 4 STP/g VS) (0.10 LCH 4 STP/d) EH (0.24 LCH 4 STP/g VS) (0.10 LCH 4 STP/d) Control (0.22 LCH 4 STP/g VS) (0.10 LCH 4 STP/d)
95 Table 5 1. Summary of anaerobic digestion of microalgae incorporating different pretreatments S.no Algae type Type of reactor Pretreatment Methane Yield (L/g VS) % increas e in methane HRT (day s) Reference Type Dura tion Para meter Before pre treatment (control) After pre treatme nt 1 Chlorella+ Scenedes mus Serum bottle batch Ultrasonic 180 s Energy = 0.23MJ/kg 0.336 0.385 14.50% 23 Cho et al., 2013 2 Chlorella vulg aris Serum bottle batch Ultrasonic 270 s Energy = 0.2 MJ/kg 0.25 0.288 13.2% 25 Park et al., 2013 3 Chlorella vulgaris High pressure thermal+ acid catalysis 0.33 h T emperatu re =160 C Pressur e= 6bar 0.24 0.43 78.5 29 Mendez et al., 2014 4 Mixed c ulture enriched in Scenedes mus Serum bottle batch High pressure thermal hydrolysis 0.5 h T emperatu re=17 0 C Pressur e= 8 bar 0.18 0.33 81.00% 35 Keymer et al., 2013 5 Mixed culture (grown on WW, 90% algae+10% bacteria) Batch serum bottle experime nts Low temperature pretreatment 15 h T emperatu re=95 C 0.105 0.169 60.90% 43 Passos et al., 2013
96 Table 5 1 continued S.no Algae type Type of reactor Pretreatment Methane Yield (L/g VS) % increase in methane HR T (day s) Reference Type Duration Parameter Bef ore pretreatm ent (control) After pretreat ment 6 Nannochlorop sis salina Batch Thermal 2 h T=120 C 0.2 0 0.56 185.0% 49 Schwe de et al., 2013 7 Nannochlorop sis oculata Batch serum bottle Thermal 3.5 h T =90 C 0.24 0.32 33.3% 12 Marsolek et al., 2013 8 Chlorella+ Scenedes mus(Fresh water) Serum bottle batch Thermal (co substrate Anaerobic sludge) 0.5 h T=120 C 0.336 0.405 20.00% 23 Cho et al., 2013 9 Scenedesmus (Fresh water)+bac teria minor Semi continuou s Thermal 3 h T=90 C 0.126 0.283 124% 33 Fernandez et al.,2012 10 Mixed culture (Wastewater) Serum bottles batch Microwave Energy = 1.079X10^ 3 0.117 0.209 78.63% 46 Passos et al., 2013
97 Table 5 1 continued S.no Algae type Type of reactor Pretreatment Methane Yield (L/g VS ) % increase in methane HR T (day s) Reference Type Duration Parameter Before pretreatm ent (control) After pretreat ment 11 Mixed culture (Fresh water) Semi continuou s Microwave 180 s Energy = 1.08 MJ/kg 0.17 0 0.270 5 9 .00 % 20 Passos et al., 2013 12 Nannochlorop sis oculata Semi continuou s Ultrasonicator HPTH TAH ETAH ETTH 300 s 1.5 h 1.5 h 4 h 4 h 0.28 0.26 0.39 0.48 0.5 0.22 0.28 0.26 0.39 0.48 0.5 26% 18% 77% 118% 127% 4 Present study
98 Table 5 2. Rate of methane production from digestion of pretreated N.oculata Type of pretreatment Rate of methane production (L/d) None (Control) 0.1 0 0.05 Photocatalysis (TiO 2 PT) 0.72 0.02 U ltrasonication 0.11 0.02 Acid catalyzed th ermal and enzyme treated (ETAH) 0.21 0.05 Thermal and enzyme treated (ETTH) Enzyme treated (EH) 0.26 0.02 0.10 0.03 Acid catalyzed thermal treated (TAH) High pressure thermal treated (HPTH) 0.12 0.03 0.10 0.03
99 CHAPTER 6 CONCLUSIO NS The present study proposes broad scope of anaerobic digestion of a c urrently cultivated terrestrial biomass (sugarbeet) and a future aquatic biomass (microalgae, N.oculata). Frozen sugarbeets yields 49% higher amount of methane than fresh beets and 15% higher amount of methane than airtight stored beets. Maximum fraction of total methane production (95%) during digestion of airtight stored sugarbeets was attained in half the amount of time than obtained during digestion of frozen sugarbeets Freezing lar ge amounts of sugarbeets is an expensive option. Airtight storage is a cheaper alternative of storing sugarbeets and storing conditions are similar to plastic tube silos used for large amounts of sugarbeets storage in fields. Sugarbeets stored in piles at places experiencing long winters would be the optimum storage for feedstock for biogas production. Places experiencing shorter winters could utilize plastic tube silo. Fresh sugarbeets are not suitable for biomethane production. Size reduction of sugarbeet s has no effect on ultimate methane yield. Not surprisingly there is an improvement in rate of methane production after size reduction of sugarbeets but this would be at a cost from additional unit operation and increased energy consumption. Further, a pi lot scale two stage batch anaerobic digestion system for frozen whole beet was proposed which consists of solids and filter digester and exchange of liquid at regular interval between the two reactors assists in faster digestion of sugarbeets and regulatio n of pH. Digestion time of sugarbeets was reduced by 40% in two stage system. Problem of acidification in solids digester is controlled by sequencing of liquid between solids and filter digester.
100 Biomethane potential of a microalgae, N.oculata was determi ned in a semi continuous system co digesting with table sugar at mesophilic conditions. Initiation of methane production was observed from day one in N.oculata digestion with average yield of 0.22 L/g VS. Different pretreatment techniques such as, thermal acid hydrolysis, ultrasonication were employed including two never tried pretreatment for microalgae digestion. The novel pretreatment is photocatalysis by titanium dioxide and enzymatic saccharification. Titanium dioxide is a cost effective and easy to use technology, which consumes less energy than its radiation counterparts. TiO2 PT N.oculata gives a decent yield of 0.408 L@STP methane/g VS loaded, with maximum rate of methane production as 0.72 L@STP/d. TiO2 PT shows 81% increase in methane yield from untreated N.oculata. This novel pretreatment technique was 3 times faster in terms of methane production than well studied technology of ultrasonication and 7 times faster than conventional thermal and acid hydrolysis techniques. Enzyme hydrolysis showed 131% increase in methane production from untreated N.oculata and 27% higher methane production from TiO2 PT N.oculata but owing to much higher rate of methane production and cost effectiveness, this novel technique of photocatalysis looks promising for fut ure energy production ventures.
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112 BIOGRAPHICAL SKETCH Samriddhi Buxy was born in Madhya Pradesh, India. She received her Bachelor of Engineering degree in Chemical Engineering with honors from Pt. Ravisha nkar Shukla University in 2007. Thereafter, she was enrolled as graduate school at University of Florida. She worked as research assistant at bioprocess laboratory at University of Florida. After graduation, she plans to work in the field of bioprocess and environmental engineering.