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T E M A I N S T I T U T E
Biomethanation of Red Algae from theEutrophied Baltic Sea
Rajib Biswas
M A S T E R ’ S T H E S I S
Environmental Science Master Programme
August 27, 2009
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Supervisor:
Prof. Jörgen EjlertssonDepartment of Water and Environmental Studies
Linköping UniversitySE-581 83 Linköping
Sweden.&
Research and Development Director, Vice PresidentScandinavian Biogas Fuels AB
Väderkvarnsgatan 14SE-753 29 Uppsala
Sweden.
Examiner:
Prof. Bo SvenssonDepartment of Water and Environmental Studies
Linköping UniversitySE-581 83 Linköping
Sweden.
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© Rajib Biswas, February 2009
Pictures and Illustrations: Rajib Biswas
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Abstract
In the semi-enclosed Baltic Sea, excessive filamentous macro-algal biomass growth as a result ofeutrophication is an increasing environmental problem. Drifting huge masses of red algae of thegenera Polysiphonia, Rhodomela, and Ceramium accumulate on the open shore, up to five tones ofalgae per meter beach. During the aerobic decomposition of these algal bodies, large quantities ofred colored effluents leak into the water what are toxic for the marine environment.
In this study, feasibility of anaerobic conversion of red algae Polysiphonia, rich in nitrogen and phos-phorous, was investigated. Biogas and methane potential of Polysiphonia, harvested in two differentseasons [October and March], was investigated through three different batch digestion experimentsand laboratory scale CSTR [continuous stirred tank reactor] at mesophilic (37oC) condition. Au-toclavation [steam and heat] and ultrasound pretreatments were applied in order to enhance thebiodegradation. In STR, anaerobic codigestion of algal biomass with SS [sewage sludge] was appliedwith a gradual increase in organic loading rate [1.5-4.0 g VS/L/day] and operated for 117 days at 20days HRT [hydraulic retention time]. Reactor digestate was analyzed four times over the period todetermine the nutrients and heavy metals content.
It is concluded that the methane potential of algae harvested in October is almost two-fold than thatof algae harvested in March, probably due to it’s higher [more than double] nitrogen richness. Anincrease in biogas yield was observed upto 28% and VS reduction was increased from 37% to 45%due to autoclave pretreatment. Ultrasound pretreatment had no effect on biodegradation of algae.In batch digestion, maximum methane yield 0.25 m3/kg VSadded at 273oK, was obtained from algae[harvested in October] pretreated in autoclave.
Codigestion of algae with SS worked well in STR with a comparatively lower OLR. At a higher OLR,methanogens were inhibited due to increased VFAs accumulation and decreased pH. A maximumbiogas yield 0.49 m3/kg VSadded at 310oK , was obtained from algae [harvested in October] pretreatedwith autoclave. The methane content of the produced biogas was 54%. Average [over a shortperiod, day 99-107, reactor showed steady performance] maximum biogas yields from untreatedalgae obtained 0.44 m3/kg VSadded at 310oK and the VS reduction was calculated 32%. Digestate,to be used as a fertilizer, was found NH4-N, N, P, K, S and Na rich and only Cadmium level wasabove the maximal limit among the heavy metals. The sand content in algae during harvestingwas considered as a factor to disrupt the operation. Codigestion of Polysiphonia algal biomass withsubstrate with higher C:N ratio like paper mill waste should be more appropriate to increase themethane and biogas yields. It is inconclusive whether AD process is a good method to dewater redalgae or not, but large scale harvesting of algae will definitely contribute to curb eutrophication ofthe Baltic Sea through decreasing N and P level.
Key words: Anaerobic Digestion, Baltic Sea, Biogas, Codigestion, Eutrophication, Nutrients, Pre-treatment, Red Algae.
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Preface
This thesis is the final part to the fulfillment of Master of Science in Environmental Science Degreefrom the Department of Water and Environmental Studies (TEMA), Linköping University, Sweden.The practical work of this thesis was carried out at the Department of Water and Environmen-tal Studies (TEMA Institute) in Linköping University, Sweden and had a close collaboration withScandinavian Biogas Fuels AB from November 2007 to July 2008.
For any questions about this thesis, please don’t hesitate to contact me. AND we all should considerone thing...
“Nature does nothing uselessly...”
Aristotle (384 BC - 322 BC),Politics Greek critic, philosopher, physicist, & zoologist
Thanks for reading my thesis...
Rajib Biswas
Cell- +46(0)[email protected]
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Contents
1 Introduction 11
1.1 General introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.2 Aim and hypotheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2 Background 13
2.1 Biogas: A green energy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.2 Anaerobic digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3 The Baltic Sea eutrophication . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.4 Red algae at the Baltic Sea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3 Materials and methods 23
3.1 Experimental overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.2 Preparation of substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
3.3 Experimental setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
3.4 Analytical procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
4 Results and discussion 35
4.1 Batch experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
4.2 Stirred reactor experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.3 Results from similar studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
4.4 Concluding discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
5 Conclusion 49
A Calculation on VS reduction 56
B Raw data 585
List of Figures
2.1 A schematic figure [modified] of anaerobic digestion of organic material. [23, 24] . . . 15
2.2 Collected Polysiphonia red algae for this study. . . . . . . . . . . . . . . . . . . . . . 22
3.1 Preparation of experimental bottles for incubation. . . . . . . . . . . . . . . . . . . . . 29
3.2 The laboratory-scale anaerobic digesters used for this study, equipped with steeringequipments and gas meters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
4.1 Accumulated biogas in bottles [error bars show the standard deviation] of the firstbatch experiment investigating biogas potential of OA [−■−], OA pretreated in au-toclave [−▲−] and Whatman paper [−♦−]. Control [− ⋇ −] shows accumulation ofbiogas produced from the inoculum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
4.2 Specific methane yields (i.e., methane produced from inoculum was substracted) in thefirst batch experiment investigating biogas potential of OA [−■−], OA pretreated inautoclave [−▲−] and Whatman paper [−♦−]. Error bars show the standard deviationand yields are corrected at STP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
4.3 Specific methane yields (i.e., methane produced from inoculum was substracted) in thesecond batch experiment investigating methane potential of OA [−×−, −⋇−, −•−],NA [−♦−, −■−, −▲−] (3 replications of each). . . . . . . . . . . . . . . . . . . . . . . 38
4.4 Accumulated specific methane yields (i.e.; methane produced from inoculum was ex-cluded) in the third batch experiment investigating biogas potential of NA [−■−], NApretreated with ultrasound [−▲−] and Whatman paper [−♦−] (up to day 40). Givendata are averaged over three samples [error bars show the standard deviation, almostunseen] and corrected at STP. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
4.5 Biogas yields in the reactors D9 [−♦−] and D10 [−■−] and CH4 amounts in producedgas of the reactors D9 [■] and D10 [■] between Day 99-117. Here both reactorsshowed steady performance. D10 was fed with OA treated in autoclave from Day 107-117 (a sharp increase in gas yields). The gas amount given here as observed at 37±2°C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.6 Effect of OLR on total biogas yields in D9 [■, −♦−] and D10 [■, −■−] over theexperimental period. Data are given here obtained from incubation room at 37 °C. . . 42
4.7 High VFAs accumulation affected on total gas production in the reactor D10 [■, −■−]between Day 54-77 while D9 [■, −♦−] shows normal production. The daily producedgas amount given here as observed at 37±2 °C. . . . . . . . . . . . . . . . . . . . . . . 43
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LIST OF FIGURES 7
4.8 Destruction of volatile solids over the experimental period in reactor D9 [♦] and D10[■]. Arrow shows the VS reduction after 3 retention time. . . . . . . . . . . . . . . . . 44
List of Tables
2.1 Calorific value of biogas and natural gas (modified) [9]. . . . . . . . . . . . . . . . . . . 14
3.1 Experimental design (STR) and digester operating conditions for the study of theanaerobic codigestion of Polysiphonia algal biomass with primary sludge [PS]. . . . . 24
3.2 Experimental design for the batch digestion experiments. . . . . . . . . . . . . . . . . 27
3.3 Preparation of samples for standard curve. . . . . . . . . . . . . . . . . . . . . . . . . . 33
4.1 Results obtained from the batch digestion experiments. Data shown here are specificyields from test material [subtracted mean biogas and methane production in controls]and averaged over three replications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4.2 Specific biogas and methane yields (mL/added g VS) in the batch digestion experi-ments by days. The yields are averaged over three replications and corrected at STP. . 39
4.3 A summary of reactors performance data [data are averaged over the given period] forthe experiment at a 20 days HRT. The gas production given here is at 37±2 °C, asobserved in the incubation room. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
4.4 The maximal content of metals in the digestate [According to the Swedish qualityassuring system SPCR 120] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.5 The characteristics and nutrients content of algae and digestate from reactor D9 andD10 based on the analyses reports. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
4.6 Results from previous studies on anaerobic digestion of marine biomass in differentconditions and scales. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
B.1 Performance data of the reactor experiment during feeding with OA at a 20 days HRT(Day 01-17). The gas production given here is at 37±2 °C, as observed. . . . . . . . . 59
B.2 Performance data for the experiment during feeding with NA at a 20 days HRT (Day18-34). The gas production given here is at 37±2 °C, as observed. . . . . . . . . . . . 60
B.3 Performance data for the experiment during feeding with NA at a 20 days HRT (Day35-54). The gas production given here is at 37±2 °C, as observed. . . . . . . . . . . . 61
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LIST OF TABLES 9
B.4 Performance data for the experiment during feeding with NA at a 20 days HRT (Day55-77) while gas production rate of digester D10 deteriorated seriously because of highaccumulation of VFAs. The gas production given here is at 37±2 °C, as observed. . . 62
B.5 Performance data for the experiment during feeding with OA in D10 and with NA inD9, at a 20 days HRT (Day 99-107). The gas production given here is at 37±2 °C, asyields observed at the incubation room. . . . . . . . . . . . . . . . . . . . . . . . . . . 63
B.6 Performance data for the experiment during feeding with OA pretreated with autoclaveat 121 °C for 30 minutes, at a 20 days HRT (Day 108-117). The gas production givenhere is at 37±2 °C, as yields observed at the incubation room. . . . . . . . . . . . . . 64
Abbreviations
NA Experimental term for Algae collected in March 2008 (new algae)
OA Experimental term for Algae collected in October 2007 (old algae)
D9 Experimental term of control reactor
D10 Experimental term of test reactor
g gram
g/L gram per liter (1 g/L = 1 kg/m3 )
lb pound (1 lb = 0.4536 kg)
m3 cubic meter (1 m3 = 35.31 cu ft)
mL milliliter
mL/g milliliter per gram (1 mL/g = 1 L/kg = 0.001 m3/kg)
OLR Organic Loading Rate
TS Total Solids
VFAs Volatile Fatty Acids
VS Volatile Solids
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Chapter 1
Introduction
1.1 General introduction
Anaerobic digestion of marine algae to provide renewable energy is an attractive possibility. Algalcells convert and store solar energy through their photosynthetic activities. They can be degraded tofor methane through anaerobic digestion. Moreover, the whole digestion process could be acceleratedby existing advanced technologies (e.g. codigestion, pretreatment etc.) in order to increase the biogasyield. Further, the digestate can be used as a fertilizer on arable land to improve soil quality sincedigestates are considered as promising sources of N, P, K and S and other essential macro and micronutrients for plants in available forms.
Eutrophication [Section 2.3] in coastal and inland waters caused by increased input of nutrients ororganic matter resulting in excessive planktonic and filamentous algal biomass growth is one of themost significant environmental problems well described both globally and regionally [1, 2, 3, 6, 8,35, 36, 37, 39, 38, and references therein]. In particular, the Baltic Sea is naturally vulnerable tothe environmental degradation as the portions are almost enclosed by landscape and the shallowbrackish sea characterized by cold temperature. Besides, the pollution of the Baltic Sea is highbecause of the intense pressure from human activities compared to the other sea areas on the globe.This sea is continuously affected by the pressure from ca 85 million people in 14 countries. In thesemi-enclosed Baltic Sea, where the nutrient load has strongly increased from its natural level, thishas led to marked changes in the coastal ecosystems [1]. This has resulted in increased planktonbiomass and increased amount of filamentous algae; decreased transparency of water body, changesin community structure and abundance of benthic communities and anoxia/hypoxia of deeper basins[2]. Consequently, large masses of filamentous red algae of the genera Polysiphonia, Rhodomela, andCeramium are regularly washed up on beaches of the central Baltic Sea [3] and during the summerit cover shallow bottoms close to the shore. Winds and currents move the masses towards the shoreand large masses of algae accumulate on the beaches up to five tones of algae per meter recorded [4].
During the decomposition of these algal bodies, large quantities of red colored effluents leak intothe water which is toxic for human health and the marine environment. Several organohalogeniccompounds are produced by red algae, many of them being similar to toxic commercial productswith acute effects on the central nervous system and also with chronic endocrine effects [5].
In spite of the international co-operation for the last three decades aiming to curb the eutrophicationand to protect the Baltic Sea environment, eutrophication is still not under control. The eutrophica-tion problem and toxic algal bodies may not be managed properly in a sustainable way except by anintegrated effort, where harvesting of algal bodies and e.g. converting them into biogas is initiated.This would possibly become a profitable means both from economic and environmental aspects.
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CHAPTER 1. INTRODUCTION 12
Biogas production by the anaerobic digestion (AD) process is a promising means of achieving multipleenvironmental benefits such as producing an energy carrier from renewable resources i.e., methane.This process results in reduction of the emission of greenhouse gases, nitrogen oxides, hydrocarbonsand particles by replacing fossil fuels. In addition, utilization of substrates such as toxic algal biomass,hazardous waste, agricultural byproducts, municipal waste etc. has various ancillary benefits as thiswill prevent leaching colored effluents into the water (in case of toxic red algae) and prevents methane,ammonia, phosphorus and nitrogen etc. leaching during their uncontrolled aerobic and/or anaerobicdecomposition or landfilling. Also, large scale harvesting of algal biomass would contribute to curbeutrophication by limiting the nutrient availability (N, P) in the Baltic Sea.
My thesis was designed to investigate the biogas potential of abundant red algae from the eutrophiedBaltic Sea. The experiments were carried out in laboratory scale stirred digesters [Section 3.1.2] andas three non-parallel batch experiments [Section 3.1.1]. Additionally, several methods, i.e. codigestionof algae with primary sludge from a waste water treatment plant, increasing organic loading rate(OLR), pretreatments of algal biomass with ultrasound and autoclavation, were applied in order tostudy the effects on the biogas production and degradation of the organic fraction.
1.2 Aim and hypotheses
This thesis aims to utilization of abundant algal biomass of the eutrophied Baltic Sea as a renewablesource of energy as well as to a sustainable management of the toxic red algae to eliminate it’senvironmental impact on the coastal ecosystem.
Hypotheses are:
^ Red algae can be codigested with sewage sludge.
^ Large scale harvest of red algae will influence the nitrogen and phosphorous balance in theBaltic Sea.
