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Evaluation of Cellruptor pre-treatment on biogas yield from various substrates
SELVAKUMAR THIRUVENKADAM
Supervisor: Mr Andreas Berg
Research Manager
SCANDINAVIAN BIOGAS FUELS AB SE-581 83 Linköping SWEDEN
Examiner: Prof. Gen Larsson
Head of Div. Bioprocess Technology Department of Biotechnology
KTH ROYAL INSTITUTE OF TECHNOLOGY SE-106 91 Stockholm SWEDEN
ABSTRACT
In this thesis work, Cellruptor pre-treatment was evaluated in order to increase biogas yield. Initially, the effects of residence time (30, 60, 90, 120 and 180 min) and substrate release (rapid/non-rapid) from the draining port of Cellruptor on biosludges were investigated to find the optimum operating conditions of Cellruptor. Under these optimum operating conditions, the effect of Cellruptor pre-treatment on batch reactors of various substrates and semi-continuous digester of biosludge were investigated at mesophilLF WHPSHUDWXUH Ü& 7KH YDULRXV VXEVWUDWHV LQ EDWFK UHDFWRUV LQFOXGH
biosludge, dewatered sludge, digested sludge, fibre sludge, hay, maize silage, minced meat, orange peel, seaweed and yeast. From the initial study, 90 min residence time and rapid release of pre-treated substrate from draining port were found to be optimum operating conditions of Cellruptor. From the batch experiments, Cellruptor pre- treatment showed maximum and minimum increase of methane yield in hay (32%) and dewatered sludge (2%) respectively. The semi-continuous digester experimental results showed increase in biogas production by 22.4% from Cellruptor pre-treatment of biosludge at HRT of 15 days and OLR of 2.0 g VS/L/day. With further studies, Cellruptor pre-treatment may be deployed in large-scale biogas plants to improve biogas yield.
Keywords: Cellruptor, pre-treatment, biogas, methane, biosludge, mesophilic, batch
Contents
ABSTRACT I
CONTENTS II
1 INTRODUCTION 1
1.1 Aim 2
1.2 Hypothesis 2
1.3 Strategy 2
2 BACKGROUND 3
2.1 Biogas ă for a sustainable environment 3
2.2 Anaerobic Digestion 3
2.2.1 Microbiology and Biochemistry 4
2.2.2 Environmental factors 5
2.2.3 Solid Characteristics 6
2.2.4 Operational Parameters 6
2.2.5 Control Parameters 7
2.3 Pre-treatment Techniques of substrates 7
2.3.1 Cellruptor 9
2.4 The substrates 9
2.4.1 Biosludge 9
2.4.2 Dewatered Sludge 9
2.4.3 Digested Sludge 10
2.4.4 Fibre Sludge 10
2.4.5 Hay 10
2.4.6 Maize Silage 10
2.4.7 Minced Meat 10
2.4.8 Orange Peel 11
2.4.9 Seaweed 11
2.4.10 Yeast 11
3 METHODS AND MATERIALS 12
3.1 Cellruptor 12
3.1.1 Process conditions 12
3.2 Batch Experiment 13
3.2.1 The substrates & Experimental set-up 13
3.2.2 Batch start up 13
3.2.3 Analysis 16
3.3 Semi-continuous digester experiment 17
3.3.1 The substrates 17
3.3.2 Digester configuration 17
3.3.3 Digester Operation 17
3.3.4 Analysis 18
3.4 Software 18
4 RESULTS 20
4.1 Batch experiments 20
4.1.1 Batch set A 20
4.1.2 Batch set B 21
4.1.3 Batch set C, D and E 22
4.2 Semi-continuous digester experiment 26
5 . DISCUSSION 28
5.1 Batch experiments 28
5.1.1 Biosludge 28
5.1.2 Dewatered Sludge 29
5.1.3 Digested Sludge 29
5.1.4 Fibre sludge 29
5.1.5 Hay 30
5.1.6 Maize Silage 30
5.1.7 Minced Meat 30
5.1.8 Co-digestion of minced meat with digested sludge 31
5.1.9 Orange Peel 31
5.1.10 Seaweed 31
5.1.11 Yeast 32
5.2 Semi-continuous digester experiment 32
5.2.1 Biogas production and Methane content 32
5.2.2 pH, VFA and VS reduction 33
5.3 Evaluation of Cellruptor Pre-treatment 33
6 . CONCLUSION 35
7 REFERENCES 36
ACKNOWLEDGEMENT 42
APPENDIX A: Statistics Sweden 43
APPENDIX B: Batch raw data 44
APPENDIX C: Statistical data 47
1 INTRODUCTION
For the past few decades, the increasing global scarcity of petroleum and petroleum- derived fuels has led to intensive research on finding new alternative energy sources for power generation and transportation all over the world. Among the proposed alternative fuels, biogas has received much attention in recent years for gas engines and could be one remedy in many countries to reduce their oil imports. The European Union (EU) renewable energy policy has set a target to produce renewable energy, which meets 20% of European energy demand, by 2020, while biogas contributes 25%
share of this renewable energy (Nielsen and Oleskowicz-Popiel). Among the EU nations, Germany remains top in biogas production, where major amount of biogas (85%) is produced from municipal solid waste methanisation plant, decentralised agricultural plant and centralised co-digestion plant (XU2EVHUY¶(5 .
