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

1

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.

Copyright

The publishers will keep this document online on the Internet – or its possible replacement – for aperiod of 25 years starting from the date of publication barring exceptional circumstances.

The online availability of the document implies permanent permission for anyone to read, to down-load, or to print out single copies for his/her own use and to use it unchanged for non-commercialresearch and educational purposes. Subsequent transfers of copyright cannot revoke this permission.All other uses of the document are conditional upon the consent of the copyright owner. The publisherhas taken technical and administrative measures to assure authenticity, security and accessibility.

According to intellectual property law the author has the right to be mentioned when his/her workis accessed as described above and to be protected against infringement.

For additional information about Linköping University Electronic Press and its procedures for publi-cation and for assurance of document integrity, please refer to its www home page: http://www.ep.liu.se/.

© Rajib Biswas, February 2009

Pictures and Illustrations: Rajib Biswas

2

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.

3

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)704174553rajib14@gmail.comwww.biogasexpert.com1

1The main contents of the website still under processing, will be available soon!

4

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

6

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

8

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

10

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.

11

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.

13

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.

�

���

������ ��� ���

�

�����

����� ���

�

�����

������ ��

��� � �� �� ��

���

�

��

���

���

���

���

���

� � �� �� �� �� �� �� ��

�� �

�����������

������������ ����� ���� ������������ �����

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

�

���

������

��� ���

�

��� ������ ��

���

�

��� ����

��� ��

���

�

��

���

���

���

���

���

���

���

���

� � �� �� �� �� �� �� ��

�� �

�����������

���������������� ��������������������

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

�

����������

�����������

�

���������� ����� �����

�

��������

���� ����

�

�����

������

��������

�

�����

�����������

����

�

������

�����������

����

�

��

���

���

���

���

� �� �� �� �� �� ��

� ��

�� ��������

���������������� ��������������������

������������������ !��"������������ !��� �#$����%�!�&'#�!��#$��������� ��("�

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

� ��

���

���

������ ��� ���

��� �� �� � �� ��� ���

�

� �� �� �� �� ��� ���

������

���������������������

� � �� �� �� �� �� �� �� ��

� ��

�� ��������

���������������� ��������������������

������������������������������������ ����������������������!

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

Bibliography

[1] Weckström K. (2006): Assessing recent eutrophication in coastal waters of the Gulf of Finland(Baltic Sea) using subfossil diatoms. Journal of Paleolimnology 35:571–592.

[2] Gray J.S., Wu R.S. and Or Y.Y. (2002): Effects of hypoxia and organic enrichment on thecoastal marine environment. Marine Ecology Progress Series. 238:249–279.

[3] Eklund B., Svensson A. P., Jonsson C. and Malm T. (2005): Toxic effects of decomposing redalgae on littoral organisms. Estuarine, Coastal and Shelf Science. 62:621–626.

[4] Malm, T., Råberg, S., Fell, T., Carlson, P. (2004): Effects by beaches cleaning on littoral waterquality, microbial food web and macro faunal biodiversity. Estuarine, Coastal and Shelf Science.60:339–347.

[5] Sauviat, M.P., Pages, N. (2002): Cardiotoxicity of lindane, a gamma isomer of hexachlorocyclo-hexane. Journal de la Societe de Biologie. 196:339–348 (in French).

[6] Rönnberg C., Bonsdorff E. (2004): Baltic Sea eutrophication: area-specific ecological conse-quences. Hydrobiology. 514: 227–241.

[7] Börjesson P., Mattiasson B. (2008): Biogas as a resource-efficient vehicle fuel. Trends in Biotech-nology. 26 (1) DOI:10.1016/j.tibtech.2007.09.007.

[8] Crouzet, P., Leonard, J., Nixon, S., Rees, Y., Parr, W., Laffon, L., Bøgestrand,J., Kristensen, P., Lallana, C., Izzo, G., Bokn, T., Back, J. & Lack, T.J. (1999):Nutrients in European ecosystems. In: Thyssen, N. (Ed.), Environmental AssessmentReport no-4. European Environmental Agency. p. 82. Date Accessed on 12-06-2007.http://reports.eea.europa.eu/ENVIASSRP04/en/enviassrp04.pdf

[9] Angelidaki, I., Ellegaard, L. & Ahring B.K. (2003): Applications of the anaerobic digestionprocess. Advances in Biochemical Engineering/Biotechnology, Biomethanation II. 82:1-33.