^ Ultrasound and autoclave pretreatments of algal biomass will increase the total biogas produc-tion.
^ The biogas process/anaerobic digestion is a good method to dewater red algae.
^ 60% of the volatile solids (VS) of the algae is degradable and converted into biogas.
To prove the hypotheses, the following questions will be answered:
1. What are the methane and biogas yields per added amount of treated or untreated algae?
2. Will pretreatment of algae with ultrasound at 10 kWhL-1 and autoclavation for 30 minutesincrease the digestibility as well as the biogas and methane yields?
3. Can the organic loading rate (OLR) of a digester gradually be increased with 1.5 g and 3.0 gVS algae L-1D-1 and be codigested with primary sludge at a rate of 0.5 g VS L-1D-1 ?
4. What is the rate of volatile solids reduction of the algal biomass in biogas reactors?
5. What are the qualities of digestate (e.g., nutrients, heavy metals) from a biogas reactor fedwith algae?
Chapter 2
Background
2.1 Biogas: A green energy
Biogas is a mixture of CO2 and the inflammable gas CH4, which is produced by bacterial conversionof organic matter under anaerobic (oxygen-free) conditions [11]. Several studies have showed thatthe biogas released from anaerobic biodegradation [Section 2.2] of organic material contains 55 to 75% methane, 25%-45% carbon dioxide and other traces gases in minute quantities, i.e. N2, NH3, H2,H2S and O2, usually less than 1% of total gas volume [9]. These proportions, as well as the biogasyields, are largely determined by the raw materials digested and the digestion technology applied.For instance, the digestion of a raw material with a high fat content can provide a higher gas yieldand a higher proportion of methane than the digestion of a raw material rich in carbohydrates [12].
The microbial conversion of organic matter to methane, which can be burned for heat generation,is a process that is becoming increasingly attractive as a method of waste treatment and resourcerecovery [10]. After the rise of energy prices in 1970s, the process received renewed attention dueto the need to find alternative energy sources to reduce the dependency on fossil fuels [9]. Dueto the environmental advantages, the interest in biogas process still remained in spite of the fuelsprice decreased in early 1985. Recently this has become more evident due to the concern on thegreenhouse gas emissions to the atmosphere. Plant biomass assimilate and store atmospheric CO2through their photosynthetic activity. Therefore, when biomass is degraded in the AD process therecovered biogas may be burnt without the occurrence of any additional CO2 emission into theatmosphere. In contrast, fossil fuels combustion increases the overall level of CO2 since it has beensequestered in the earth since many millions of years.
The biogas, apart from being used for heat and electricity production and as a vehicle fuel, canalso be distributed on the natural gas grid [13]. Biogas has to be upgraded at least to 96-97% CH4content for the use as a motor vehicle fuel or before being injected into the natural gas grid. Biogashas a lower calorific value than natural gas and in specific applications such as automotive fuel [9].Table 2.1 shows the upper and lower caloric value of biogas in comparison to natural gas.
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CHAPTER 2. BACKGROUND 14
Table 2.1: Calorific value of biogas and natural gas (modified) [9].Gas composition Biogas 65% CH4 Biogas 55% CH4 Natural gas
Upper calorific value KWh/m3 STPa 7.1 6.0 12.0
Lower calorific value KWh/m3 STPa 6.5 5.5 10.8
aSTP (standard temperature and pressure), i.e. the volume at 0 oC and 1 bar pressure.
Biogas has several advantages from an environmental and resource efficiency perspective comparedto other biomass based vehicle fuels, which have so far been introduced [7]. Indirect environmentalbenefits occur, e.g. anaerobic digestion of crop residues and manure reduces the plant nutrientleaching from arable land, the use of digestates as a fertilizer reduces the need for chemical fertilizersleading to a more sustainable use of phosphorus and lower production of energy-demanding nitrogenfertilizers [13].
In the case of algal biomass, however, indirect environmental benefits are added to those of energyrecovery. During the degradation of algal bodies on the beaches, considerable methane emissions tothe atmosphere may take place. Furthermore, leaching of toxic red effluents into the water, decreasesthe quality of beaches which in turn affects the ecosystem and the use of beaches for recreation. Largescale harvest will balance N and P levels of the water body. Additionally, N, K and P in digestatesafter the anaerobic digestion of marine algae would become a source of nutrients for arable land.
The production and use of biogas is increasing in Sweden and now exceeds the use of natural gas asvehicle fuel [14]. In January 2007, the European Commission adopted new guidelines for an ambitiousenergy policy for Europe with a binding target of increasing the level of renewable energy in the EUfrom the current level of <7% to 20% by 2020 [7]. The biogas potential in Sweden is estimated to besome 50 PJ/year, which is ten times higher than the current production and corresponds to 3–4% ofthe current energy consumption in Sweden [15].
It is noteworthy that Sweden is the world leader in biogas system utilization. In Sweden, the presentattention to the alternative energy sources, particularly biogas production, is highly appreciable.Nevertheless, the overall scenario represents the need for improvement of biogas systems, implemen-tation of new plants and need for further incentives to reach profitability.
2.2 Anaerobic digestion
Anaerobic digestion (AD) is a naturally occurring biochemical process, where organic matter isdegraded to CO2 and CH4 by subsequent oxidations and reductions in absence of oxygen. Thisprocess occurs in the environment (e.g. sediments, wetlands, swamps, paddy field etc.), in intestinaltracts of higher animals and insects, in landfills and is applied in anoxic bioreactors [21, 20, 22].
A main feature of AD is its high degree of organic matter reduction capability in comparison withaerobic degradation. In addition, energy conversion during the digestion process in form of CH4makes the process economically profitable. Currently, the AD process is mainly utilized in foursectors of waste treatment [9]:
1. Primary and secondary sludge produced during aerobic treatment of municipal sewage.
2. Industrial waste-water produced from biomass, food procession or fermentation industries.
3. Livestock waste to recover energy and improve manure qualities for agricultural purposes.
CHAPTER 2. BACKGROUND 15
4. The organic fraction of municipal solid waste (OFMSW)
In this study, it is hypothesized that the AD process will become a technology to produce greenenergy carrier and, to dewater marine toxic algae of the Baltic Sea which accumulate on the sea shore.Another reason for utilizing biomass to generate energy is that the solid remainder from anaerobicdegradation can be used as organic fertilizer [17]. Consequently, the digestates are expected to befree from heavy metals and toxic substances for it’s further use as a nutrient source for arable land.Previous studies have shown that the marine algae consist of polysaccharides (alginate, laminaranand mannitol), with zero lignin and low cellulose content, which should make them an easy materialto convert into methane by anaerobic digestion processes [16].
2.2.1 Microbiology of anaerobic digestion
The general microbiology of the AD process is well known, while there is a lack in our knowledgeconcerning the difference microorganisms involved. It has been reported that only a few percent ofbacteria and archaea have so far been isolated, and almost nothing is known about the dynamics andinteractions between these and other microorganisms [22]. However, the anaerobic microbiologicaldecomposition in AD process is a process in which micro-organisms derive energy and grow bymetabolizing organic material in an oxygen-free environment resulting in the production of methane(CH4) [23]. The process can be subdivided into the following four phases and each phase requiredits own characteristic group of micro-organisms [Figure 2.1].
Figure 2.1: A schematic figure [modified] of anaerobic digestion of organic material. [23, 24]
CHAPTER 2. BACKGROUND 16
2.2.1.1 Hydrolysis
Hydrolysis is the first phase of anaerobic digestion process where hydrolytic and fermentative micro-organisms are responsible for the conversion of non-soluble biopolymers to soluble organic com-pounds [17, 23]. This part of the process may occur without methanogenesis. The hydrolytic andfermentative bacterial groups ideally breaks down biopolymers (C100 -C10,000) into soluble organiccompound such as mono- and oligomers (C10-C100) e.g., sugars, amino acids, long chain fatty acidsetc. In case of more complex biopolymers, pretreatments of organic materials are often needed toaccelerate this phase. The polymers are unavailable for intracellular metabolism because of theirsize and morphology and are, thus, degraded by extracellular enzymes such as lipases, cellulases,amylases or proteases [25].
2.2.1.2 Acidogenesis
In this phase, acidogenic bacteria convert soluble organic compound such as mono- and oligomers(C10-C100, e.g., sugars, amino acids, long chain fatty acids etc.) occurs, to fermentation products,i.e. fatty acids, alcohols, H2 and CO2. However, hydrolytic, fermentative and acidogenic activitymay be performed by the same bacterium [17].
2.2.1.3 Acetogenesis
The third phase of AD process is the acetogenesis where fermentation products i.e. mainly fattyacids and alcohols are converted into acetate, CO2 and H2 by acetogenic bacteria [23]. This bacterialgroup is termed as acetogens and are obligate hydrogen producer. Later, those products are used bymethanogens to produce CH4.
H2 concentration is an important factor regulating the metabolic activities in both methanogenesisand acetogenesis. Biogas formation from the fermentation products is thermodynamically possibleonly when the hydrogen concentration is below a threshold concentration; thus, H2 is barely de-tectable in the biogas formed. At the same time, the biological activity of methanogens requires acontinuous supply of hydrogen to carry out the redox reaction. The relationship between the ace-togens and methanogens is syntrophic, supported by a process called interspecies hydrogen transferor interspecies electron flow. Additionally, low acetate level (usually 10-4 and 10-1 M) is required foracetogens to convert products into acetate [32, and references therein].
2.2.1.4 Methanogenesis
Methanogenesis is the final step in anaerobic decomposition of biomass in AD. This is, however, thesensitive part of the process, where microorganisms show most sensitivity with the system’s chemicaland physical environment. The two major pathways of methanogenesis are known as acetotrophic andhydrogenotrophic. Some 60-70% CH4 is produced via the acetotrophic pathway [24]. Methanogenscan use a limited number of substrates of which H2/CO2, formate and acetate are the most common,why methanol, ethanol, isopropanol, methylated amines, methylated sulfur compounds and pyru-vates can also be used under specific conditions [24, and references therein]. Thus, there are threemetabolic pathways: acetotrophic (acetate metabolized), hydrogenotrophic (H2/CO2 metabolized)and methylotrophic (methylated one-carbon compound metabolized). Often the hydrogenotrophicmethanogens are able to use formate, while those using acetate for methane formation can only useacetate. However, Methanosarcina is metabolically and physiologically most versatile which possessall three known pathways for methanogenesis.
When acetate-utilizing methanogens are inhibited by e.g., ammonia, sulfides etc., bacteria will oxidizeacetate to H2 and CO2 which is then the source of methane [33]. Thus, there is a syntrophic
CHAPTER 2. BACKGROUND 17
relationship between acetogens and methanogens and an interspecies hydrogen transfer or interspecieselectron flow takes place [32, and references therein].
However, in methanogenesis, mainly acetate is converted to CH4 and CO2 as the end products inanaerobic degradation of organic matter. Some other or even same methanogens use CO2 and H2for their metabolic activities and convert them to CH4 and H2O :
CH3COOH → CH4 + CO2
CO2 + H2 → CH4 + H2O
However, the performance of any certain methane-forming species is regulated by several factorssuch as accumulation of VFAs, hydrogen pressure, buffering capacity, bicarbonate concentration inliquid phase and CO2 concentration in the gas phase, pH, ammonia concentrations and other toxicsubstances, nutrients availability, and other environmental factors, such as temperature, light etc [22,and references therein]. Important factors are discussed in the following section 2.2.2.
2.2.2 Factors influencing methanogenesis
Several studies show that the AD process is affected by many factors. The most important factorsare temperature, pH, substrate composition and toxins [9]. Variation in those parameters affects theprocess resulting in the e.g. accumulation of VFAs and low biogas production.
2.2.2.1 pH and temperature
Each of the microbial groups involved in anaerobic degradation has a specific pH optimum and cangrow in a specific pH range [9]. For methanogenic and acetogenic organisms, an optimum pH range isbetween 6.5 and 7.5, while acidogenesis and hydrolysis has their optima around 6. However, the pHof an anaerobic digester and its optimization largely depends on the characteristics of the substratesused. It is noteworthy that, the optimum ranges given for anaerobic digestion processes in differentstudies may be contradictory. The favorable conditions for microbial growth of a process and itsstability are often related to the characteristics of the inoculum used. This means, if the microbesgrow well at pH 8 during the start-up then the process is more likely stable at a pH of 8.
Three types of AD process conditions are defined as related to temperature:- psychrophilic (10 -20oC), mesophilic (20 - 40oC) and thermophilic (50 - 60oC). Temperature directly influences thebacterial growth and conversion rate of organic materials. Most anaerobic reactors are carried outwithin the mesophilic and thermophilic ranges [9]. The microbial growth within the psychrophilictemperature range is slow resulting in long retention time. Although the reactor operational energyinput is high for biodegradation of organic waste under thermophilic conditions, there are several ad-vantages: i.e.: fast digestion rate, short retention time, high volumes waste treated in comparativelysmall digester volumes, high hydrolysis rate of particular matter, efficient destruction of pathogensetc.
Both pH and temperature factors influence methanogenesis heavily. In addition, toxic compoundsconcentration (e.g., ammonia, sulfide) are also influenced by pH and temperature. For instance, inhigh temperature and higher pH, ammonia concentration is higher and more toxic to the methanogensthan optimum conditions.
CHAPTER 2. BACKGROUND 18
2.2.2.2 Bicarbonate alkalinity
The pH of a reactor system is primarily controlled by the bicarbonate concentration. CO2 producedduring the biological conversion of biopolymers reacts with ammonia released from the degradation ofnitrogen-containing organic matter and, thus, produce bicarbonate alkalinity. In this case ammoniaacts as a strong base to react with the CO2. It is a better idea to control a system’s pH by measuringbicarbonate alkalinity instead of measuring only pH. When bicarbonate concentration is low, pH maydecrease quickly because of the low buffering capacity. The buffering mechanism is shown below:
CO2-3 + H+ ⇌ HCO-
3(carbonate to bicarbonate)
HCO-3 + H+ ⇌ H2CO3 (bicarbonate to carbonic acid)
OH- + H+ ⇌ H2O (hydroxide to water)
However, alkalinity regulates the presence/availability of acidity (H+). This becomes, when thealkalinity of a system is reduced and the buffering capacity of the solution gets weaker.
2.2.2.3 Accumulation of VFAs
Volatile fatty acids (major VFAs are acetate, propionate, butyrate, isobutyrate, valerate and iso-valerate) accumulation in a reactor could lead to a decrease of pH, methane production and eventemporary or complete cessation of methane formation. However, VFAs are important substratesthat are readily used by methanogenic microorganisms [29, 30]though high VFAs accumulation affectsthe methanogens in the anaerobic process [17, and references therein]. If the VFAs accumulation ex-ceeds the utilization capacity by methanogens, excess VFAs, which are not uptaken by methanogens,will start to accumulate what will lead to decrease in pH.