In Sweden, biogas is been produced since 1940 from sewage treatment plants. The HQHUJ\FULVLVGXULQJ ¶VLQFUHDVHGFRQFHUQLQ biogas production from other organic substrates. Biogas production from sugar refinery plants and paper mills were initiated during this period. Every Swedish municipality constructed biogas plants at their sewage treatment facility to enhance WKH ELRJDV SURGXFWLRQ ,Q ¶V extraction of methane gas from landfills were innovated to minimise these methane emission to the atmosphere. Large scale anaerobic co-digestion of various organic substrates such as agricultural waste, food waste, slaughterhouse waste, etc., was developed during
¶V This led to continuous research and development in the field of biogas technology.
With the aim of becoming wRUOG¶VILUVWRLO-free economy by 2020, Swedish government has implemented new renewable energy policies to promote the renewable energy production in Sweden (Swedish energy agency, 2011; EREC, 2011). Being a leader of biogas-to-vehicle-fuel revolution, amount of biogas delivered as a vehicle fuel is substantially higher than natural gas supplies in Sweden (SCB, 2011; Appendix A).
According to Avfall Sverige (Swedish Waste Management), 317,440 MWh of biogas was produced by anaerobic digestion of green and food waste in 2009, which is equivalent to 35 million litres of petrol. Table 1 represents the primary biogas production in 2009 from Germany (leading biogas producer in EU), Sweden and EU (EurOEVHUY¶(5
Considering perspectives of bioenergy systems and waste management, the biogas production from various waste materials has been gaining more attention in the last couple of years. Further increase in biogas production can be accomplished by improving the biodegradability on pre-treating the substrates. An ample scope on
research and applications of anaerobic digestion and various pre-treatment strategies has been made in this thesis with emphasis on combining both topics to enhance the biogas production from various waste materials.
Table 1: Biogas production in Germany, Sweden and EU, in 2009
Region
Primary biogas output (ktoe)
Landfills Sewage sludge* Others** Total
Germany 265.5 (6%) 386.7 (9%) 3561.2 (85%) 4213.4
Sweden 34.5 (31%) 60.0 (55%) 14.7 (14%) 109.2
EU 3001.6 (36%) 1003.7 (12%) 4340.7 (52%) 8346.0
* Urban and Industrial sludge
** Municipal solid waste methanisation plant, decentralised agricultural plant and centralised co-digestion plant
1.1 Aim
This thesis work evaluates the Cellruptor pre-treatment to enhance biogas production from various substrates.
1.2 Hypothesis
Cellruptor pre-treatment will improve anaerobic digestion process.
1.3 Strategy
¾ Optimum Cellruptor operating conditions were analysed after investigating the effect of cellruptor residence time variation and rapid/non-rapid release, on methane yield of biosludge.
¾ Batch experiments were performed to evaluate the cellruptor pre-treatment on biogas production from various substrates, namely: biosludge, digested sludge, dewatered sludge, fibre, hay, maize, minced meat, orange peel, seaweed and yeast.
¾ A semi-continuous digester experiment was also carried out to study the pre- treatment effect on methane yield from biosludge.
2 Background
2.1 Biogas ă for a sustainable environment
Biogas is a renewable energy source, comprising of methane (50-80%), carbon dioxide (20-50%), and traces of other gases such as hydrogen, carbon monoxide, and nitrogen.