[10] Zinder S. H. (1984): Microbiology of Anaerobic Conversion of Organic Wastes to Methane:Recent Development. ASM News. 50 (7): 294-298.

[11] Raven R.P.J.M., Gregersen K.H. (2004): Biogas plants in Denmark: successes and setbacks.Renewable and Sustainable Energy Reviews. 11 (2007): 116–132.

[12] Berglund, M and Börjesson, P. (2006): Assessment of energy performance in the life-cycle ofbiogas production. Biomass and Bioenergy. 30 (3): 254- 266.

[13] Börjesson, P. & Berglund, M. (2006): Environmental systems analysis of biogas systems-Part I:Fuel-cycle emissions. Biomass & Bioenergy. 30 (5): 469-485.

[14] EurObservER (2007): Biofuels Barometer. Systemes Solaires. 179: 63– 75.

[15] Börjesson, P. & Berglund, M. (2007): Environmental systems analysis of biogas systems—PartII: The environmental impact of replacing various reference systems. Biomass & Bioenergy. 31:326–344.

52

BIBLIOGRAPHY 53

[16] Alberto V. F., Gisela V., Nelson A. & Antonio V. (2008): Evaluation of marine algae as a sourceof biogas in a two-stage anaerobic reactor system. Biomass & Bioenergy. 32: 338-344.

[17] Ahring BK. (2002): Perspectives for anaerobic digestion. Advances in Biochemistry Engineer-ing/Biotechnology. 81: 1–30.

[18] Benabdallah E. T., Dosta J., Ma´ rquez-Serrano R., Mata-A´ lvarez J. (2007): Effect of ul-trasound pretreatment in mesophilic and thermophilic anaerobic digestion with emphasis onnaphthalene and pyrene removal. Water Research. 41: 87-94.

[19] Oremland, R.S. (1988): The biogeochemistry of methanogenic bacteria. In: Zehnder, A.J.B.(ed.) Biology of Anaerobic Microorganisms. (pp. 641 - 706) J. Wiley and Sons, N.Y.

[20] Boone D.R. (1991): Ecology of methanogenesis. In: JE Rogers and WB Whitman (eds.). Mi-crobial Production and Consumption of Greenhouse Gases: Methane, Nitrogen Oxides, andHalomethanes. (pp. 57-70) American Society for Microbiology, Washington.

[21] Crill, P.M., R.C. Harriss, & K.B. Bartlett. (1991): Methane fluxes from terrestrial wetlandenvironments. In: Rogers J.E. and Whitman W. (eds.) Microbial Production and Consumptionof Greenhouse Gases: Methane, Nitrogen Oxides, and Halomethanes. (pp. 91-109) AmericanSociety for Microbiology, Washington, D.C.

[22] Hofman-Bang, J., Zheng, D., Westermann, P., Ahring, B. K. & Raskin, L. (2003): Molecu-lar Ecology of Anaerobic Reactor Systems, B. K. Ahring (Ed.), Biomethanation, Advances inBiochemical Engineering/Biotechnology, 81: 151-203. Springer-Verlag, Inc.

[23] de Mes, T. Z. D., Stams, A. J. M., Reith, J. H. & Zeeman, G. (2003): Methane production byanaerobic digestion of wastewater and solid wastes. In: Reith, J. H., Wijffels, R. H., Barten, H.(Eds.), Bio-methane & Bio-hydrogen. Status and Perspectives of Biological Methane and Hydro-gen Production. (pp. 58–102) Dutch Biological Hydrogen Foundation, Petten, The Netherlands.

[24] Stams A. J. (1994): Metabolic interactions between anaerobic bacteria in methanogenic envi-ronments. Antonie Van Leeuwenhoek. 66: 271–294.

[25] Kaseng, K., Ibrahim, K., Paneerselvam, S.V. & Hassan, R.S. (1992): Extracellular Enzymesand Acidogen Profiles of a Laboratory-Scale Two-Phase Anaerobic Digestion System. ProcessBiochemistry. 27(1): 43-47.

[26] Karakashev, D., Batstone, D. J. & Angelidaki, I. (2005): Influence of environmental condi-tions on methanogenic compositions in anaerobic biogas reactors. Applied and EnvironmentalMicrobiology. 71: 331-338.

[27] Lettinga, G. & Haandel, A.C. (1993): Anaerobic digestion for energy production and environ-mental protection, in Renewable energy; Sources for fuels and electricity, T.B. Johansson, et al.,Editors. Island press, California: London. p. 817-839.