Since methanogenic activities are low at low pH, acetate and H2 utilization by methanogens willdecrease. This may result in further accumulation of VFAs and decrease of pH. Accumulation ofhigher molecular weight VFAs may lead to complete cessation of methanogenic activities. Usuallyfeeding is reduced or suspended, when the VFAs accumulation occurs in order to abate the effect.
2.2.2.4 Toxicity
Inhibition of specific microorganisms by e.g. toxic compounds inherently included in a substrate orformed during its degradation will lead to instability of the process. Although anaerobic microor-ganisms are less sensitive to toxic compounds than are aerobic microorganisms, the growth rate ofthe anaerobes makes the re-establishment of a microbial community more time consuming. Toxicityof an anaerobic system widely depends on the characteristics of substrate(s) used, their intracel-lular effects. Generally, inhibitory compounds of an AD system include ammonia, sulfide, oxygen.For both ammonia and sulfide, toxic effects are dependent on pH and temperature - the higher thetemperature and pH, the higher the toxicity [17, 31]. The adaptation to an inhibitory compoundis, however, possible over time if the concentration of the toxic compounds can be kept constant.However, likely a suboptimal gas yield will be obtained [17].
CHAPTER 2. BACKGROUND 19
2.2.2.5 Nutrients
Nutrients for anaerobes are grouped as macronutrients and micronutrients. Two macronutrientsessential in all biological treatment process are nitrogen (in form of ammoniacal-nitrogen NH4
+-N) and phosphorus (in form of orthophosphate-phosphorus HPO4
--P) [68]. Methanogens possessseveral unique enzyme systems leading to the need for some micronutrients, e.g. cobalt, iron, nickeland sulfide. Out of these micronutrients, methanogens need some other obligatory micronutrientssuch as selenium, tungsten, molybdenum as additional trace elements to complete the metabolism.Additional micronutrients of concern are calcium, magnesium, barium and sodium. The shortage ofmacro- and micronutrients may lead to suboptimal growth of microbes in anaerobic digestion.
2.2.3 Codigestion concept
The hypothesis behind codigestion is that addition of biosolids will improve the biodegradation ofalgae and enhance the biogas production. The suggested optimum C/N ratio for anaerobic digestionis in the range of 20:1 to 30:1 [34, 56, and references therein]. The unbalanced nutrient compositionof the algal sludge (low C/N ratio) was regarded as a limiting factor for its use in an anaerobicdigestion process [56]. However, the C/N ratio was about 5.3/1 in algal sludge reported in previousstudies [56]. The analyses reports show that algae (new and old) has a C/N ratio varying from 13/1and 20/1. Therefore, the addition of primary sludge from a sewage treatment plant may provide thenitrogen as well as other nutrients required for an optimal anaerobic digestion of algal biomass.
2.2.4 Pretreatment
The hydrolysis and fermentation steps of an anaerobic digestion process are often regarded as ratelimiting as a result of the extent at which the substrate is possible to hydrolyze [9]. To enhance theoverall degradability of substrate, different pretreatment techniques are being introduced [69]. Thecore function of different pretreatments is to break down the complex biopolymers, disrupt cell wallsand bring out the chemical substances from polymers. In this study, autoclavation and ultrasoundpretreatments were applied.
2.2.4.1 Pretreatment with autoclave
Steam and heat pretreatments have been applied in several studies to open up cellular structuremaking cell components accessible to hydrolytic enzymes. This is one of the proven mechanisms toaccelerate the AD process.
2.2.4.2 Pretreatment with ultrasound
Numerous studies reveal that ultrasonic pretreatment affects anaerobic digestion [51, 52, 53], and forsome substrates the biogas yields increase substantially. In other cases the effect is an increase of theprocess rate occur as a result of pretreatment with ultrasound. This means that a shorter retentiontime (RT) may be applied [17]. Ultrasound basically acts on particular material by decreasingparticles size, which leads to an increase of the exchange area between liquid phase and particles.In ultrasound pretreatment, extremely intense hydro-mechanical forces accelerate disintegration ofbio-solids [69]. Increased microbial activities take place due to this disintegration resulting in higherbiogas yields and higher organic solid reduction.
CHAPTER 2. BACKGROUND 20
2.3 The Baltic Sea eutrophication
The Baltic Sea is almost enclosed by land with a narrow and shallow straits connected with NorthSea around Denmark and Sweden. More than 200 large rivers characterized by cold temperaturebring fresh water into the Baltic Sea, which makes it the world’s biggest brackish sea. Exchange ofwater with the open sea is very limited. It typically takes about 25-30 years for all the water in theBaltic Sea to be replaced [44]. The Baltic Sea habitats and species are threatened by eutrophicationand elevated amounts of toxic substances from agriculture and industrial waste stream regulated byhuman activities. At present, sixteen million people live in nine countries along the coast of theBaltic Sea. A total of 85 million people live in the 14 countries in its catchment [39].
Eutrophication has become a widespread matter of concern especially in coastal and inland watersduring the last 50 years [6]. Eutrophication can be defined as ‘‘the enrichment of water by nutrients,especially compounds of phosphorus and nitrogen causing an accelerate growth of algae and higherforms of plant life to produce an undesirable disturbance to the balance of organisms present in thewater and to the quality of the water concerned’’ [8]. Numerous papers explained and discussed thecauses, consequences and definitions of eutrophication [35, 36, 37, 38]. Industrialization, intensifyingagricultural production and rapid urbanization increase the rate of eutrophication.
Nitrogen inputs to the Baltic Sea have increased four-fold and phosphorus inputs eight-fold since themid-19th century [40] as a result of over-fertilization with phosphorus (P) and nitrogen (N) [39, andreferences therein]. Discharges of both N and P from sewage treatment plants are also significantcontributors to eutrophication [41]. Interestingly, scientists have demonstrated that eutrophicationis mainly regulated by the P level in the water body. Because, when the P level is high in thewater, cyanobacteria can fix atmospheric N to balance the N:P level suitable for their growth. Ex-tensive nitrogen removal may stimulate nitrogen-fixing Cyanobacteria, if not otherwise limited byphosphorus [39].
External source of nutrient input in the Baltic Sea is heavy nitrogen-fixation. Prokaryotic microor-ganisms, including cyanobacteria contain the necessary genes for nitrogen fixation. In a ecosystem,nitrogen-fixing organisms are extremely important for supplying food (e.g., amino acids - useful ni-trogen) to depending living organisms. But excessive nitrification as result of mass development ofnitrogen fixers may cause severe problem as they supply so much nitrogen that they aggravate localor regional eutrophication. Excessive nitrogen fixers generate undesirable excess of biomass in thewater which exceeds the ecosystem’s ability to assimilate. According to MARE (Marine Researchon Eutrophication) [42], Aphanizomenon, Nodularia and Anabaena are the most nitrogen producinggenera among the nitrogen-fixing cyanobacterial genera in the Baltic Sea. Aphanizomenon sp. (ear-lier often called Aphanizomenon flos-aquae) and Nodularia spumigena are the two most importantspecies of nitrogen-fixing cyanobacteria in the Baltic Sea. Both species are heterocystous, filamentousand colonial. Surface blooms, which are patchy and episodic, are generally dominated by Nodulariabut Aphanizomenon has a larger biomass. Although both genera are toxic, it has not been estab-lished whether Baltic Anabaena strains are toxic. The highest abundance of the genera is seen in thesummer period though they occur the year round in the water. Toxic blue-green algal blooms canrepresent a considerable health risk for people and animals, and people are advised not to swim inbloomy water [43].
The sources of nutrients causing eutrophication in the Baltic Sea are often classified into pointsources (settlements, industrial plants or fish farms), diffuse sources (agriculture, forestry, dispersedsettlements, storm water), or airborne pollution (emitted from traffic or fossil fuels combustion forpower and heat generation) directly deposited into the sea [43].
However, continuous excessive nutrient inputs disrupt the natural balance of the Baltic Sea seriously.As a result, intense algal growth with abnormal algal blooms, adverse effects on communities of faunaand flora, additional undesired organic matter production, increase in oxygen consumption, oxygendepletion resulting in death of benthic organisms (lifeless areas on the seabed) are often reported.The excessive growths of algae, as a result of eutrophication, make the water less transparent. Large
CHAPTER 2. BACKGROUND 21
quantities of algae eventually end up on the seabed where their decomposition uses up oxygen. Thiscan lead to anaerobic condition near the seabed. Moreover, subsequent decay of high plant biomasscauses an increase in oxygen consumption which may lead to anoxic conditions in bottom watersand sediments, since the biological oxygen consumption exceeds the supply of oxygen by diffusionby orders of magnitude [45]. When the uppermost sediments on the seabed become anaerobic inthis way, they release nutrients, particularly phosphorus, back into the water through a phenomenonknown as internal loading [43]. Today roughly one-third of the bottom of the Baltic is practicallydead, and the deepest basins mostly contain hydrogen sulphide instead of oxygen [46].
Consequently, filamentous macroalgae (red, brown and green) are reported abundant in the BalticSea and proliferated as a result of eutrophication. At the end of the summer, filamentous algaeproduce thick, loose mats covering shallow bottoms close to the sea shore. Huge masses of algaeare accumulated on the beaches by winds and currents movements. Malm et al. [3, 4] recordedextended banks at the shores of south eastern Sweden, amounting up to five tonnes of algae per meterbeach. The quality of beaches are declined, affecting tourism and severe environmental degradationis reported.
2.4 Red algae at the Baltic Sea
2.4.1 Toxicity of red algae
Filamentous red macroalgae of the genera Ceramium, Polysiphonia, and Rhodomela make up mostof the algal biomass along the open coasts of the central Baltic proper [3, and references therein].During the aerobic decomposition of the accumulated red algae on the beaches, large amount ofred colored effluents leak and gradually mixed into the water. Several studies revealed that marinemacroalgae produce a number of organohalogen compounds and many of these compounds are similarto toxic commercial products like pesticides. Some of them are claimed to have acute effects on thecentral nervous system and chronic endocrine effects [5], carcinogenic and nerve toxic effects [3, andreferences therein]. The extracts from accumulated filamentous red algae (Polysiphonia, Rhodomelaand Ceramium) increase mortality in crustaceans, fish and fish larvae in the Baltic Sea [47, 4, 3, andreferences therein]. The red macroalgae under the family Rhodomelacea is dominating in the centralBaltic Sea.
2.4.2 Characteristics and productivity
The term algae refer to a large and diverse assembly of eukaryotic organisms that contain chlorophylland can carry out oxygenic photosynthesis [63]. Marine algae consist of polysaccharides (alginate,laminaran and mannitol), with zero lignin and low cellulose content, which make them an easymaterial to convert to methane by anaerobic digestion processes [16]. Typically, algae are unicellularand microscopic, but assembled to multicellular organisms they constitute seaweeds. The mostcommon groups of macroalgae are red algae (Rhodophyta), brown algae (Phaeophyta) and greenalgae (Cholorophyta). Polysiphonia, a genus of red algae under the division Rhodophyta, with morethan 150 species, is one of the most dominated red algae at the south-east Sweden (Öland Island [4])Baltic Sea shore. This study was limited to this genus of red algae as it is most abundant andheavily overgrown on the sea shore. Filamentous and typically well branched with a length up to30 cm and polysiphonous construction are basic characteristics of Polysiphonia. The life cycle oftriphasic (three phases) red algae Polysiphonia, consists of a sequence of a gametangial (gametesproducing), carposporangial (carpospores producing) and tetrasporangial (tetraspores are producedfrom meiosis) phases.
CHAPTER 2. BACKGROUND 22
(a) Old red algae (OA) (b) New red algae (NA)
Figure 2.2: Collected Polysiphonia red algae for this study.
However, the algae used for the study were not 100% Polysiphonia. It appeared to be mixed withother seaweeds, but less than at 10%. Newly produced new algae (NA) appeared to be darker andwith a shorter thallus than older sample (OA).
Chapter 3
Materials and methods
3.1 Experimental overview
The anaerobic digestion of collected red algal biomass Polysiphonia were carried out in three separatebatch experiments in 330 mL glass bottles [see 3.1.1] with a working volume of 100 mL and smallstirred reactors [see 3.1.2] with an active volume of 4 L and once a day feeding. Batch experimentsand reactor experiments were carried out during the period of November 2007 to July 2008 and Aprilto July 2008, respectively. All digestion experiments were performed at mesophilic conditions (37°C)as previous studies showed maximum methane yield and production rate at this temperature [50, 51].The details on individual experiments are described below.
3.1.1 Digestion experiments (batch)
Methane potential of organic matter is measured by batch methods. The basic approach is toincubate a small amount of material to be treated with an anaerobic inoculum and measure themethane generation, usually by simultaneous measurement of gas volume and gas composition [48].The study on biogas potential of algal biomass has followed this general procedure by Shelton andTiedje [49] with some modifications. Three individual series of experiments were carried out todetermine the methane potential of the substrates at different conditions.
^ 1st batch experiment (Expt.-1) aimed at a determination of methane potential of old algae(OA) and the effect of heat and stream (autoclave) pretreatment (121 °C for 30 minutes).
^ 2nd batch experiment (Expt.-2) addressed the methane potential of algae collected at twodifferent seasons, NA and OA.
^ 3rd batch experiment (Expt.-3) was set up to determine the effect of ultrasonic pretreatment(ultrasound) of NA on biogas and methane yields.
The test materials were weighted to measure the TS and VS [Section 3.4.1 and 3.4.2] and approxi-mately 2.5 g VS/L of algae organic matter was transferred into experimental serum bottles, whereinoculum, solutions and water were mixed with the substrate at oxygen free condition to produce avolume of 100 mL. The serum bottles were then sealed with EPDM (ethylene propylene diene M-class) stoppers and capped with aluminum screw caps. The experimental bottles were then placed ina climate room for the incubation at 37°C. Gas pressure in serum bottles was measured to calculatethe biogas production. Gas samples were collected each time of pressure measurements to determine
23
CHAPTER 3. MATERIALS AND METHODS 24
the methane concentration by gas chromatography (GC) [Section 3.4.6]. Pressure measurement andanalysis of methane concentration were performed twice a week for the two first weeks, then once aweek and finally less frequent. Gas production and methane concentration was followed over 35, 51and 61 days for Expt.-1, Expt.-2 and Expt.-3, respectively.
3.1.2 Digestion experiment (STR)
The aim of this part of the study was to evaluate biogas yields, process stability and codigestibility ofalgae and primary sludge in laboratory scale stirred tank reactors (STR). The effects of an increasingorganic loading rate as well as of pretreatments of substrates were also applied to monitor reactorperformance. In this experiment, Polysiphonia algal biomass was codigested with primary sludgecollected from Himmerfjärdsverke water treatment plant, which is the fourth largest wastewatertreatment plant in Sweden. This digestion experiment was carried with an intermittent steering,i.e. 4 times a day stirring for 15 minutes each via at 400 rmp automatically operated by electronicregulators. Feeding occurred once-a-day. Two digesters were operated with the same working volumeof 4 L [Section 3.3.2.1] under mesophilic conditions (37±2°C) in the dark.