In large scale, biogas can be used for production of heat and/or steam, electricity, chemicals and fuel cells whereas in small scale, it remains as an alternative energy source in rural communities, which meets the basic need of cooking and lighting. Once upgrading biogas to high purity level adequate to vehicle fuel standards, it can be used as vehicle fuel similar to natural gas. Biogas can be produced by many ways which includes pyrolysis, hydrogasification and anaerobic digestion, while anaerobic digestion remains as a most promising technology for developing a sustainable environment. At the environmental level, biogas production forbids the release of greenhouse gas (methane) into the atmosphere and also replaces the chemical fertilizers with nutrient rich digestate. (Engler et al., 1998)
2.2 Anaerobic Digestion
Anaerobic digestion is a biological process which is capable of converting almost all types of organic materials into methane and carbon dioxide. Some existing sources of methane emissions are wetland soils, oceans, rumen of ruminant animals, and the lower intestinal tracts of humans, landfills, and sewage digesters. Microbial production of methane from organic matter has become an attractive method of waste treatment and resource recovery, and this is carried out by action of complex anaerobic flora consisting of bacteria, fungi, protozoa and archaeal methanogens. Anaerobic process also offers an effective means of pollution reduction, which is superior to that achieved via conventional aerobic process due to the fugitive volatile emissions taking place before degradation in aerobic treatment plants leading to air pollution. Methane produced by anaerobic fermentation of biomass is a clean, renewable fuel.
Three basic points about anaerobic digestion process are:
(i) Slow growing anaerobic bacteria and archaeal methanogens are the most important microbial community involved in biogas production process;
(ii) A higher level of metabolic specialization could be seen in this process than aerobic process;
(iii) Most of the substrate free energy is converted to terminal product methane. At the end of digestion, the end product contains less microbial biomass than aerobic decomposition and, therefore, disposal of digested sludge after digestion may not be a problem but it also depends on the feedstock characteristics. (Nagamani and Ramasamy, 1999)
As practiced for several years, interest in anaerobic digestion in many countries has widely focused on the economic recovery of fuel gas from municipal sludge, cattle, industrial and kitchen wastes and agricultural surpluses (Demirbas et al., 2011).
2.2.1 Microbiology and Biochemistry
Hydrolysis, acidogenesis, acetogenesis and methanogenesis are four important steps of anaerobic digestion process. The model of microbial groups involved in this four-step flow of carbon from complex polymers to biogas consists of five groups. During the process of anaerobic digestion (Figure 1), complex polymers are broken into simple products by enzymes produced by fermentative bacteria (Group 1), which ferment the substrate to short chain fatty acids, hydrogen and carbon dioxide. Fatty acids, longer than acetate are catabolized to acetate by obligate hydrogen producing acetogens (Group 2). Hydrogen, carbon dioxide and acetate are the major products produced by these two groups after digestion of the substrate. Hydrogen and carbon dioxide can be converted into acetate by hydrogen oxidizing acetogens (Group 3) or methane by carbon dioxide reducing, hydrogen oxidizing methanogens (Group 4). Acetate is also converted into methane by acetotropic methanogens (Group 5) (Show et al., 2010).
Figure 1: Steps in Anaerobic Digestion
2.2.2 Environmental factors
Nutrients
Carbon, nitrogen and phosphorus are the macro nutrients that nourish the microbial growth. Generally, these nutrients are available in sufficient quantities in municipal sewage and sludge. Microbial community also relies on micro nutrients such as sulphur, vitamin and trace of minerals (iron, cobalt, nickel, molybdenum, selenium). All nutrients should be available in sufficient quantities as the microbial activity depends on the multiplicative factor of all essential nutrients. There should also be a balanced proportion of carbon and nitrogen and the optimum proportion ranges between 20:1 to 30:1 (C:N ratio) (Davidsson, 2007).
Temperature
Digestion temperature remains a crucial factor in anaerobic processes and it has to remain constant throughout the process. The operational temperature ranges of anaerobic digestion process are classified as mesophilic (30-Ü& WKHUPRSKLOLF -
Ü& RUSV\FKURSKLOLF -Ü& FRQGLWLRQV7KHPHVRSKLOLFSURFHVVLVPRVWFRPPRQO\
used as the thermophilic process has disadvantages such as process instability, lowered effluent quality, low methane production per unit substrate and high energy requirement for heating, and maintenance (Duran and Speece, 1997, Vindis et al., 2009).