[28] Schnurer, A., Zellner G. & Svensson B.H. (1999): Mesophilic syntrophic acetate oxidation duringmethane formation in biogas reactors. FEMS Microbiology Ecology. 29 (3): 249-261.

[29] Wang, Q., Kuninobu, M., Kakimoto, K., Ogawa, H. I. & Kato, Y. (1999): Upgrading of anaerobicdigestion of waste activated sludge by ultrasonic pretreatment. Bioresource Technology. 68:309–313.

[30] Park C., Lee C., Kim S., Chen Y. & Chase H.A. (2005): Upgrading of anaerobic digestionby incorporating two different hydrolysis processes. Journal of Bioscience and Bioengineering.100(2): 164-167.

[31] Koster, I.W. & Koomen, E. (1988): Ammonia inhibition of the maximum growth rate (µm)of hydrogenotrophic methanogens at various pH-levels and temperatures. Applied Microbiologyand Biotechnology. 28: 500-505.

BIBLIOGRAPHY 54

[32] Bagi, Z., Acs, N., Balint, B., Horvath, L., Dobo, K., Perei, K. R., Rakhely, G. & Kovacs,K. L. (2007): Biotechnological intensification of biogas production. Applied Microbiology andBiotechnology. 76(2): 473-482.

[33] Schink, B. (1997): Energetics of syntrophic cooperation in methanogenic degradation. Microbi-ology and Molecular Biology Reviews. 61 (2): 262-280.

[34] Stroot P.G., McMahon K.D., Mackie R.I. & Raskin L. (2001): Anaerobic codigestion of munic-ipal solid waste and biosolids under various mixing conditions – I. Digester performance. WaterResearch. 35(7): 1804-1816.

[35] Gray, J. S. (1992): Eutrophication in the sea. In Colombo, G. C., I. Ferrari, U. V. Ceccherelli& R. Rossi (eds), Proc. 23rd EMBS. Olsen & Olsen, Fredensborg. 3–13.

[36] Nixon, S. W. (1995): Coastal marine eutrophication: A definition, social causes, and futureconcerns. Ophelia. 41: 199-219.

[37] Jørgensen, B. B. & Richardson, K. (1996): Eutrophication in coastal marine ecosystems. Coastaland Estuarine Studies 52. American Geophysical Union, Washington, DC. 1–272.

[38] Cloern, J. E. (2001): Our evolving conceptual model of the coastal eutrophication problem.Marine Ecology Progress Series. 210: 223–253.

[39] Boesch D., Hecky R., O’Melia C., Schindler D. W. & Seitzinger S. (2006): Eutrophication ofSwedish Seas. (Final Report, Swedish Environmental Protection Agency, Stockholm Sweden).

[40] Larsson U., Elmgren, R. & Wulff, F. (1985): Eutrophication and the Baltic Sea: causes andconsequences. Ambio. 14: 9-14.

[41] Naturvårdsverket. (2003): Ingen övergödning: Underlagsrapport till fördjupad utvärding avmiljömålsarbetet. Rapport 5319, ISBN 91-620-5319-1. Naturvårdsverket, Stockholm.

[42] Hägerhäll, A. B. (2001): Nitrogen Fixation in the Baltic Sea: Brief Guide for Environmen-tal Managers. Report from a workshop arranged by MARE. Date accessed on 2007-06-20.http://www.mare.su.se/dokument/nitrogenfix.pdf .

[43] Ministry of Environment (MoE), Finland. (2004): Curbing eutrophication in theBaltic Sea. Background briefing document for the Meeting of the Finnish Com-mission on Sustainable Development 8.12.2004. Date Accessed on 01-07-2007.http://www.environment.fi/download.asp?contentid=43542&lan=en.

[44] HELCOM (The Helsinki Commission), Date accessed on 2008-08-31.http://www.helcom.fi/environment/intro/en_GB/introduction/.

[45] Meyer-reil, L. & Köster, M. (2000): Eutrophication of Marine Waters: Effects on BenthicMicrobial Communities. Marine Pollution Bulletin, Vol. 41, pp. 255-263.

[46] Swedish Environmental Protection Agency (Swedish EPA), Date accessed 12-05-2007,http://www.naturvardsverket.se.

[47] Rajasilta, M., Laine, P. & Eklund, J. (2006): Mortality of herring eggs on different algal sub-strates (Furcellaria spp. and Cladophora spp.) in the Baltic Sea - an experimental study, Hy-drobiologia. 554: 127-130.