The two digesters were labeled as D9 and D10, D9 was operated as control reactor; i.e., no significantchanges in digester operation conditions occurred. D10 was the test reactor, receiving increasedorganic load and pretreated substrate [Table 3.1].
Table 3.1: Experimental design (STR) and digester operating conditions for the study of the anaer-obic codigestion of Polysiphonia algal biomass with primary sludge [PS].
Parameters Duration D9 D10
Digester active volume Over the perioda 4L 4LHRT Over the period 20 days 20 daysMixing Level Over the period Intermittentb IntermittentDigestion condition Over the period Mesophilic (37±2 °C) Mesophilic (37±2 °C)Design OLRc Day 01-20 1.5 g Algae + 0.5 g PS 1.5 g Algae + 0.5 g PS
Day 20-24 1.5 g Algae + 0.5 g PS 2.0 g Algae + 0.5 g PSDay 25-33 1.5 g Algae + 0.5 g PS 2.5 g Algae + 0.5 g PSDay 34-104 1.5 g Algae + 0.5 g PS 3.0 g Algae + 0.5 g PSDay 105-120 1.5 g Algae + 0.5 g PS 3.0 g Algae (pretreated)d
+ 0.5 g PS
aFrom April 3 to July 28, 2008 (117 days).bMinimal and intermittent, designed stirring speed -400 rpm. 4 times a day, 15 minutes/time.cOrganic Loading Rate, units g VS/L active volume/day.dThe study was initially designed to digest algae pretreated with ultrasound. But lately autoclave pretreatment was
performed.
The reactors were fed seven days a week, mainly between 10.00-13.00, while withdrawal of sludgewas performed five days a week (Monday-Friday). The liquid volume in the digesters was adjustedto 4L on every Monday. The following measurements were performed:
Biogas yields: The biogas production was determined daily by gas meters [Figure - 3.2] equippedwith a digital volume indicator.
pH: pH of the reactor materials was determined at least twice a week and whenever necessary.
VFAs accumulation: The concentration of VFAs of the reactor material was measured twice aweek or more frequently.
CHAPTER 3. MATERIALS AND METHODS 25
Gas composition: Produced biogas from both reactors were captured in plastic balloons once aweek to determine the CH4, CO2, O2 and H2S concentrations using a portable gas analyzer[Section 3.4.6].
VS reduction: TS and VS of the digesters’ slurry were measured once a week to determine the VSreduction [Section A].
Digestate nutrients: The final digestate from the reactors was also analyzed at the termination ofthe experiment to evaluate the nutrients and heavy metals concentration. These analyses weredone by Analycen, Lidköping, Sweden.
3.2 Preparation of substrates
3.2.1 Collection of Polysiphonia
The seaweeds used for this study were collected by personal from SLU research station at the Islandof Öland in Sweden two times, early autumn (October 2007) and early spring (March 2008). Algaewere packed in several layers in plastic air tight bags in a thick paper box and delivered withing oneday to our laboratory, where it was stored . Once received, the collected seaweeds were kept in therefrigerator at -20 °C temperature until use to avoid rotting.
3.2.2 Sample preparation for the experiments
The collected sea weeds (red algae) were up-to 30 cm in size and in order to increase the biodegrad-ability, it was necessary to make a homogenized substrate for anaerobic digestion. The algae werecut into small pieces with scissor and after that the small pieces [2-5 cm] of algal body were minced,with a meat mincer (manufactured by Braun). The minced and homogenized seaweed was then keptin refrigerator at -20 °C in polythene bags for further use. Before using algae as feeding, TS and VSwere measured for every algae bag thawed.
3.2.3 Feedstock for reactors
3.2.3.1 Old algae (OA)
Old algae (OA) refer to the algae collected in October 2007. Filamentous and well branched with ahigher length than that of New algae, greenish - brown color were main visual characteristics of Oldalgae. The organic matter content of these algae was lower than new algae (NA). However, the sandcontent seemed lower in OA than that of NA as experienced during homogenization of the biomass.The characteristics of algae are given in table 4.5.
3.2.3.2 New algae (NA)
Algal biomass collected in March 2008 was termed as new algae (NA). The collection was performedat same place on the Island of Öland and by the same person. Filamentous and branched but thenshorter the OA. Their color was dark reddish with comparatively higher sand content as experienceduring preparation of samples. A comprehensive characteristic is given in the table 4.5.
CHAPTER 3. MATERIALS AND METHODS 26
3.2.3.3 Primary sludge
The primary sludge was collected twice a month from Himmerfjärdsverket in southern Sweden andstored at 4-7°C for maximum one week before use. When received, the primary sludge container wasshaken by hand and then the sludge was homogenized with a hand mixer (manufactured by Braun)and stored. TS and VS of newly collected sludge were determined ofter the mixer and before use, ifstored from more than one week. TS and VS values of received primary sludge varied between 3.6%to 4.9% and 74.7% to 81.6% respectively.
3.2.4 Pretreatment of samples
3.2.4.1 Heat and steam
To determine the effects of autoclavation, a series of experiments were carried out with the substratepretreated in autoclave at 121 °C for 30 minutes. About 100g homogenized OA was transferred to aserum bottle with an average volume of 330 mL and placed in the autoclave. The pretreatment wascarried out just before using the substrate for the batch and reactor experiments.
3.2.4.2 Ultrasound pretreatment
Ultrasound pretreatment was applied on homogenized NA to study the effects on biogas yields andVS reduction rates in the batch Expt.-3. Upon a positive result the ultrasound pretreatment wouldbe introduced for STR digestion.
A custom-made sonicator was provided by Scandinavian Biogas Fuels AB. This is a single transducerultrasonic laboratory reactor, where sludge flow is controlled by a screw pump with variable speed.An oscilloscope is used to measure the current and the voltage of the ultrasound load (electrical outputpower). A combination of energy input and pumping speed gives the actual treatment energy.
Treatment energy (Wh/L) =Ultrasonic input load (W )Sludge flow (L/h)
200g of homogenized NA with 100 mL tap water (to make the substrate pumpable) was ultrasonicatedat 19 kHz with a treatment energy ranging 1-5 Wh/L.
3.3 Experimental setup
3.3.1 Batch experiments
The batch experiments were designed as given in the table 3.2. Each included three replicates andfollowing controls:1. Positive controls with Whatman paper (Whatman filter paper, 18.5 cm, 3 qualitative, Kebo LabAB),2. Inoculum only3. External methane standards were also introduced for experimental validation.
The method is a biological test method using inoculum from full-scale biogas plants (varying quality)as described by Hansen T. L., et. al. [48].
CHAPTER 3. MATERIALS AND METHODS 27
Table3.2:
Expe
rimentald
esignfortheba
tchdigestionexpe
riments.
Par
amet
erE
xpt.
-1E
xpt.
-2E
xpt.
-3
Exp
erim
enta
lpe
riod
Nov
’07-
Jan
’08
Apr
-Jun
’08
May
-Jul
’08
Dur
atio
nof
incu
bati
on35
days
51da
ys61
days
Tot
alnu
mbe
rof
take
nm
easu
rem
ents
54
8
TE
STSU
BST
RA
TE→
OA
Aut
ocla
ved
OA
Wha
tman
Pap
erO
AN
AW
hatm
anP
aper
NA
Ult
raso
nica
ted
NA
Wha
tman
Pap
er
Pre
trea
tmen
t→
Non
eA
utoc
lave
aN
one
Non
eN
one
Non
eN
one
Ult
raso
nicb
Non
eO
LR
(gV
SL
-1)
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
TS
(%)
12.4
812
.65
98.6
116
.41
14.6
996
.91
10.0
9.9
98.7
VSc
(%T
S)60
.89
59.9
399
.46
58.0
168
.39
99.6
075
.274
.299
.5L
iqui
dvo
lum
ein
seru
mbo
ttle
s(m
L)
100
100
100
100
100
100
100
100
100
Subs
trat
edi
gest
ed(g
)3.
253.
350.
252.
562.
650.
253.
003.
000.
25
Not
e:T
hree
repl
icat
ions
wer
epe
rfor
med
for
each
subs
trat
esin
each
and
ever
yba
tch
expe
rim
ents
inor
der
tom
inim
ize
the
expe
rim
enta
ler
rors
[i.e.
-3
incu
bati
onbo
ttle
sfo
rO
Ain
Exp
t.-1
].A
vera
geT
San
dV
Sva
lues
are
give
nhe
rein
the
tabl
e3.
2.
a At
121
°Cfo
r30
min
utes
b Wit
hul
tras
onic
equi
pmen
tat
19kH
zan
den
ergy
inpu
t1-
5W
h/L
.c M
easu
red
duri
ngpr
epar
atio
nof
bott
les
CHAPTER 3. MATERIALS AND METHODS 28
3.3.1.1 Characteristics of medium
An anaerobic medium was used in the digestion experiments in order to maintain osmotic pressure,reducing conditions and pH stable and suitable for microbial activity in high rate. Each serum bottle(except 3 bottles of external methane standards) was filled with the following medium in appropriatetime during the bottle preparation-
W3 Saline solution (MgCl2.6H2O, NH4Cl, NaCl, CaCl2.2H2O) i.e. a mineral nutrient solution, 2mL in each vial for ionic strength and nutrient supply.
W7 (100 mM) Sulphide solution (Na2S.9H2O) in order to create the reducing environment needed,0.3 mL in each vial.
3.3.1.2 Inoculum
Inoculum was taken from a laboratory reactor, run by Scandinavian Biogas Fuels AB, fed by sewagesludge and paper-mill residues and stored with an anaerobic headspace until use. 20 mL was trans-ferred into the experimental bottles (except 3 bottles of external methane standards). Preparationof the experimental bottles in every series were completed within 6 hours.
3.3.1.3 Preparation of experimental bottles
Serum bottles, with an average volume of 332 mL were used. After cleaning, bottles were kept inroom temperature until the remaining water disappear from bottles. In every series of experimentsthe experimental bottles were prepared following the steps below-
1. 3L of Milli-Q water was boiled for 20 minutes in a glass pot and then kept in ice water forcooling under a continuous flushing with N2 to maintain the oxygen free condition.
2. The empty serum bottles were flushed with N2 continuously, while transfers of substrate, in-oculum solutions (see above).
3. While till flushing with N2, the required amount of boiled MilliQ water was transferred intothe bottles to adjust the amount to 100 mL. Then the bottles were immediately sealed withthe stoppers and aluminum crimps.
4. The N2 gas phase was exchanged for N2/CO2 (80%/20%) by nine evacuations and fillings ofthe bottles [Fig-3.1].
CHAPTER 3. MATERIALS AND METHODS 29
(a) Substrates were inserted in bottles (b) Evacuation and filling with nitrogen and carbondioxide.
Figure 3.1: Preparation of experimental bottles for incubation.
5. Over pressure inside the bottles was released by inserting needle. However, in the Expt.-1and Expt.-2, the overpressure in the external methane standard bottles was not released afterflushing to keep the over pressure inside the vials.
6. 0.3 mL of W7 was injected in all bottles except those aimed for external methane standards.
3.3.1.4 Positive control preparation
Whatman paper (2.5 g VS/L; Whatman filter paper, 18.5 cm, 3 qualitative, Kebo Lab AB), i.e.paper made of 100% cellulose, was used as positive control in experiment. Three replications of thissubstrate were prepared in the same manner as the other substrates. As the theoretical and practicalgas production of Whatman paper is known, it is suitable for a validation and performance of theexperiments. The theoretical methane production from completely digested cellulose [(C6H10O5)n]is calculated as follows: the molecular weight of C6H10O5 is 162 g/mol, thus, 1 g of carbohydratescorresponds to 1/162 mol = 0.006173 mol. If C6H10O5 is assumed to be completely oxidized to CO2and all electrons ending up in H2, then:
C6H10O5 + 7H2O → 6CO2 + 12H2
12H2 + 3CO2 → 3CH4 + 6H2O
The combination of the two equations above gives:
C6H10O5 + H2O → 3CH4 + 3CO2
Thus, 3 mol. CH4 is formed from 1 mol C6H10O5, why the 0.006 mol C6H10O5 gives 0.019 mol CH4in a complete digestion. From the gas law, the corresponding volume of CH4 is calculated:
PV = nRT
Where,P = Pressure in Pascal (Pa) and normal atmospheric pressure 101325 Pa.V = Volume in m3
n = Number of molesR = Gas constant (8.314)T = Temperature in K (310°K is equivalent to 37°C)
CHAPTER 3. MATERIALS AND METHODS 30
So, using the equation, from 1 g C6H10O5 , the maximum methane yield could be-
^ 471 mL CH4 at 37°C/310°K
^ 414 mL at 0°C/273°K.
3.3.1.5 External methane standard
Another way to validate the gas and methane measurements of the experimental bottles is by deter-mining the methane loss from methane standard bottles. Besides the experimental bottles, a seriesof methane bottles was incubated in same manner with the test bottles. Inoculum was not addedin this series of bottles. With the 100 mL of boiled MilliQ water, 10 mL of pure CH4(99.99%) wasinjected into the external methane bottles at the final stage of bottle preparation. A 10 mL syringe(BD Plastipak, Sweden) with a needle of 0.4*25 mm (Sterican 27 G * 1½, B.Braun Melsungen AG)was used to collect methane from cylinder. The overpressure after filling with N2/CO2 (80%/20%)and 10 mL CH4 of these three bottles was kept unreleased for Expt.-1 and Expt.-2. It was difficultto maintain high pressure inside the bottles during the pressure measurement. In the Expt.-3, thepressure was released like other experimental bottles, but in that case 20 mL of CH4was injected ineach bottles prior incubation to have a good CH4 concentration inside the vials.
3.3.1.6 Experimental modification
The set up for the third batch experiment addressing the effect of ultrasonic pretreatment of Polysi-phonia, on biogas and methane yields was modified compared to the previous ones [Table 4.1]:incubation period was 61 days; eight pressure measurements and methane analyses were performed;low pressure inside the serum bottles, gas releasing from the bottles was postponed until next mea-surement. Moreover, the homogenized NA were diluted, with 100 Milli-Q water/200g algae, to makesubstrate pumpable through the tube of sonicator. The actual OLR was 2.2 g VS/L.
3.3.2 Reactor experiment
3.3.2.1 Digester setup
Two laboratory-scale anaerobic digesters (Scott-Duran glass of 5L, Germany), with a working volumeof 4L in this study. They were equipped with two openings of each: one for feeding and withdrawalof sludge, one for a central stirrer in a rubber stopper, which had a tubing for gas outlet [Figure 3.2].
(a) Digesters with stirrers. (b) Digesters, placed in incubationroom.
(c) Gas meters, placed in incubationroom.
Figure 3.2: The laboratory-scale anaerobic digesters used for this study, equipped with steeringequipments and gas meters.