Psychrophilic conditions are seldom used due to the slow microbial growth.
pH
The enzymatic activity of methanogenic bacteria is regulated within a specific pH range and the maximum activity is achieved at optimum pH. In mesophilic anaerobic process, the desired pH range for methanogens are 6.6-7.6 (optimum pH around 7.0) and 6.6-7.8 (optimum pH 6.8) under low solids (1-2%) and high solids (90-96%) sludge respectively (Lay et al., 1997). The inhibition of methane formation might also occur, when the pH is lesser than 6.3 or higher than 7.8 during digestion of high solids sludge (Liu et al., 2008).
Alkalinity
The accumulation of volatile fatty acids (VFA) and the production of carbon dioxide during anaerobic digestion can result in a pH drop, which may cause process instability and inhibition of methanogenesis. Thus, the addition of external alkalinity source (buffering agents) leads to achieve stable pH and may improve the rate of anaerobic digestion (Couderc et al., 2008). The bicarbonate of the liquid phase and carbon dioxide in gas phase stabilizes the system pH by producing alkalinity, which counteracts the pH reduction by accumulation of VFAs (Appels et al., 2008).
Moisture Content
Water is important as the nutrients get dissolved in it, which in turn facilitate the diffusion transport of these dissolved substances across the bacterial cell membrane.
Thus addition of water increases the rate of hydrolysis, whereby decreasing the rate of
solids accumulation (Couderc et al., 2008). Lay, et al. (1997) investigated the effect of moisture content of digesting sludge on biogas production.
Toxic Substances
Volatile fatty acids (VFAs), free ammonia, hydrogen, hydrogen sulphide, heavy metals, chlorinated compounds and detergents are few toxic substances that inhibit the anaerobic process. These substances are either produced during the digestion process or already present in the substrates and hence, few substrates need to be pre-treated before AD, to remove toxic substances (Show et al., 2010).
2.2.3 Solid Characteristics
Total Solids
Total solids (TS) are the amount of dry matter remaining after the removal of moisture FRQWHQWE\GU\LQJDWÜ&DQG76DUHFRPSRVHGRIYRODWLOHDQGIL[HGVROLGV,QFUHDVHLQ
TS % of the substrate fed into the reactor has no effect either in TS or VS removal (Fongsatitkul et al., 2010).
Volatile Solids
Volatile solids (VS) are the amount of organic matter lost on combusting dry solids at
Ü& 2UJDQLF ORDGLQJ UDWH LV GHWHUPLQHG E\ 96 DQDO\VLV DV LW JLYHV DQ DSSUR[LPDWH
amount of organic matter present in the waste.
2.2.4 Operational Parameters
Organic Loading Rate
Organic loading rate (OLR) is the measure of organic material fed into the digester and this depends on volatile solids content and methane potential of the substrate. Feeding the digester above optimum OLR may lead to accumulation of inhibitory substances, disturbing the process stability or low VS-reduction.
Hydraulic Retention Time
Hydraulic retention time (HRT) is the average residence time of the liquid inside the digester and the optimum HRT for most mesophilic anaerobic digester ranges between 15 to 30 days (Davidsson, 2007).
Temperature (Refer 2.2.2) Stirring
The constituents in the reactor must be mixed well to increase contact between the substrate and microorganisms. It provides a uniform sludge concentration across the UHDFWRU WR DFKLHYH PD[LPXP GLJHVWLRQ E\ LPSURYLQJ PLFURRUJDQLVPV¶ DFFHVVLELOLW\ WR substrates. Optimum mixing should be maintained to avoid the disruption of microorganism. Different substrates in co-digestion process should be mixed well before entering the AD process.
2.2.5 Control Parameters
Volatile Fatty Acids
Volatile fatty acids (VFA) are intermediates formed during the digestion process and when VFAs get accumulated in high quantities, they inhibit Methanogenesis. The most prominent inhibitory VFAs are acetic and propionic acid. At increasing temperatures, accumulation of VFA decreases the pH value and when pH falls below 6.0, AD process gets inhibited (Nielsen and Angelidaki, 2008).
Volatile Solids Reduction
Volatile solids constitute the organic portion of total solids and these reduce during the digestion process, as they are converted to biogas. Volatile solids reduction is directly related to the biogas yield (Appels et al., 2008).
Methane Potential
Based on economical aspect of AD, it is important to know the methane potential of the substrates. Many techniques such as biochemical methane potential (BMP), dynamic respiration rate (DR4) and chemical oxygen demand (COD) test are available to determine the methane yield. The most common BMP test is a batch test for 28 days, which is likely to provide information useful for execution of CSTR (Shanmugam and Horan, 2009).