[48] Hansen T. L., Schmidt J. E., Angelidaki I., Marca E., Jansen J. I. C., Mosbaek H. & ChristensenT. H. (2004): Method for determination of methane potentials of solid organic waste. WasteManagement. 24: 393–400.

[49] Shelton D.R. & Tiedje J.M. (1984): General method for determining anaerobic biodegradationpotential. Applied and Environmental Microbiology. 47 (4): 850-857.

[50] Samson, R. & Leduy, A. (1986): Detailed study of anaerobic digestion of Spirulina maxima algalbiomass. Biotechnology and Bioengineering. 28: 1014-1023.

BIBLIOGRAPHY 55

[51] Benabdallah E. H. T., Dosta, J., Márquez, S. R. & Mata, A. J. (2007): Effect of ultrasoundpretreatment in mesophilic and thermophilic anaerobic digestion with emphasis on naphthaleneand pyrene removal. Water Research. 41: 87-94.

[52] Tiehm, A., Nickel, K., Zellhorn, M. & Neis, U. (2001): Ultrasonic waste activated sludge disin-tegration for improving anaerobic stabilization. Water Research. 35(8): 2003-2009.

[53] Bougrier, C., Albasi, C., Delgenes, J.P. & Carrere, H. (2006): Effect of ultrasonic, thermal andozone pre-treatments on waste activated sludge solubilisation and anaerobic biodegradability.Chemical Engineering and Processing. 45: 711–718.

[54] Swedish Standard Institute – SIS, Characterization of sludges – Determination of dry residueand water content. SS-EN 12880:2000.

[55] European Standard. Characterization of Sludges – Determination of the loss on ignition of drymass. EN 12879:2000.

[56] Y. Hong-Wei & Brune, D. E. (2007): Anaerobic co-digestion of algal sludge and waste paper toproduce methane. Bioresource Technology. 98 (1): 130-134.

[57] Golueke, C. G., Oswald, W. J. & Gotaas H. B. (1957): Anaerobic Digestion of Algae. Appliedand Environmental Microbiology. 5(1): 47-55.

[58] Cecchi, F., Pavanb, P. & Mata-Alvarez, J. (1996): Anaerobic co-digestion of sewage sludge:application to the macroalgae from the Venice lagoon. Resource, Conservation and Recycling.17: 57-66.

[59] Gunaseelan V. N. (1997): Anaerobic digestion of biomass for methane production: A review.Biomass and Biornergy. 13: 83-114.

[60] Hansson, G., (1981): Methane Fermentations: End Product Inhibition Thermophilic MethaneFormation and Production of Methane from Algae. Ph.D. Dissertation, Dept. of Technical Mi-crobiology, University of Lund, Sweden.

[61] Hanisak, M. D. (1981): Methane production from the red seaweed Gracilaria tikvahiae. In Proc.10th Int. Seaweed Symp., ed. T. Levring. Walter de Gruyter, Berlin. pp. 681-686.

[62] Pind P. F, Angelidaki I., Ahring B. K, Stamatelatou K. & Lyberatos G. (2003): Monitoring andcontrol of anaerobic reactors. Advances in biochemical engineering/biotechnology. 82:135-82.

[63] Svein Jarle Horn, S. J. (2000): Bioenergy from brown seaweeds. Ph.D. Dissertation, Departmentof Biotechnology, Norwegian University of Science and Technology NTNU.

[64] Marinari, S., Masciandaro, G., Ceccanti, B. & Grego, S. (2000): Influence of organic and mineralfertilizers on soil biological and physical properties. Bioresource Technology. 72: 9-17.

[65] Debosz, K., Petersen, S.O., Kure, L.K. & Ambus, P. (2002): Evaluating effects of sewage sludgeand household compost on soil physical, chemical and microbiological properties. Applied SoilEcology. 19: 237-248.

[66] Odlare, M. (2005): Organic residues - a resource for arable soil. Doctoral thesis, Department ofMicrobiology. Swedish University of Agricultural Sciences. Uppsala.

[67] Odlare, M., Pell, M. & Svensson, K. (2008): Changes in soil chemical and microbiologicalproperties during 4 years of application of various organic residues. Waste Management 28:1246–1253.

[68] Kayhanian, M. (1994): Performance of a high-solids anaerobic digestion process under variousammonia concentrations. Journal of Chemical Technology & Biotechnology. 59: 349–352.

[69] Taherzadeh M. J., Karimi K. (2008): Pretreatment of lignocellulosic wastes to improve ethanoland biogas production: A review. International Journal of Molecular Sciences. 9: 1621-1651.

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.