CHAPTER 3. MATERIALS AND METHODS 31
Stirrer was a 3 bladed propeller [Figure 3.2] powered by Switchmode Power Supply PSU24-075, type-MACOO-R1, manufactured by JVL, Denmark with 24 VDC power supply. The propeller was set 5.5cm up from the bottom of the reactor, which indicates one third of the liquid volume. The motorswere run initially at 400 rpm. The speed was increased up to 600 rpm in the reactor D10 after 30days of operation, to avoid a foam layer.
The gas meters used [Figure 3.2], originated from TuTech Hamburg-Harburg Technical University(Germany). The gas meters were calibrated for ca twelve hours for validation of their accuracy.Usually, the gas meters were replaced with newly calibrated ones once a week. Readings from thedigital display of the gas meters were taken regularly on a daily basis and reset to zero after feeding.
3.3.2.2 Inoculum
The inoculum was collected in two plastic containers from digester 3 at the sewage treatment plant inLinköping and directly transported to the laboratorium and stored in the incubation room for aboutone hour prior to the inoculation reactors. After inoculation, the reactors placed in the incubationroom at 37°C.
3.3.2.3 Feed portion
The feed for all the reactors were a mixture of homogenized algae (Section 3.2) and primary sludgeaccording to designed OLR. Since the designed HRT was 20 days, and total active volume (V) of thereactors were 4L, the volume of exchanged sludge (R) per day was calculated following the equationbelow-
R = VHRT = 4000mL
20Days = 200mL/Day.
The volume of exchanged sludge represents the volume of feed for the reactors. The same amount ofsludge was withdrawn from reactors to keep the active volume constant at 4.0L. The designed g VSalgae and primary sludge was supplied with water up to 200g. Usually feed was prepared for fourdays at a time and kept in the refrigerator.
3.3.2.4 Start-up
The reactors units were set up and placed in the incubation room. Both reactors were inoculated atthe same time on April 02, 2008. First feeding was introduced on the following day (day 1) as givenin the table 3.1. Since the start-up period is sensitive to microbial adaptation in the reactors, pHand VFAs were determined more often during the start-up period. However, the pH value 7.4 forboth reactors D9 and D10 and no VFAs found in the inoculum during the first retention period. Thestirring equipments were installed on day 4. Until then, manual mixing was performed by shakingthe digesters for about two minutes by hand.
To evaluate the performance and role of the inoculum during the start-up, the digesters were operatedat the same. Both digesters were fed with similar feed portions, mixture of 1.5 g VS of OA and 0.5g VS primary sludge (PS) .
After 17 days of reactors operation, the feeding was changed to NA. OLR was increased as designedfrom the Day 18. A thin foam layer was observed in the digesters on Day 21, why the stirrer speedwas increased to 400 rpm at the same stirring schedule as before. After 2 days, the layer was dissolvedin the reactor D9, but in D10, the thin foam layer remained and increased later on.
CHAPTER 3. MATERIALS AND METHODS 32
On Day 35, the OLR was increased further to 3.0 g VS , while the primary sludge loading wasunchanged. In the period Day 35-54, the test reactor D10 showed slight deterioration (foamingoccurred and VFAs accumulated), while control reactor D9 was stable. In order to recover stableconditions of D10, the stirring speed was increased to 500 rpm on Day 42. The aim of this modificationwas to break down the thick layer and dissolve it in the sludge. On Day 54, the reactor D10experienced accumulation of VFAs: i.e. acetate, propionate, isobutyrate and isovalerate respectively.No VFAs were found in reactor D9 during that period.
3.4 Analytical procedures
3.4.1 Determination of total solids (TS)
Total solid (TS) is the dry fraction of a substrate is the organic and inorganic matter/fixed solidssuch as minerals. The analysis includes sample homogenization achieve representative subsamplesand followed the Swedish Standard SS-EN 12880:2000 [54]. Sample is dried to constant mass in anoven at (105±5) °C for 20 hours. The difference in mass before and after the drying process is usedto calculate the dry residue and the water content. Oven dried (105 °C) crucibles were used andplaced in desiccator with an active drying agent silica gel. To determine TS of a substrate, 0.5-5.0gwas taken depending on the type of substrate, so that the dry matter obtained has a minimummass 0.5g, in each crucibles. Analytical balance has been used with an accuracy of 1 mg. Theoven was thermostatically controlled with forced air ventilation and capable of maintaining the settemperature.
3.4.2 Determination of volatile solids (VS)
The organic fraction of a substrate is given as Volatile Solids (VS). The sampling and the analyticalprocedure followed in the European Standard EN 12879:2000 [55]. The principle was: ‘Samples ofdried substrates are heated in a furnace at (550±25)°C. The difference in mass before and after theignition is used to calculate the loss of ignition’. The dry samples (after 105°C) in the crucibles wereignited at (550±25)°C for two hours in muffle furnace.
3.4.3 pH measurement
The pH meter, used for this study, was manufactured by Christan Berner AB, model WTW InolabpH 730. pH values of the digested sludge of the reactors were measured twice a week. The pH meterwas calibrated once a week and buffer solution was changed to obtain valid results.
3.4.4 Determination of VFAs
Accumulation of volatile fatty acids was determined by GC-FID manufactured by Hewlett Packard,model HP 5880A. VFAs (acetic, propionic, butyric, isobutyric, valeric, isovaleric). N2was used as acarrier gas. Oven temperature initial, final and post value were set to 80, 175 and 200 respectively.
CHAPTER 3. MATERIALS AND METHODS 33
3.4.5 Pressure measurement in batch experiments
The pressure inside the bottles was measured in the climate room at a temperature of 37°C by use ofa 5 mL glass syringe with a needle of 0.6×25 mm (Sterican 23 G×1, B.Braun Melsungen AG). Theneedle was introduced through the rubber stopper and a volume of 2 mL was withdrawn into thesyringe. The bottle, the needle was pulled up into the rubber stopper to close it while still keepingthe exact 2 mL volume in the glass syringe. Then the piston of the syringe was released to let thegas expand to atmospheric pressure. The volume increase within the syringe was used to calculatethe overpressure of the bottle accordingly:
P = (GasVExpt. bottle) + (VNeedle) + (VSample)(GasVExpt. bottle)
×
(GasVafter equalising + VNeedle)(VSample + VNeedle)
Just after the pressure measurement, 1 mL sample gas was transferred to a glass bottle (30.7 mL)capped with rubber stopper for later methane concentration analysis. Thereafter, the overpressurein the experimental serum bottles was released inserting a needle of 0.6×25 mm (Sterican 23 G×1,B.Braun Melsungen AG). However, in the Expt.-3, when pressure was found too low to measurewith the glass syringe, pressure of some bottles was not released. The volume of discharged gas wascalculated every time of pressure measurement.
3.4.6 Gas sampling and methane analysis
3.4.6.1 Gas chromatography
The gas sample bottles of the batch experiments were analyzed for methane concentration by GC-FID (HP 5880A) equipped with a Poraplot T column. Nitrogen was used as carrier gas (30 mLmin-1). The temperatures of the injector, column and detector were 150, 150 and 80 °C, respectively.Three standard methane bottles were prepared [Table 3.3]. CH4 concentration of standard bottles(an average volume of 123.4 mL) was measured at the beginning of measurement with GC-FID. 2and 10 mL syringe (BD plastic) with a needle of 0.4×25 mm (Sterican 27 G×1½, B.Braun MelsungenAG) used during collection of CH4from a cylinder (99.99%). The standard curve, with calculatedslope and intercept, was used to calculate the methane concentration of the gas sample bottles.
Table 3.3: Preparation of samples for standard curve.
Standard Empty volumeof glass bottle
Injectedvolume of CH4
Injectedvolume of N2
Injected volumefrom standard-2
CH4 con-centration
1 123.4 0 0 10 0.103%2 123.4 2 20 0 1.376%3 123.4 4 10 0 2.911%
0.3 mL of sample was injected into the head of GC using a 1 mL plastic syringe (BD plastic) equipped0.4×25 mm needle (Sterican 27 G×1½, B.Braun Melsungen AG). One syringe has been used for onesample to avoid contamination. Three injections were performed from each sample bottle. Theresults (area) obtained from the GC were used for calculation of methane concentration afterward.The standard deviation of the average output for each gas sample GC were <2%.
CHAPTER 3. MATERIALS AND METHODS 34
3.4.6.2 Portable gas analyzer
CH4, CO2, O2 and H2S contents of the biogas produced in the reactors were measured at roomtemperature once a week with a portable gas analyzer GFM series, manufactured by Gas Data,mainly configured to measure biogas. However, it was not possible to measure the H2S concentrationover 2000 ppm with this equipment.
Chapter 4
Results and discussion
4.1 Batch experiments
4.1.1 Experiment 1:OA, autoclave pretreatment of OA and Whatman paper
In the first batch experiment, the methane potential of the OA collected in October 2007 [Figure 2.2]was investigated. In addition, effects of pretreatment by autoclavation (121°C for 30 minutes) on thebiogas and methane yields were investigated.
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Figure 4.1: Accumulated biogas in bottles [error bars show the standard deviation] of the first batchexperiment investigating biogas potential of OA [−■−], OA pretreated in autoclave [−▲−] andWhatman paper [−♦−]. Control [−⋇−] shows accumulation of biogas produced from the inoculum.
The experiment lasted for 35 days. Three replications of each investigated material were used andthe results given [Table 4.1] are averaged over the three samples. The accumulated biogas producedin experimental bottles are shown in the graph [Figure 4.1].
35
CHAPTER 4. RESULTS AND DISCUSSION 36
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Figure 4.2: Specific methane yields (i.e., methane produced from inoculum was substracted) inthe first batch experiment investigating biogas potential of OA [−■−], OA pretreated in auto-clave [−▲−] and Whatman paper [−♦−]. Error bars show the standard deviation and yields arecorrected at STP.
The methane yields were 211 (±7), 251(±2) and 370 (±19) mL/added g VS for OA, Autoclaved OAand Whatman paper respectively. The methane yields of OA correspond well to the results fromprevious studies on anaerobic digestion of different sea weeds found in literature [Table 4.6]. Moreover,Whatman paper gave 89% of the theoretical methane yields calculated in the section 3.3.1.4. Theresults show that there is an effect of autoclavation, which increased the yield by ca 20%.
4.1.2 Experiment 2:OA, NA and Whatman paper
The aim of the second batch experiment was to determine the seasonal effect on biogas and methaneyields of Polysiphonia. In this experiment, biogas potential of OA, NA and Whatman paper wereinvestigated. The incubation period was 51 days and 4 measurements were taken to determinethe biogas and methane yields of the substrates in anaerobic digestion. The other experimentalprocedures remained the same as in the first experiment. Detailed results are given in the table 4.1.
CHAPTER 4. RESULTS AND DISCUSSION 37
Table4.1:
Results
obtained
from
theba
tchdigestionexpe
riments.Datashow
nhe
rearespecificyields
from
test
material[subtracted
meanbiog
asan
dmetha
neprod
uctio
nin
controls]
andaverag
edover
threereplications.
Par
amet
erE
xpt.
-1E
xpt.
-2E
xpt.
-3
Subs
trat
eA
lgae
(Old
)A
utoc
lave
dO
AW
hatm
anP
aper
Alg
ae(O
ld)
Alg
ae(N
ew)
Wha
tman
Pap
erA
lgae
(New
)U
ltra
soun
dA
lgae
(New
)W
hatm
anP
aper
Pre
trea
tmen
tN
one
Aut
ocla
vea
Non
eN
one
Non
eN
one
Non
eU
ltra
soni
cbN
one
OL
Rc
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
Exp
erim
enta
lco
ndit
ions
TS
(%)
12.4
812
.65
98.6
116
.41
14.6
996
.91
10.0
9.9
98.7
VS
(%T
S)60
.89
59.9
399
.46
58.0
168
.39
99.6
075
.274
.299
.5Su
bstr
ate
dige
sted
(g)
3.25
3.35
0.25
2.56
2.65
0.25
3.00
3.00
0.25
Bio
gas
prod
uced
inbo
ttle
at31
0K
(mL
)10
7(±
5)14
0(±
3)21
6(±
11)
131
80(±
3)11
7(±
3)63
(±2)
77(±
3)21
5(±
1)C
H4
prod
uced
inbo
ttle
at31
0K
(mL
)59
(±2)
72(±
1)98
(±5)
63(±
3)35
(±3)
78(±
2)27
(±1)
26(±
1)94
(±2)
Yie
lds
Bio
gas/
adde
dg
VS
at27
3K
(mL
)38
1(±
18)
486
(±9)
771
(±38
)47
326
6(±
9)42
8(±
12)
247
(±9)
308
(±12
)77
3(±
5)C
H4/a
dded
gV
Sat
273
K(m
L)
211
(±7)
251
(±2)
370
(±19
)22
9(±
12)
116
(±9)
285
(±7)
102
(±5)
104
(±6)
351
(±7)
CH
4%
55.5
51.6
48.0
48.3
43.8
66.7
42.6
33.6
43.5
VS
redu
ctio
n(%
)d37
4577
5237
9552
4392
Cal
cula
tion
sB
ioga
s/g
VS
redu
ced
at27
3K
(mL
)10
2610
8210
0890
972
445
147
272
283
6C
H4/g
VS
redu
ced
at27
3K
(mL
)56
955
948
349
331
730
119
5124
438
0
Itis
note
wor
thy
that
VS
Red
ucti
onca
lcul
atio
nin
batc
hex
peri
men
tsis
kind
ofsh
aky
onit
sou
tcom
es.
The
seV
Sre
duct
ion
rate
sar
eca
lcul
ated
toge
tan
over
all
unde
rsta
ndin
g.
a At
121
°Cfo
r30
min
utes
b 19
kHz
wit
ha
trea
tmen
ten
ergy
1-5
Wh/
L.
c Org
anic
Loa
ding
Rat
e(g
VS/
L)
d VS
redu
ctio
nca
lcul
atio
nis
show
nin
the
App
endi
xA
.
CHAPTER 4. RESULTS AND DISCUSSION 38
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Figure 4.3: Specific methane yields (i.e., methane produced from inoculum was substracted) in thesecond batch experiment investigating methane potential of OA [−×−, − ⋇ −, − • −], NA [−♦−,−■−, −▲−] (3 replications of each).
In the second experiment, methane yields for OA and NA were 229 (±12) and 116 (±9) mL / addedg VS; approximate VS reductions were 52% and 37% respectively. The methane potential of OA andNA were surprisingly different. As the results showed that OA gives almost double methane yieldsthan that of NA.
4.1.3 Experiment 3:NA, ultrasound pretreatment of NA and Whatman paper
In this experiment, methane yields for NA, Ultrasound treated NA and Whatman paper were 102(±5), 104 (±6) and 351 (±7) mL / added g VS respectively [Figure 4.4].
No effect of the ultrasound pretreatment was seen in this experiment. The methane yields of NA inthe third experiment, however, correspond well with the obtained methane yields of NA in secondexperiment. Whatman paper gave 85% of the theoretical methane yield.