2.3 Pre-treatment Techniques of substrates
The digestion process is affected by the non-degradable constituents and rigid cell wall of the substrate which cause the cell constituents inaccessible for the anaerobic microorganisms and, hence, the anaerobic digestion is limited by hydrolysis rate (Rivard et al., 1998). The microbial consortia in the reactor tend to multiply by metabolizing the organic matter and forms biomass. The reduction of substrate biomass is an important factor to enhance biogas production and this can be achieved by cell lysis. Hence, an effective pre-treatment aims to enhance the biogas production by
improving the substrate accessibility to the microbial community and consequently, accelerating the rate of anaerobic digestion.
During recent years, many studies have been made on various mechanical pre- treatment techniques that disrupt cells by application by force, including:
High pressure homogenizer
The High pressure pump compresses the sludge up to several bars (up to 900 bar) and then, the sludge undergoes a sudden depressurization in the homogenizing valve forming cavitation bubbles. An irreversible disruption of the cell membrane happens during the explosion of these bubbles (Rai and Rao, 2009).
Ultrasonic homogenizer
Cavities or microbubbles are formed due to the repetitive compression and rarefaction of the ultrasonic waves, when passed through sludge medium. The cell wall and membranes are disrupted due to the powerful mechanical shear force generated during the collapse of many microbubbles (Khanal et al., 2007). This principle is an adaptation from Pulsed electric field technology, which has notable significances in medical field (imaging device), food industry (extraction of vegetable oils), etc.
Thermal Hydrolysis
Cell rupture is achieved by effect of heat produced at high temperature (160-Ü&IRU
30-60 min) leading to increase in sludge digestion and soluble COD (Carrere et al., 2008).
Freezing and Thawing
Freeze/thaw pre-treatment disrupts the cell membrane physically by forming ice crystals. They cause irreversible rupture of cell floc by reducing the bound water content (Gao, 2010).
Gamma-irradiation
Gamma radiations disrupt the cell membrane and release the soluble organic compounds, which influences the hydrolysis step in the digestion process (Lafitte- Trouque and Forster, 2002).
Besides the above mentioned mechanical pre-treatments, viz. chemical pre-treatment (Acid or alkaline hydrolysis, Ozone pre-treatment) (Perez-Elvira et al., 2006), Biological pre-treatments (Yunqin et al., 2010) and combination of pre-treatments such as Microsludge® (combination of chemical and mechanical pre-treatment) are available to increase the digestion rate. Even though, there is existence of pre-treatments in commercial level such as Microsludge®, %LR7+(/<62SHQ&(/®&DPELWKHUPDO
hydrolysis process and Crown® GLVLQWHJUDWLRQV\VWHPWKHUH¶VVWLOODVHDUFKIRUDEHWWHU
pre-treatment based on economical and operational grounds. One such promising technology is Cellruptor.
2.3.1 Cellruptor
Eco-Solids International Ltd. (Hampshire, UK) has developed this simple cell disintegration technology, Cellruptor and, reported 28% increase in biogas production during the commercial trial period at Yorkshire wastewater treatment plant (WWTP) (Yorkshire, UK). Unlike other pre-treatment techniques, Cellruptor just require low energy (maximum 10 bar pressure) to disrupt the cells.
Principle
A soluble gas such as CO2 is compressed to the sludge at 10 bar pressure and this soluble gas diffuses to the cell through the cell wall. During a rapid depressurization, the diffused CO2 causes cell expansion leading to an irreversible rupture of cell wall. At large scale, biogas containing 40% CO2 can be passed to the Cellruptor making the process very more economical than spending for a compressed gas tank.
2.4 The substrates
2.4.1 Biosludge
Biosludge is the outcome of the secondary (biological) treatment of sewage treatment plants. It is also called as excess sludge, activated sludge, waste activated sludge (WAS) or surplus activated sludge (SAS). Resulted due to overproduction of microorganisms, biosludge contain rich biomass, extracellular polymeric substances (EPS) with more than 95% water (Yin et al., 2004). The biomass comprises of Bacteria, fungi, protozoa, and rotifers. Generally, the TS and VS are around 7-10 g/L and 70-80% respectively.