CHAPTER 4. RESULTS AND DISCUSSION 39
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Figure 4.4: Accumulated specific methane yields (i.e.; methane produced from inoculum was ex-cluded) in the third batch experiment investigating biogas potential of NA [−■−], NA pretreatedwith ultrasound [−▲−] and Whatman paper [−♦−] (up to day 40). Given data are averaged overthree samples [error bars show the standard deviation, almost unseen] and corrected at STP.
In all batch experiments, most gas production occurred during the first 20 days from the start ofexperiment and after day 30 the gas production had ceased [Figure 4.2, 4.3, 4.4]. The experimentswere not run with the same incubation period and same number of measurements.
Table 4.2: Specific biogas and methane yields (mL/added g VS) in the batch digestion experimentsby days. The yields are averaged over three replications and corrected at STP.
Expt. No./ Day 23 Day 24 Day 29 Day 35Substrate Biogas CH4 Biogas CH4 Biogas CH4 Biogas CH4
1. OA 361 (±15) 181 (±10) - - - - 381 (±18) 211 (±7)1. Autoclaved OA 454 (±12) 234 (±8) - - - - 486 (±9) 251 (±2)
2. NA - - 229 (±9) 106 (±9) - - - -2. OA - - 433 209 (±3) - - - -
3. NA - - - - 212 (±9) 103 (±3) - -3. NA with ultrasound - - - - 273 (±12) 102 (±5) - -
Total biogas yields may vary in different experiments between treated and untreated substratesbecause of different incubation period. However, accumulated methane at day 35 for untreated bothNA and OA in different experiments was the same (usually yields may vary ±10%).
4.1.4 Effect of pretreatments
4.1.4.1 Heat and steam (autoclavation)
The effect of heat and steam pretreatment on OA was analyzed. OA pretreated in autoclave gave 28%and 19% more biogas and methane yields respectively than that of untreated OA in this experiment[Figure 4.2, 4.1] and, thus, a corresponding increase in VS reduction observed, from 37% to 45%[Table 4.1].
CHAPTER 4. RESULTS AND DISCUSSION 40
4.1.4.2 Ultrasound
The ultrasound pretreatment applied on NA didn’t affect the methane yields as observed in thirdbatch experiment [Figure 4.4]. However, an increased biogas production was observed [Table 4.1] inAlgae pretreated with ultrasound compared to untreated Algae.
4.2 Stirred reactor experiment
4.2.1 Biogas and methane yields
During the start-up, biogas yields from the two digesters were similar except at day 06, 15, 16 and17. It was assumed that the yields differed because of the gas meters as the yields became similarafter replacing. The average gas yields from reactors D9 and D10 in day 01-17 were 480 and 450mL/added g VS respectively.
The gas yields from OA in stirred digesters were identical with the gas yields obtained from batchexperiments. It is noteworthy that no VFAs were found in the digesters during this initial period.The pH of both reactors were identical. A summary of reactors performance data is given below[Table 4.3].
From Day 18, an increase in OLR was introduced in the reactor D10. The effect of higher the OLRis described in the section below [Section 4.2.3]. The specific gas production [mL/added g VS] wasinitially affected by higher OLR. Additionally, an accumulation of VFAs was observed after a periodof receiving higher OLR.
Figure 4.5: Biogas yields in the reactors D9 [−♦−] and D10 [−■−] and CH4 amounts in producedgas of the reactors D9 [■] and D10 [■] between Day 99-117. Here both reactors showed steadyperformance. D10 was fed with OA treated in autoclave from Day 107-117 (a sharp increase in gasyields). The gas amount given here as observed at 37±2 °C.
Between days 85 and 107, D10 was fed with OA and at about same OLR as introduced for D9. It wasalready determined that OA had a higher biogas potential than NA. In the reactor experiment, thehigher yields are achieved from OA in reactor D10 than yields in reactor D9 fed with NA [Figure 4.5].
CHAPTER 4. RESULTS AND DISCUSSION 41
Table4.3:
Asummaryof
reactors
performan
ceda
ta[dataareaverag
edover
thegivenpe
riod]
fortheexpe
rimentat
a20
days
HRT
.The
gasprod
uctio
ngivenhe
reis
at37±2
°C,a
sob
served
intheincu
batio
nroom
.
Period
[Day
s]
Parameter
Units
Digester
01-17
18-34
35-54
55-77
77-85
86-98
99-107
108-117
Test
substrate
D9
OA
NA
NA
NA
NA
NA
NA
NA
D10
OA
NA
NA
NA
NA
OA
OA
Pretreateda
OA
Actua
lOLR
bgVS/
L/Day
D9
1.47
1.44
1.34
1.43
1.98
1.41
1.12
1.16
D10
1.75
2.33
3.09
1.63
2.06
1.54
1.51
1.42
TotalP
rodu
cedGas
mL
D9
3812
2974
2860
2583
2453
2396
2845
2885
D10
4127
3126
3792
2442
2680
3563
3359
3491
Gas
Produ
ctionRate
mL/
addedgVS
D9
477
371
314
317
308
303
349
351
D10
454
289
264
368
326
438
412
455
Metha
ne%
D9
-48.0
53.3
44.9
--
50.0
53.8
D10
-44.5
43.8
38.8
55.7
-51.8
49.1
CO
2%
D9
-32.6
36.8
25.1
--
34.3
36.5
D10
-32.6
33.2
25.0
35.1
-38.2
33.9
O2
%D
9-
3.9
2.0
5.6
--
3.0
2.2
D10
-4.7
4.7
6.4
1.7
-2.0
3.4
H2S
ppm
D9
-125.5
275
1500
--
1969
2900
D10
-67.5
1200
833
1426
-2000
2600
pHS.U.
D9
7.29
7.29
7.25
7.36
7.24
7.23
7.25
7.22
D10
7.27
7.23
7.34
7.01
7.31
7.27
7.26
7.23
TSin
Digesters
%D
92.73
3.58
4.26
4.16
4.06
4.42
4.38
3.87
D10
3.30
4.09
6.79
6.28
4.75
4.86
4.99
4.69
VSin
Digesters
%D
959.1
59.0
66.9
71.1
72.0
70.7
71.7
74.0
D10
58.8
59.0
65.6
69.5
70.0
64.9
60.6
59.3
VSReductio
n%
D9
60.0
44.4
35.6
33.8
29.8
26.4
21.1
32.1
D10
61.8
54.4
42.9
31.9
27.6
29.1
24.2
30.2
a OA
pretreated
with
autoclaveat
121
°Cfor30
minutes
b Determinationof
TSan
dVSof
algaein
prepared
food
portionwas
carriedou
tfrom
thesamples
ofdefrostedalgaeused
aswell.
OLR
ofalgaegivenhere
only.The
totalO
LRinclud
ed0.5g
VSprim
aryslud
ge/L
/day.
CHAPTER 4. RESULTS AND DISCUSSION 42
During this period, the gas yields obtained from D9 and D10 were 350 and 410 mL/added g VSrespectively. The pH in both reactors remained in a range of 7.2-7.3 and no volatile fatty acidsaccumulated in the digesters. Overall, the performance of both reactors was excellent at the relativelylow OLR. The methane contents in the biogas were 51 and 54% at D9 and D10. However, the biogascontained a considerable amount of oxygen (~5%) which might correspond to presence of N2 in theproduced gas which amount was not detected.
4.2.2 Effect of pretreatment on biogas yields
The detail performance data over the period of this experiment are given in appendix B. On Day108, D10 was fed with OA pretreated by autoclavation in the same way as for the batch tests[Section 2.2.4.1]. In the graph [Figure 4.5], a sharp increase in specific gas production is seen fromday 108, which indicates the effect of autoclavation. The gas production rate increased up to 490mL/added g VS and VS destruction went up to 31%. Average gas production rates over days 108-117in D9 and D10 were 351 and 455 mL/added g VS [Table 4.3]. VFAs accumulation was disappearingcompletely and an apparent steady state was achieved for both reactors. The detailed performancedata of the reactors over the last period [Day 108-117], see table B.6 in Appendix B.
4.2.3 Effect of increased OLR
From Day 18, higher OLR was introduced in D10. Though total biogas production increased, agradual decrease in specific biogas yields (mL/added g VS) took place immediately. It was assumedthat this happened due to the increasing OLR of NA. Since the pH and VFAs were in acceptablerange for the operation during that period, the influence of increasing organic loading rate could beconsidered as the reason of the low gas production.
Figure 4.6: Effect of OLR on total biogas yields in D9 [■, −♦−] and D10 [■, −■−] over theexperimental period. Data are given here obtained from incubation room at 37 °C.
The average gas production rates over the period Day 35-54 were 340 and 270 mL/added g VS inD9 and D10, respectively. From the Day 61 [Figure 4.6], the total biogas production ceased and theaccumulation of VFAs increased. To recover the reactor performance, feeding was interrupted andstrategy was modified as described below [Section 4.2.4].
CHAPTER 4. RESULTS AND DISCUSSION 43
4.2.4 Accumulation of VFAs
Between days 55 and 77, the total gas production decreased considerably in D10 down to 93 mL/addedg VS on day 62, due to the accumulation of VFAs, while an apparent steady state was achieved inD9. Feeding was interrupted (reduced) from Day 62-77 and extra care was needed to maintain thepH in between the optimal range. The pH was decreased to 6.2 from 7.2 in reactor D10. A summaryof the reactors performance in those days (55-77) is reported in the table B.4 in Appendix B.
Reactor pH was recovered gradually by adding 2.0 g Ca(OH)2 to D10 on Days 62, 70 and 71.Additionally, it was fed by the liquid withdrawn (200 mL) from D9 on Days 70, 71 and 72. OLR waskept lower by just feeding with the primary sludge portion. From Day 77 the gas production ratestarted to increase in reactor D10 [Figure 4.7].
Figure 4.7: High VFAs accumulation affected on total gas production in the reactor D10 [■, −■−]between Day 54-77 while D9 [■, −♦−] shows normal production. The daily produced gas amountgiven here as observed at 37±2 °C.
From Day 78-84, OLR for D10 followed as designed for D9 [i.e., OLR for both digesters were identical]l.During the period, the average gas production rate for D9 and D10 were 310 and 340 mL/added gVS, respectively. pH ranged in between 7.2-7.3 for both reactors and no VFA accumulated. Overallconditions after the period assured stable performance of both reactors.
4.2.5 VS reduction
The calculation of VS reduction is shown in Appendix A. At the beginning, the VS reduction wascomparatively higher in both reactors [Figure 4.8] than the VS reduction rate during the termination.Gradually, the reduction rate started to decrease from ~60% to ~31% in both reactors over theexperimental period. Actual VS reduction was obtained after 3 retention time (from the week 8).
CHAPTER 4. RESULTS AND DISCUSSION 44
(a) VS reduction [%].
Figure 4.8: Destruction of volatile solids over the experimental period in reactor D9 [♦] and D10 [■].Arrow shows the VS reduction after 3 retention time.
4.2.6 Digestate as a fertilizer
There are two products in anaerobic digestion process: biogas and digestate. Optimal managementof both products could ensure good economy for the process. The digestate contains organic matterand plant nutrients (N, P, K and Mg) and these positively affect soil quality by improving the soilstructure, increasing the water-holding capacity and stimulating the microbial activity [64, 65]. Theend result is not only an increase in soil quality, but also higher crop yields and better grain qualityin comparison with unfertilized soil, or equivalent effects after application of artificial fertilizers [66].Moreover, the biogas residue increase the substrate induced respiration, the proportion of activemicroorganism, and the nitrogen mineralization, as well as potential ammonia oxidation [67].
Table 4.4: The maximal content of metals in the digestate [According to the Swedish quality assuringsystem SPCR 120]
Heavy metal Limits for heavy metals when using digestate as a fertilizer in agricultural crops(mg/kg DM)
According to the Swedish quality assuringsystem SPCR 120
For the use in organic farming accordingto EEG. no 2092/91
Lead, Pb 100 45Cadmium, Cd 1 0.7Copper, Cu 600 70Chrome, Cr 100 70Mercury, Hg 1 0.4Nickel, Ni 50 25Zinc, Z 800 200
Nevertheless, there are risks in using digestate on arable lands because of the concentration of heavymetals and organic pollutants. This is why, digestate must be certified and the concentration of
CHAPTER 4. RESULTS AND DISCUSSION 45
plant nutrients, heavy metals content and pathogens present have to be declared. In this paper, anevaluation of the digestate quality was made, considering the nutrient contents and heavy metalslevel. According to the Swedish quality assuring system SPCR 120, the maximal content of metalsin the digestate is given in the table 4.4.
The major components of digestates have been analyzed four times over the period of runningthe reactors. Digestates found rich in nutrients mainly nitrogen, ammonia, chloride, phosphorus,aluminum, calcium, iron, potassium, magnesium, sodium and sulfur. In addition, a considerableportion of micro nutrients is also available in the mixture of digestate as an excellent source ofnutrients for arable land. The detail results are reported in the table 4.5.
Digestate usually contains heavy metals and organic pollutants. According to the Swedish qualityassuring system SPCR 120, the maximal content of metals in the digestate is given in table 4.4. Inthis experiment, heavy metals content in the digestate mostly were below the maximum limits exceptfor Cadmium (Cd). However, the digestate has a greater NH4-N , N, P, K content.
4.3 Results from similar studies
There are a few studies on the biogas potential of algal biomass given in the literature, but noneaddressing the methane potential of Polysiphonia. An early investigation was made by Golueke, et.al. (1957) [57]. Biogas yields from algae, codigestion of algae with sewage sludge showed rates of0.381 and 0.493 m3/added kg VS in 35°C and 50°C, respectively.
A range of 0.13-0.2 m3 CH4/added kg VS was produced from Gracilaria tikvahiae in semi-continuousdigesters at 30°C [61]. This species is alga from Rhodomelaceae family, branched, 12-15 cm long,morphologically similar with Polysiphonia. Also methane yield of 0.25-0.35 m3/added kg VS of Ulva,Cladophora and Chaetomorpha mixture at 35°C in semi-continuous fermentation [60]. In full scalebiogas plant, biogas yields were reported 0.240 m3/added kg VS, where methane content in producedbiogas was 61.4% and VS reduction achieved 28.7%, using macro algae from the Venice lagoon [58].
Results from my experiments obtained from both batch and STR experiment had higher yields incomparison to the results obtained by Hanisak [61], mainly yields from the OA in both STR andbatch experiments. However, marine biomass would be a renewable source of mass energy, manywould have been attracted to study this area. A summary from previous studies and results is givenin the table 4.6.
4.4 Concluding discussion
The aim of this study was to evaluate red algal biomass collected from the eutrophied Baltic Sea asa desired substrate for bioreactor. The substrate (Algae) used for this study, was collected from thesea shore. While reactor operation was terminated, a thick layer of sand was found at the bottomof the digester. However, it is difficult to draw a conclusion whether the presence of sand in thesubstrate affected the performance of the reactors.