2.4.2 Dewatered Sludge
Dewatered sludge is the waste activated sludge with less water content. The water is removed from the excess sludge before storage at anaerobic conditions to avoid the hydrolysis process. Anaerobically stored dewatered sludge has proven to enhance biodegradability due to earlier breakdown of polyacrylamides (PAM) to soluble substrates during anaerobic storage, with the anaerobic storage acting as a pre- treatment technique (Xu et al., 2010). Dewatered sludge used in this work was dewatered waste activated sludge.
2.4.3 Digested Sludge
Digested sludge is the outcome of tertiary treatment of sewage treatment plants. The digested sludge has reduced mass, odour and pathogens due to complete anaerobic digestion of primary and secondary sludge (Ek, 2005). The TS and VS are around 20-40 g/L and 50% respectively and, digested sludge had showed improved dewaterability after thermal and alkaline pre-treatments than conventional process (Carballa et al., 2009).
2.4.4 Fibre Sludge
Fibre sludge, a waste material generated from lignocellulosic bio refineries, such as paper and pulp industries. These wastes are either dumped into the soil or burnt out, causing environmental pollution and hence, these wastes can be used for biogas production because of their high polysaccharide and low lignin content (Cavka et al., 2010).
2.4.5 Hay
Besides their use as animal fodder, hay can be also for biogas production. However, its use for biogas should be controlled to protect the biodiversity and the methane yield of 255-327 mL/g VS from hay of size range of 0.5‒20 mm (Stewart et al., 1984). Menind and Normak (2009) found a negative correlation between biogas yield, particle size and lignin content during grinding pre-treatment of hay.
2.4.6 Maize Silage
Maize silage, an animal fodder, can be an ideal substrate for anaerobic digestion because of high carbohydrate and low lignin content. The presence of rapidly degrading organic content leads to initial increase in biogas production, which may limit the loading rate. Anaerobic digestion of maize silage (30.8% TS, 94.1% VS) at mesophilic temperature yielded, 0.347 m3 CH4/kg TS (ãSDONRYiHWDO ).
2.4.7 Minced Meat
Meat and other animal by-products are likely to be potential biogas producers because of the high fat and protein content. Recent studies on anaerobic co-digestion of animal by-producWV ZLWK VHZDJH VOXGJH DW PHVRSKLOLF WHPSHUDWXUH Ü& KDYH shown improved methane production (Luostarinen et al., 2009, Luste and Luostarinen, 2010).
Thermochemical pre-treatment (Ü& DQG 1D2+ (Wu et al., 2009) of animal by- products have enhanced the efficiency of AD process while pasteurization, sterilization
and alkali hydrolysis showed no improvement in methane production (Hejnfelt and Angelidaki, 2009).
2.4.8 Orange Peel
A large amount of solid wastes from the fruit processing industries are commonly landfilled because to avoid the expensive treatment of these wastes. One such waste is the Orange Peel, a lignocellulosic biomass (cellulose (%): 13.61±0.6, hemicellulose (%):
6.10±0.2, lignin (%):2.10±0.3) (Ververis et al., 2007), containing high organic content (ca 90-95% TS) which makes it a suitable feedstock for anaerobic digestion. However, the antimicrobial agents (peel oil and limonin) may inhibit the digestion process (Naparaju and Rintala, 2006).
2.4.9 Seaweed
Seaweed is multicellular marine algae, which cause social problems in coastal regions due to its high accumulation resulting from marine eutrophication. Results on biogas production from seaweeds in laboratory tests at mesophilic (Moen et al., 1997, Kerner et al., 1991) and thermophilic conditions (Hansson, 1983) have been reported. Nkemna and Murto (2010) reported the effect of heavy metals removal from seaweed on biogas production in batch tests and UASB reactors. Mussgnug, et al. (2010) investigated six Germany dominant microalgae species (cyanobacteria, freshwater and saltwater algae) for biogas production with drying pre-treatment and they also concluded that a suitable cell disruption method is of great importance to enhance the biogas production. The seaweed used in this study was filamentous red alga of genera Polysiphonia, Rhodomela and Ceramium.
2.4.10 Yeast
Yeast residue, a solid waste from beer brewery industries can be considered as a suitable substrate for biogas production because of its high organic content. Yeast cells have a rigid cell wall constituting mainly of polysaccharides, namely glucans and mannans, which has to be ruptured to make the cell constituents accessible for anaerobic digestion. Cell wall lysis may be achieved by pre-treatment methods like enzymatic pre-treatment (Mallick et al., 2010), horizontal bead mill (Heim et al., 2007), autolysis(Shotipruk et al., 2005) and a combination of enzymatic pre-treatment with high pressure homogenizer (Baldwin and Robinson, 1994) %DNHU¶V \HDVW ZDV XVHG LQ
this thesis work.
3 Methods and Materials
3.1 Cellruptor
The Cellruptor equipment used in this work was obtained from Eco-Solids International Ltd. (Hampshire, UK) and is shown in Figure 2. The assembled equipment consist of three main units: 8 L pressure cylinder with sampling and drain ports, compressed CO2 tank air cylinder and a collecting bucket. In order to ensure that the right pressure is maintained in the cylinder, there was an extra gas meter attached near the sampling port of the high pressure cylinder, apart from the gas regulator near the gas cylinder.
3.1.1 Process conditions
The pressure that was operated during this thesis work was 10 bar and the residence time ranged from 30 to 180 min. The equipment was handled according to the PDQXIDFWXUHU¶V LQVWUXFWLRQV (FR-Solids International Ltd., Hampshire, UK). All pre- treatment run took place at room temperature and the working volume for every run of pre-treatment was between 1.5-2.0 L.
Figure 2: Cellruptor
3.2 Batch Experiment
3.2.1 The substrates & Experimental set-up
Ten different substrates were studied in five sets (A-E) of batch experiments and Table 2 summarizes all the substrates with their collection place and solids content. Both batch sets A and B utilized biosludge from wastewater treatment plants (WWTP), but the difference lies in the aim of each one. Batch set A was designed to study the effect of residence time (30, 60, 90, 120 and 180 min) while batch set B aimed to evaluate the effect of substrate release (rapid/non-rapid) from cylinder draining port on biogas production. Remaining batch sets C, D and E were designed to evaluate the effect of Cellruptor pre-treatment of various other substrates on biogas production, under operating conditions of 10 bar pressure and 90 min residence time.
Substrates as starting material for pre-treatment
Cellruptor needs the substrates in slurry form and hence, substrates with high TS content (>10%) and in non-slurry form were diluted with water or digested sludge. The non-treated substrates (or control) were also diluted, as to eliminate the influence of dilution factor and pre-wetting over biogas production. Few substrates were processed in the following way prior to Cellruptor pre-treatment: dry substrates (maize and hay) were manually scissored into small pieces (~1-2 cm); orange peel was mashed using a food processor; seaweed was initially washed with water to remove sand particles and then scissored to shorter fragments (~2-3 cm).
3.2.2 Batch start up
The mandatory solutions in all batch bottles, irrespective of the substrate type, are inoculum, nutrient solution, Na2S solution and Milli-Q water. Inoculum was prepared by mixing the digested sludge from Nykvarn sewage treatment plant (Linkoping, Sweden) with the sludge collected from various semi-continuous stirred tank reactors (CSTR) at Scandinavian Biogas Fuels AB. The nutrient solution comprising of NH4Cl, NaCl, CaCl2.2H2O and MgCl2.6H2O while Na2S solution (0.1 M) acts as a reducing agent ensuring low redox potential by complete removal of residual oxygen. Milli-Q water is the double distilled water prepared from the Millipore System (Millipore, Billerica, USA). The amount of loading substrate and milli-Q water in each bottle was calculated based on OLR and assumed methane potential of each substrate.