In the second batch experiment, the OA used for the study, was proven to have a higher biogaspotential than that of NA. Since the total nitrogen content in OA was more than double than of NA,it can be hypothesized that the C:N ratio was not high enough in NA to support the same yield.The preserved amount of OA was not sufficient to run both reactors over the experimental period.It was speculated that, if both reactors were fed with OA over the period, the start-up period wouldbe shorter. Since OA had a higher VS reduction rate, digester would accept the increasing OLR 3.0g VS/L/Day for OA as well.
CHAPTER 4. RESULTS AND DISCUSSION 46
Table4.5:
The
characteris
ticsan
dnu
trientscontentof
alga
ean
ddigestatefrom
reactorD
9an
dD
10ba
sedon
thean
alyses
repo
rts.
Substrates
Digestate
Day
26Day
68Day
75Day
110
Com
ponent
Unit
OA
NA
D9
D10
D10
D9
D10
D9
D10
Exp
erim
ental
Fractio
nGiven
ref./
instrument
TotalS
olids
%13.1
14.1
4.1
4.0
6.6
4.2
4.8
3.7
4.6
±10%
SS-E
N12880
VolatileSo
lids
%TS
69.3
59.1
62.1
63.6
70.3
70.8
68.6
71.4
61.7
±10%
SS-E
N12879
TotalN
itrogen
(Kjeldah
l)%
TS
5.3
2.3
5.9
6.0
6.8
9.8
1012
6.7
±10%
SS-E
N13342
Ammon
ium
Nitr
ogen
%TS
0.76
0.21
2.0
2.3
2.0
2.4
2.3
2.2
1.5
±10%
KLK
1965:1,5
:35mod
Chloride
%TS
<0.38
2.1
2.7
2.8
<0.053
<1.2
<1.0
3.0
2.6
±8%
Silvernitratetit
r.,Lidfett0A
.01
Pmg/kg
TS
3200
4300
11700
13900
7300
8700
8700
8200
8500
±15%
ICP-A
ES
Al
mg/kg
TS
1100
1400
4200
5000
3500
4500
4300
3400
3400
±15%
ICP-A
ES
Bmg/kg
TS
210
270
220
250
220
250
240
190
260
±15%
ICP-A
ES
Ca
mg/kg
TS
15900
11600
21300
23500
22200
21600
28100
21400
21300
±15%
ICP-A
ES
Femg/kg
TS
3000
2500
18100
23500
9300
10800
11900
9900
8700
±15%
ICP-A
ES
Kmg/kg
TS
9200
24200
19200
18200
9200
10100
9700
9500
21300
±20%
ICP-A
ES
Mg
mg/kg
TS
5600
6000
6400
6700
5600
6200
6300
4900
7100
±15%
ICP-A
ES
Mn
mg/kg
TS
310
390
330
370
320
370
330
280
330
±15%
ICP-A
ES
Na
mg/kg
TS
17000
12600
14900
15000
13800
15200
15100
15000
14900
±20%
ICP-A
ES
Smg/kg
TS
18000
11600
12800
15000
16900
19500
18400
21400
13800
±20%
ICP-A
ES
Pb
mg/kg
TS
7.4
3.0
8.8
1011
1311
--
±25%
ICP-M
SCd
mg/kg
TS
8.2
0.41
3.8
4.2
8.1
9.8
8.9
--
±15%
ICP-A
ES
Cu
mg/kg
TS
336.9
98130
85110
110
8661
±15%
ICP-A
ES
Cr
mg/kg
TS
8.3
2119
4242
4050
--
±15%
ICP-A
ES
Hg
mg/kg
TS
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
<0.05
--
±25%
ICP-A
FS(K
allfö
rång
ning
)Ni
mg/kg
TS
229.8
2034
4244
4829
23±15%
ICP-A
ES
Znmg/kg
TS
270
40310
360
360
450
420
360
180
±15%
ICP-A
ES
CHAPTER 4. RESULTS AND DISCUSSION 47
Table4.6:
Results
from
previous
stud
ieson
anaerobicdigestionof
marinebiom
assin
diffe
rent
cond
ition
san
dscales.
Substrate
Con
dition
sSc
ale
Unit
Yield
VSredu
ction
Ref.
°CHRT
OLR
aBiogas
CH
4%
Algae
3530
-La
bcu
ft/add
edlb
VS
6.1
-36.4
[57]
Algae
5030
-La
bcu
ft/add
edlb
VS
7.9
-54.0
[57]
BGA
b35
3020
Lab(C
STR)
m3/k
gVSad
ded
-0.35
-[50]
Macroalgae
3715
1.7
Plant
cm
3/k
gTVS
0.24
61.4%
28.7
[58]
M.
pyri
fera
3518
1.6
CST
Rm
3/k
gVSad
ded
-0.28-0.31
NR
d[59,
andreferences
therein]
L.sa
ccha
rina
3524
1.65
CST
R(sem
i)m
3/k
gVSad
ded
-0.23
NR
[59,
andreferences
therein]
L.hy
perb
orea
3524
1.65
CST
R(sem
i)m
3/k
gVSad
ded
-0.28
NR
[59,
andreferences
therein]
Asc
ophy
llum
nodo
sum
-24
1.75
NR
m3/k
gVSad
ded
NR
0.110
NR
[59,
andreferences
therein]
Gra
cila
ria
tikva
hiae
29-35
NA
NA
Batch
2Lm
3/k
gVSad
ded
NR
0.220
75.7
[60]
Gra
cila
ria
tikva
hiae
29-35
30-60
0.54
CST
Rm
3/k
gVSad
ded
NR
0.13-0.20
26-48
[60]
Gra
cila
ria
tikva
hiae
29-35
30-60
0.54
CST
Rm
3/k
gVSad
ded
NR
0.13-0.20
26-48
[60]
Ulv
asp
.29-35
NA
NA
Batch
2Lm
3/k
gVSad
ded
NR
0.220
70.1
[59,
andreferences
therein]
Mac
rocy
stis
pyri
fera
371
32ph
asee
mL/
gdryalgaef /
d181.4(±
52.3)
50-6
5%NR
[16]
Dur
ville
aan
tarc
tica
371
Do
2ph
ase
mL/
gdryalgae/d
179.3(±
80.2)
50-6
5%NR
[16]
Ulv
a+
Cla
doph
ora
+C
haet
omor
pha
3512-15
2-2.5
SemiC
ontin
uous
m3/k
gVSad
ded
-0.25-0.35
50-55
[61]
a kgTVS/
m3/d
ayb B
lueGreen
Algae,S
piru
lina
max
ima.
c Pilo
tplan
twith
1m
3working
volumedigester.
d Not
repo
rted
e Two-ph
asean
aerobicdigestionsystem
,which
consistedof
anan
aerobicsequ
encing
batchreactor(A
SBR)an
dan
upflo
wan
aerobicfilter(U
AF)
,The
results
show
that
70%
ofthe
totalb
iogasprod
uced
inthesystem
was
generatedin
theUAF.
f The
algaespecieswerewashed,
driedat
701C
for24
h,then
crushedan
ddissolvedin
300m
Lof
distilled
water
before
feedinginto
thebioreactors.
CHAPTER 4. RESULTS AND DISCUSSION 48
Co-digestion of algal biomass with primary sludge worked well though particular influence of primarysludge could not be specified. The algae employed had a high nutrient content with a C:N ratio inthe range of 12-20. The biogas yield of the STR [Section 4.2.1] during continuous operation found insame range compared to yields in batch experiments as calculated [Section 4.1]. However, codigestionof algal biomass with high C:N ratio substrate (e.g., waste products from paper and pulp industries)should be a good combination for anaerobic digestion and higher methane yield.
Maximum methane yield 0.25 m3/kg VS added at 273 K, was obtained from OA pretreated inautoclave in batch experiment. However, the methane potential of untreated OA achieved fromthe batch experiments was 0.21-0.23 m3/kg VS added. It is noteworthy that NA found just halfpotential in methane yield than that of OA. In the STR experiment, the biogas yields from OA andNA [Table 4.3, 4.1] correspond to yields in batch experiment. However, biogas yield from NA in STRwas comparatively higher than in batches, this might be due to codigestion with primary sludge.
However, 1 metric ton dry algae contains 53 [Algae(old)] and 23 [NA] kg N and 3.2-4.3 kg P respec-tively [Table 4.5]. This indicates that harvesting of algae from the Baltic Sea would surely contributeto balance the nutrients and contribute to curb eutrophication. Moreover, both N and P amountsin digestate increased more than two folds after a certain digestion period to value as a fertilizer forarable land.
Chapter 5
Conclusion
The questions asked in the section 1.2 are answered as follows:
^ Methane yields for untreated OA in the batch experiments, were obtained 0.21-0.23 m3/kg VSadded at 273 K [Table 4.1]. Methane yield from untreated NA in the batch experiments wereobtained 0.11-0.12 m3/kg VS added at 273 K. Average methane yield obtained from OA wasalmost double than that of NA.
^ Biogas yields for untreated OA in the batch experiments, were obtained 0.38-0.47 m3/kg VSadded at 273 K [Table 4.1]. Biogas yields from untreated NA in the batch experiments wereobtained 0.25-0.27 m3/kg VS added at 273 K.
^ Autoclave pretreatment increased biogas and methane yields by 28% and 19% respectively andincreased VS reduction rate from 37% to 45%, while almost no effect of ultrasonic pretreatmentwas observed in this experiment.
^ In STR codigestion experiment, maximum average [over a short period] biogas yields fromuntreated OA obtained 0.44 m3/kg VS added at 310 K when reactor performance achievedmost steady state [Table 4.3]. A maximum biogas yield 0.49 m3/kg VS added at 310 K , wasobtained from OA pretreated by autoclavation. The methane content of the produced biogaswas 54%.
^ The process remains stable and the reactor seemed to perform well at an OLR 1.5 g VS/L/DAlgae and 0.5 g VS/L/D of primary sludge. Process became unstable and VFAs accumulatedin the reactor with an higher OLR, as observed in this experiment. However, it is hypothesizedthat reactor would performed well in higher OLR if OA used instead of NA over the period.
^ VS reduction rate was ~32% for both reactors after 112 days operation though the rate was~62% at the beginning.
^ The digestate was found rich in nutrients (N, P, K, S etc.) [Table 4.5]. All heavy metals contentin the digestate remained under the limit according to Swedish quality assuring system SPCR120, except cadmium (Cd).
Following conclusions were drawn from this study:
^ Codigestion of algae with primary sludge is an advisable practice and it can be carried out inexisting biogas plants. However, codigestion of Polysiphonia algal biomass with substrate withhigher C:N ratio like paper mill waste should be more appropriate to increase the methane andbiogas yield. To confirm this hypothesis, further studies are needed.
49
CHAPTER 5. CONCLUSION 50
^ Large scale harvest of algal biomass from the Baltic Sea will surely contribute to reduce N andP levels which regulate eutrophication.
^ Ultrasound pretreatment of algal biomass had no effect on the biogas yield, so this hypothesisis rejected. However, autoclave pretreatment had positive effects on biogas yields and VSreduction rate.
^ Removal of the sand content from the substrate is likely necessary.
^ From these limited experiments, I can not draw a conclusion whether anaerobic digestionprocess is a good method to dewater red algae or not. For this, further study is needed.
^ After a 3 retention time of reactors operation, when reactors performance achieved the moststeady state, VS reduction rate found ~32%.
^ The economy of the process was not analyzed. But still it is possible to predict that the wholeprocess will be economically profitable considering the biogas yields and nutrient rich digestate,mainly N and P. The value of N and P fertilizer is increasing day-by-day and algal biomass inthe Baltic Sea is an existing source. Moreover, large scale harvest of algae will contribute tocurb eutrophication of the Baltic Sea.
Acknowledgments
This thesis would not have been possible without the help of many people and their genuine interestson this study. Many helped me, who I don’t even know, fed reactors during the weekends and otherholidays. I sincerely thank all of you who helped directly or indirectly in this study. But particularly-
^ I want to express my gratitude to Prof. Bo Svensson at TEMA, for your greater interest,ideas and your valuable time. I will remember, while university was vacant during the summer,you were there to listen my words! The discussion with you is always amusing, inspiring andenjoyable.
^ A bunch of thanks goes to Cecilia Gustafsson, Ida Andersson and Mia Wirf fromScandinavian Biogas Fuels AB, who provided assistance in numerous ways all over the period...without your help it was totally impossible to complete this study. I really enjoyed yourcompany and learned lots of technical things eventually. Congrats Cissi for being a mother!
^ I am grateful to Lena Lundman, Förste forskningsingenjör at TEMA, for collecting me Englishversion of SIS articles, all out help at the laboratory and ride me to Norrköping from the lab!
^ I would like to thank Anna Karlsson for nice discussion, suggestions and help at the labo-ratory.
^ The warmest thanks to Scandinavian Biogas Fuels AB for giving me the opportunity to useequipments and laboratory; and to those personnel who helped me at the laboratory, collectedalgae, operated sonicator.
^ My deepest gratitude goes to my parents, sisters, brothers for their unflagging love and supportthroughout my life. Thanks to all my friends who supported me over the period, especially toZoheb, for your company while I was working at TEMA during the summer. Thanks to MaleneJensen for last minute comments!
^ Finally, I am deeply indebted to my supervisor Jörgen Ejlertsson for his all out cooperationfrom preparing platform at the laboratory to result formulation and finalizing the thesis paper.I am highly motivated working with you together, your informative discussion, tie up the looseends, research ideas, your enthusiasm and interest...Wish I could be working further under yourclose supervision !!!
51
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Appendix A
Calculation on VS reduction
VS reduction indicates the percent amount of organic matter is being degraded in the reactor. Forbatch digestion, VS reduction [VSred in %] could be calculated though the calculation might not besurely correct and completed but an overall indication could be formulated -
For reactors digestion [calculated weekly basis for each reactor],
(%) VSred = 1 - QoutQin×TSoutTSin
×V SoutV Sin
Here,
^ Qout = Volume of materials withdrawal from reactor [mL/week]
^ Qin = Volume of materials in to the reactor [mL/week]
^ TSout = TS of withdrawal material from the reactor [%]
^ TSin = TS of feed material of the reactor [%]
^ VSout = VS of withdrawal material from reactor [%]
^ VSin = VS of feed material of the reactor [%]
% VSred calculation was done weekly basis since TS and VS of the reactor materials were measuredonce a week. For example, just after the second week of running reactor, VSred in the reactor D10was calculated as follows,
Qout = 1270 mL, Qin = 1400 mL, TSout = 3.21%, TSin = 6.3%, VSout = 59.06%, VSin = 70.1%.
Therefore,
(%) VSred = 1 - 12701400 ×
3.216.3 ×
59.0670.1 = 0.611 or 61.1%
56
APPENDIX A. CALCULATION ON VS REDUCTION 57
For batch digestion,
(%) VSred = 1 - V SoutV Sin
Here,
^ VSin = mass of volatile solid of the test substrate before the digestion experiment [g]
^ VSout = VSout in test bottle - VSout in control bottle
* VSout in test bottle = mass of volatile solids of test bottle after digestion [g]* VSout in control bottle = mean mass of volatile solids of control bottles after digestion [g]
For example, % VSred of Whatman paper [Test Bottle - 1] in the first batch experiment,
VSin = 0.244 g, VSout in test bottle = 0.5553 g,
VSout in control bottle = 0.5047
Hence,
(%) VSred = 1 - 0.5553− 0.50470.244 = 0.7926 or 79.26%
Now, for example, Whatman paper, if the methane yield is 370 mL/added g VS and the mean VSredis 87%. Thus the methane yield in per reduced g VS is,
3700.87 = 425.
Appendix B
Raw data
Raw data from the stirred tank reactors experiment are reported in table B.1, B.2, B.3, B.4, B.5 andB.6.
58
APPENDIX B. RAW DATA 59
TableB.1:Pe
rforman
ceda
taof
thereactorexpe
rimentdu
ringfeed
ingwith
OA
ata20
days
HRT
(Day
01-17).The
gasprod
uctio
ngivenhe
reis
at37±2
°C,a
sob
served
.
Day
Par
amet
erU
nits
Dig
este
r01
0203
0405
0607
0809
1011
1213
1415
1617
Act
ual
OL
Ra
gV
S/L
/Day
D9
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.47
1.47
1.47
1.47
1.47
1.47
1.47
1.47
1.27
D10
2.19
1.77
1.77
1.77
1.77
1.77
1.77
1.77
1.77
1.77
1.77
1.77
1.77
1.77
1.77
1.65
1.13
Gas
prod
uced
mL
D9
3194
3194
3740
3698
3488
4160
3320
3530
3362
3656
3992
4160
3773
3914
4150
4339
5140
D10
4145
4023
4310
4231
3918
3840
3840
3957
3957
4467
4623
5250
3918
3879
3644
3840
4310
Bio
gas
yiel
dm
L/a
dded
gV
SD
939
939
946
846
243
652
041
544
142
045
749
952
047
249
051
954
364
3D
1038
337
247
246
342
943
243
244
644
650
652
459
544
443
941
343
548
4pH
S.U
.D
97.
417.
267.
277.
317.
1D
107.
397.
267.
267.
337.
1T
Sin
dige
ster
s%
D9
2.49
2.96
D10
3.39
3.21
VS
indi
gest
ers
%D
958
.29
59.8
5D
1058
.47
59.0
6V
Sre
duct
ion
%D
960
59D
1063
61
Not
e:N
oV
FAs
foun
ddu
ring
the
peri
odan
dga
sco
mpo
siti
onha
dno
tbe
enan
alyz
edas
equi
pmen
tsw
ere
not
avai
labl
edu
ring
thos
eda
ys.
a Based
onT
San
dV
Sof
prep
ared
food
port
ion
dete
rmin
edla
tely
.O
LR
from
Alg
aein
the
feed
mix
ture
isgi
ven
here
only
.T
heto
tal
OL
Rin
clud
es0.
5gV
Spr
imar
ysl
udge
/L/d
ay.
APPENDIX B. RAW DATA 60
TableB.2:Pe
rforman
ceda
tafortheexpe
rimentdu
ringfeed
ingwith
NA
ata20
days
HRT
(Day
18-34).The
gasprod
uctio
ngivenhe
reis
at37±2
°C,a
sob
served
.
Day
Par
amet
erU
nits
Dig
este
r18
1920
2122
2324
2526
2728
2930
3132
3334
Act
ual
OL
Ra
gV
S/L
/Day
D9
1.27
1.27
1.37
1.37
1.37
1.51
1.51
1.51
1.51
1.51
1.51
1.51
1.51
1.51
1.51
1.51
1.25
D10
1.13
1.53
1.90
1.90
1.90
1.90
1.90
2.53
2.53
2.53
2.53
3.16
3.16
2.57
2.57
2.57
3.30
Gas
prod
uced
mL
D9
3113
2971
2641
2641
3160
2735
2735
2877
3018
3039
3100
3221
3343
2917
3221
2917
2917
D10
2978
2939
2625
2530
2765
2671
2671
3327
3327
3421
3186
3327
3468
3374
3655
3468
3421
Bio
gas
yiel
dm
L/a
dded
gV
SD
938
937
133
033
039
534
234
236
037
738
038
740
341
836
540
336
536
5D
1043
643
131
225
628
027
027
832
626
126
925
026
122
722
129
227
727
3M
etha
ne%
D9
42.5
53.4
D10
43.9
45.0
pHS.
U.
D9
7.27
7.26
7.3
7.33
D10
7.23
7.22
7.22
7.25
TS
indi
gest
ers
%D
93.
383.
334.
02D
103.
73.
84.
77V
Sin
dige
ster
s%
D9
55.5
257
.23
64.3
6D
1056
.42
60.6
059
.83
VS
redu
ctio
n%
D9
5253
29D
1045
6652
Not
e:N
oV
FAs
foun
ddu
ring
the
peri
od.
Col
lect
edbi
ogas
cont
aine
da
cons
ider
able
amou
ntof
O2,
met
hane
amou
ntw
aslo
wer
than
expe
cted
acco
rdin
gto
mea
sure
men
tw
ith
port
able
gas
anal
yzer
.
a Based
onT
San
dV
Sof
prep
ared
food
port
ion
dete
rmin
edla
tely
.O
LR
from
Alg
aein
the
feed
mix
ture
isgi
ven
here
only
.T
heto
tal
OL
Rin
clud
es0.
5gV
Spr
imar
ysl
udge
/L/d
ay.
APPENDIX B. RAW DATA 61
TableB.3:Pe
rforman
ceda
tafortheexpe
rimentdu
ringfeed
ingwith
NA
ata20
days
HRT
(Day
35-54).The
gasprod
uctio
ngivenhe
reis
at37±2
°C,a
sob
served
.
Day
Act
ual
OL
Ra
Vol
ume
gas
Yie
lds
Met
hane
Tot
alV
FAsb
pHT
Sin
dige
ster
VS
indi
gest
erV
Sre
duct
ion
Uni
tsg
VS/
L/D
aym
Lm
L/a
dded
gV
S%
mM
/LS.
U.
%%
%
Dig
este
rD
9D
10D
9D
10D
9D
10D
9D
10D
9D
10D
9D
10D
9D
10D
9D
10D
9D
10
351.
253.
3029
7838
8937
226
151
4336
1.25
3.30
2978
4088
372
274
00
7.26
7.25
371.
783.
3029
7841
2737
227
738
1.78
3.30
2553
3592
252c
241
391.
783.
3031
6141
6531
227
940
1.78
3.30
3708
4356
367
287
00
7.28
7.41
4.31
6.18
66.6
866
.85
4750
411.
423.
0028
7040
1228
426
442
1.09
3.00
2594
4127
324
295
5245
431.
093.
0029
8837
4537
326
70
07.
27.
2744
1.09
3.00
2870
3554
359
254
451.
093.
0029
4837
4536
926
746
1.09
3.00
2791
3859
349
276
471.
093.
0026
7338
9733
427
80
07.
257.
374.
146.
9166
.76
64.0
232
4348
1.09
3.00
2948
3821
369
273
491.
373.
0028
7037
6335
926
957
4350
1.37
3.00
2791
3303
349
236
00
7.19
7.36
511.
373.
0024
7732
6131
023
352
1.37
3.00
2516
3136
314
224
531.
373.
0027
1238
4733
927
554
1.37
3.00
2791
3554
349
254
012
.52
7.29
7.35
4.34
7.29
67.2
265
.87
2836
a Based
onT
San
dV
Sof
prep
ared
food
port
ion
dete
rmin
edla
tely
.O
LR
from
Alg
aein
the
feed
mix
ture
isgi
ven
here
only
.T
heto
tal
OL
Rin
clud
es0.
5gV
Spr
imar
ysl
udge
/L/d
ay.
b Tot
alV
FAs
repr
esen
tth
esu
mof
Ace
tate
,P
ropi
onat
e,B
utyr
ate,
Isob
utyr
ate,
Val
erat
ean
dIs
oval
erat
eas
mea
sure
dby
GC
.c S
udde
ndr
opof
gas
prod
ucti
onra
tew
asm
onit
ored
care
fully
and
obse
rved
that
itw
asbe
caus
eof
wor
sepe
rfor
man
ceof
gas
met
erus
ed.
How
ever
,th
ega
spr
oduc
tion
rate
was
reco
vere
dw
hen
gas
met
erw
asre
plac
edw
ith
calib
rate
don
e.
APPENDIX B. RAW DATA 62
TableB.4:P
erform
ance
data
fort
heexpe
rimentd
uringfeed
ingwith
NA
ata20
days
HRT
(Day
55-77)
whilega
sprodu
ctionrate
ofdigester
D10
deterio
rated
serio
usly
becauseof
high
accu
mulationof
VFA
s.The
gasprod
uctio
ngivenhe
reis
at37±2
°C,a
sob
served
.Day
Vol
atile
Fatt
yA
cida
Vol
ume
gas
Yie
lds
OL
Rb
Ace
tate
Pro
pion
ate
But
yrat
eIs
obut
yrat
eIs
oval
erat
epH
TS
indi
gest
erV
Sin
dige
ster
VS
redu
ctio
n
Uni
tsm
Lm
L/
gV
SA
lgae
cP
Sdm
M/L
mM
/Lm
M/L
mM
/Lm
M/L
S.U
.%
%%
Dig
este
rD
9D
10D
9D
10D
10D
9D
10D
10D
10D
10D
10D
9D
10D
9D
10D
9D
10D
9D
10
5524
7727
5931
019
73
0.5
2836
5631
0536
3738
826
03
0.5
5726
3427
1832
919
43
0.5
012
.21.
50.
30.
60.
77.
297.
2358
2516
2885
314
206
30.
559
2555
2690
319
192
3.36
0.5
6026
3425
5132
916
53.
360.
561
2673
3293
306
213
3.36
0.5
043
.40
00
07.
257.
044.
37.
569
.369
.332
3462
2870
1438
329
930
0.5
6.89
6327
1278
831
139
43.
360.
50
72.1
9.4
4.3
2.6
3.8
7.05
6426
3413
4530
287
01.
57.
246.
9465
2678
1299
335
213
01.
566
2411
1391
301
228
1.35
0.76
6729
0215
3136
318
11.
350.
7668
2411
1762
301
208
1.39
0.76
010
7.8
13.9
11.0
3.3
5.14
7.67
6.72
4.1
6.7
72.1
69.3
3333
6923
2118
5528
421
51.
390.
7670
2678
2688
328
312
0.7
0.38
6.78
7125
4417
9230
841
70.
70.
3872
2411
2895
301
673
0.7
0.38
7324
1134
4630
180
20
0.78
7424
5544
8030
614
300
0.78
7524
5535
4930
611
331.
50.
50.
280.
333.
580
01.
27.
357.
46-
4.7
-70
.136
2976
2500
3515
312
436
1.5
0.5
7724
1118
6130
023
11.
50.
50
0.35
00
00
7.26
7.35
a Pro
pion
ate,
But
yrat
e,Is
obut
yrat
e,Is
oval
erat
ew
ere
not
foun
din
D9
over
the
peri
od.
b Org
anic
Loa
ding
Rat
eas
gV
S/L
/D.
InD
9,
OL
Rw
as1.
5g
VS
ofN
A/L
/Dan
d0.
5g
VS
ofP
rim
ary
Slud
ge/L
/Dov
erth
epe
riod
(55-
77)
c NA
.d P
rim
ary
Slud
ge.
APPENDIX B. RAW DATA 63
TableB.5:Pe
rforman
ceda
tafortheexpe
rimentdu
ringfeed
ingwith
OA
inD
10an
dwith
NA
inD
9,at
a20
days
HRT
(Day
99-107
).The
gasprod
uctio
ngivenhe
reis
at37±2
°C,a
syields
observed
attheincuba
tionroom
. Day
Par
amet
erU
nits
Dig
este
r99
100
101
102
103
104
105
106
107
Act
ual
OL
Ra
gV
S/L
/Day
D9
1.34
1.09
1.09
1.09
1.09
1.09
1.09
1.09
1.16
D10
1.69
1.50
1.50
1.50
1.50
1.50
1.50
1.50
1.42
Gas
prod
uced
mL
D9
2733
3010
2560
2768
2837
2941
2872
2906
2976
D10
3323
3738
3000
3415
3415
3369
3369
3231
3369
Bio
gas
yiel
dsm
L/a
dded
gV
SD
931
234
332
034
635
536
835
936
337
2D
1038
042
837
542
742
742
142
140
442
1M
etha
ne%
D9
48.8
51.1
D10
53.9
49.6
H2S
ppm
D9
1938
>20
00D
10>
2000
>20
00pH
S.U
.D
97.
197.
267.
29D
107.
227.
267.
29T
Sin
dige
ster
s%
D9
4.38
D10
4.99
VS
indi
gest
ers
%D
971
.7D
1060
.6V
Sre
duct
ion
%D
921
D10
24
a Based
onT
San
dV
Sof
prep
ared
food
port
ion
dete
rmin
edla
tely
.O
LR
from
Alg
aein
the
feed
mix
ture
isgi
ven
here
only
.T
heto
tal
OL
Rin
clud
es0.
5gV
Spr
imar
ysl
udge
/L/d
ay.
APPENDIX B. RAW DATA 64
TableB.6:Pe
rforman
ceda
tafortheexpe
rimentdu
ringfeed
ingwith
OA
pretreated
with
autoclaveat
121
°Cfor30
minutes,at
a20
days
HRT
(Day
108-117).The
gasprod
uctio
ngivenhe
reis
at37±2
°C,a
syields
observed
attheincuba
tionroom
.
Day
Par
amet
erU
nits
Dig
este
r10
810
911
011
111
211
311
411
511
611
7
Act
ual
OL
Ra
gV
S/L
/Day
D9
1.16
1.16
1.16
1.16
1.16
1.16
1.16
1.16
1.16
1.16
D10
1.42
1.42
1.42
1.42
1.42
1.42
1.42
1.42
1.42
1.42
Gas
prod
uced
mL
D9
2976
2906
2976
2941
2803
2733
2872
2872
2941
2837
D10
3682
3601
3763
3560
3601
3237
3480
3318
3358
3318
Bio
gas
yiel
dsm
L/a
dded
gV
SD
936
135
336
135
734
033
234
934
935
734
5D
1047
946
949
046
446
942
145
343
243
743
2M
etha
ne%
D9
53.8
D10
49.1
H2S
ppm
D9
2900
D10
2600
pHS.
U.
D9
7.24
7.19
D10
7.26
7.19
TS
indi
gest
ers
%D
93.
87D
104.
824.
6V
Sin
dige
ster
s%
D9
74.0
D10
60.5
58.0
VS
redu
ctio
n%
D9
3232
D10
2931
a Based
onT
San
dV
Sof
prep
ared
food
port
ion
dete
rmin
edla
tely
.O
LR
from
Alg
aein
the
feed
mix
ture
isgi
ven
here
only
.T
heto
tal
OL
Rin
clud
es0.
5gV
Spr
imar
ysl
udge
/L/d
ay.