Batch experiments were carried out in triplicates of 320 mL glass bottles holding 100 mL liquid phase and the procedure were accordant with Scandinavian Biogas Fuels AB standard procedure described below. The substrates were weighed and loaded into their respective labelled glass bottles, which was then followed by flushing N2 gas to secure anaerobic environment in these bottles. 20 mL inoculum, 2 mL nutrient solution and milli-Q water were added to these bottles while flushing N2 gas. The bottles having been sealed immediately with EPDM rubber stoppers and aluminium caps, the gas
phase was then altered by evacuating and refilling using nitrogen/carbon dioxide mixture (N2/CO2 ; 80/20%) for more than nine times. Finally, 0.3 mL Na2S solution was injected after depressurizing the bottles completely. The bottles were then shook well DQGSODFHGLQVLGHÜ&WHPSHUDWXUHURRP
Three standard controls were used for each batch set and they were prepared (in triplicates) as mentioned below:
Inoculum Control To determine the methane production from inoculum alone and this value helps for the calculation of methane production from solely substrate in substrate bottles. (20 mL inoculum, 2 mL nutrient solution, 0.3 mL Na2S solution and 78 mL milli-Q water) Positive Control
To determine the degradation efficiency of inoculum by using cellulose filter paper. (0.5 g Whatman filtration paper Grade 3 (Whatman Ltd., UK), 20 mL inoculum, 2 mL nutrient solution, 0.3 mL Na2S solution and 78 mL milli-Q water)
Methane Control
To determine the instrument reliability by estimating the known methane amount. (50 mL methane and 100 mL milli-Q water)
Table 2: Sample collection and pre-treatment conditions of various substrates
Batch set
Substrate Collection place in Sweden
Solids content# Pre-treatment level Residence time (min)
Pressure (bar) Rapid/Non-rapid
release##
TS (%)
VS (%TS)
A
Biosludge Henriksdal WWTP, Stockholm. 4.3 71.0 30,60,90,120 & 180 min 10 bar
Non-rapid
Biosludge Municipal WWTP, Varberg. 5.3 78.9
B Biosludge Municipal WWTP, Varberg. 5.2 78.6
90 min 10 bar
Rapid and Non-rapid
C
%DNHU¶V\HDVW Supermarket, Linkoping. 28.1 93.4
90 min 10 bar Rapid
Minced meat** Supermarket, Linkoping. 40.5 97.5
Digested sludge Nykvarn sewage treatment plant, Linkoping. 2.7 66.9
Fibre sludge* Husum Pulp Plant, Husum. 29.4 66.4
D
Hay* Haga Farm, Östergötland. 93.3 90.9
90 min 10 bar Rapid
Dewatered sludge* Loudden WWTP, Stockholm. 24.7 83.8
Maize silage* Hags Farm, Östergötland. 35.2 96.8
E
Biosludge Bromma WWTP, Stockholm. 6.2 68.2
90 min 10 bar Rapid
Dewatered sludge* Henriksdal WWTP, Stockholm. 27.7 60.4
Minced meat* Supermarket, Linkoping. 40.5 97.5
Biosludge Frövi Pulp Plant, Frövi. 7.1 78.3
Seaweed* Kattegat coast, Varberg. 12.0 70.0
Orange peel* Brämhults Juice Industry, Boras. 20.7 95.6
Diluted with (*water/**digested sludge) before pre-treatment # solids content of fresh substrate ## substrate release from draining port of Cellruptor
3.2.3 Analysis
Solids content (TS & VS)
TS and VS were estimated according to the standard protocol of Swedish Standards Institute. A small amount of substrate were placed in a silica crucible and dried in hot air oven at 105ÜC for 20 hours. The crucibles were then FRROHGLQDGHVLFFDWRUDQGZHLJKHGLQD³0HOWHUFKHPLFDOEDODQFH´7KLV
procedure was performed in duplicates to obtain concordant values.
Where A - weight of silica crucible
B - weight of VLOLFDFUXFLEOHDQGVXEVWUDWHEHIRUHGU\LQJDWÜ&
C - weight of silica crucible and substrate, after drying DWÜ&
$IWHUGHWHUPLQLQJ76FRQWHQWWKHVLOLFDFUXFLEOHZDVNHSWDWÜ&IRUKRXUV
in a muffle furnace. The crucible was weighed in a matter chemical balance after cooling to room temperature.
Where A - weight of silica crucible
C - weight of silica crucible and substrate, after drying DWÜ&
D - weight of silica crucible and substrate, after ignition DWÜ&
Biogas production
The amount of biogas produced in each batch flasks were evaluated from the gas pressure measured by testo digital pressure meter (Testo AG, Lenzkirch, Germany) on 7 occasions (Day 1, 3, 7, 14, 20, 31 and 60). Having measured the gas pressure, 1 mL of biogas was withdrawn from the headspace of each flask and injected into their corresponding 31.7 mL glass vial for further analysis by gas chromatography (GC). Then, the bottles were completely depressurized expect methane control flasks. For methane control flasks, pressure is measured only on first occasion while gas sampling is done on all occasions together with other flasks.
Methane content
The methane content in the biogas was determined from the GC spectra, measured on a HP 5880A series GC system (Hewlett Packard, Houston, USA) equipped with a Flame ionization detector (FID). Separations were carried out by mobile phase (N2 gas) passing through Poraplot T column at a flow rate of 130 mL/PLQ7KH),'¶VRYHQLQMHFWRUDQGGetector temperatures were 80, 150 DQG Ü& UHVSHFWLYHO\ ,QVWUXPHQWDO FKHFN GDWD DFTXLUHPHQW DQG GDWD
