Biomethanation of Red Algae from the Eutrophied Baltic Sea

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Biomethanation of Red Algae from the

Eutrophied Baltic Sea

Rajib Biswas

M A S T E R ’ S

T H E S I S

Environmental Science Master Programme

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Supervisor:

Prof. Jörgen Ejlertsson

Department of Water and Environmental Studies Linköping University

SE-581 83 Linköping Sweden.

&

Research and Development Director, Vice President

Scandinavian Biogas Fuels AB

Väderkvarnsgatan 14 SE-753 29 Uppsala

Sweden.

Examiner:

Prof. Bo Svensson

Department of Water and Environmental Studies Linköping University

SE-581 83 Linköping Sweden.

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Copyright

The publishers will keep this document online on the Internet – or its possible replacement – for a period 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-commercial research 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 publisher has 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 work is 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

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Abstract

In the semi-enclosed Baltic Sea, excessive filamentous macro-algal biomass growth as a result of eutrophication is an increasing environmental problem. Drifting huge masses of red algae of the genera Polysiphonia, Rhodomela, and Ceramium accumulate on the open shore, up to five tones of algae per meter beach. During the aerobic decomposition of these algal bodies, large quantities of red 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 different seasons [October and March], was investigated through three different batch digestion experiments and laboratory scale CSTR [continuous stirred tank reactor] at mesophilic (37o_{C) condition.}

Au-toclavation [steam and heat] and ultrasound pretreatments were applied in order to enhance the biodegradation. In STR, anaerobic codigestion of algal biomass with SS [sewage sludge] was applied with a gradual increase in organic loading rate [1.5-4.0 g VS/L/day] and operated for 117 days at 20 days HRT [hydraulic retention time]. Reactor digestate was analyzed four times over the period to determine the nutrients and heavy metals content.

It is concluded that the methane potential of algae harvested in October is almost two-fold than that of algae harvested in March, probably due to it’s higher [more than double] nitrogen richness. An increase 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 VSaddedat 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 maximum biogas yield 0.49 m3_{/kg VS}

addedat 310oK , was obtained from algae [harvested in October] pretreated

with autoclave. The methane content of the produced biogas was 54%. Average [over a short period, day 99-107, reactor showed steady performance] maximum biogas yields from untreated algae obtained 0.44 m3_{/kg VS}

added 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 was

above the maximal limit among the heavy metals. The sand content in algae during harvesting was considered as a factor to disrupt the operation. Codigestion of Polysiphonia algal biomass with substrate with higher C:N ratio like paper mill waste should be more appropriate to increase the methane and biogas yields. It is inconclusive whether AD process is a good method to dewater red algae or not, but large scale harvesting of algae will definitely contribute to curb eutrophication of the Baltic Sea through decreasing N and P level.

Key words: Anaerobic Digestion, Baltic Sea, Biogas, Codigestion, Eutrophication, Nutrients,

Pre-treatment, Red Algae.

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Preface

This thesis is the final part to the fulfillment of Master of Science in Environmental Science Degree from theDepartment 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 with

Scandinavian Biogas Fuels ABfrom November 2007 to July 2008.

For any questions about this thesis, please don’t hesitate to contact me. AND we all should consider one 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)704174553

rajib14@gmail.com www.biogasexpert.com1

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

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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 steering

equipments and gas meters. . . 30

4.1 Accumulated biogas in bottles [error bars show the standard deviation] of the first batch experiment investigating biogas potential of OA [−■−], OA pretreated in au-toclave [−▲−] and Whatman paper [−♦−]. Control [− ⋇ −] shows accumulation of biogas produced from the inoculum. . . 35 4.2 Specific methane yields (i.e., methane produced from inoculum was substracted) in the

first batch experiment investigating biogas potential of OA [−■−], OA pretreated in autoclave [_−▲−] and Whatman paper [_−♦−]. Error bars show the standard deviation and yields are corrected at STP. . . 36 4.3 Specific methane yields (i.e., methane produced from inoculum was substracted) in the

second 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 [−■−], NA pretreated with ultrasound [−▲−] and Whatman paper [−♦−] (up to day 40). Given data are averaged over three samples [error bars show the standard deviation, almost unseen] and corrected at STP. . . 39 4.5 Biogas yields in the reactors D9[−♦−] and D10[−■−] and CH4amounts in produced

gas of the reactors D9 [

■

] and D10 [

■

] between Day 99-117. Here both reactors

showed steady performance. D10was 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 the

experimental 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 produced

gas amount given here as observed at 37±2 °C. . . 43 6

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4.8 Destruction of volatile solids over the experimental period in reactor D9 [♦] and D10

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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 the anaerobic 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 specific yields 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] for

the experiment at a 20 days HRT. The gas production given here is at 37±2 °C, as observed in the incubation room. . . 41 4.4 The maximal content of metals in the digestate [According to the Swedish quality

assuring system SPCR 120] . . . 44 4.5 The characteristics and nutrients content of algae and digestate from reactor D9 and

D10 based on the analyses reports. . . 46

4.6 Results from previous studies on anaerobic digestion of marine biomass in different conditions 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 (Day

18-34). 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 (Day

35-54). The gas production given here is at 37±2 °C, as observed. . . 61

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B.4 Performance data for the experiment during feeding with NA at a 20 days HRT (Day 55-77) while gas production rate of digester D10deteriorated seriously because of high

accumulation 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 in

D9, at a 20 days HRT (Day 99-107). The gas production given here is at 37±2 °C, as

yields observed at the incubation room. . . 63 B.6 Performance data for the experiment during feeding with OA pretreated with autoclave

at 121 °C for 30 minutes, at a 20 days HRT (Day 108-117). The gas production given here is at 37±2 °C, as yields observed at the incubation room. . . 64

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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 m}3_{= 35.31 cu ft)}

mL milliliter

mL/g milliliter per gram (1 mL/g = 1 L/kg = 0.001 m3_/kg)

OLR Organic Loading Rate TS Total Solids

VFAs Volatile Fatty Acids VS Volatile Solids

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Chapter 1

Introduction

1.1 General introduction

Anaerobic digestion of marine algae to provide renewable energy is an attractive possibility. Algal cells convert and store solar energy through their photosynthetic activities. They can be degraded to for methane through anaerobic digestion. Moreover, the whole digestion process could be accelerated by existing advanced technologies (e.g. codigestion, pretreatment etc.) in order to increase the biogas yield. Further, the digestate can be used as a fertilizer on arable land to improve soil quality since digestates are considered as promising sources of N, P, K and S and other essential macro and micro nutrients for plants in available forms.

Eutrophication [Section 2.3] in coastal and inland waters caused by increased input of nutrients or organic matter resulting in excessive planktonic and filamentous algal biomass growth is one of the most 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 to the environmental degradation as the portions are almost enclosed by landscape and the shallow brackish sea characterized by cold temperature. Besides, the pollution of the Baltic Sea is high because 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 the semi-enclosed Baltic Sea, where the nutrient load has strongly increased from its natural level, this has led to marked changes in the coastal ecosystems [1]. This has resulted in increased plankton biomass and increased amount of filamentous algae; decreased transparency of water body, changes in 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, and

Ceramium are regularly washed up on beaches of the central Baltic Sea [3] and during the summer

it cover shallow bottoms close to the shore. Winds and currents move the masses towards the shore and 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 into the water which is toxic for human health and the marine environment. Several organohalogenic compounds are produced by red algae, many of them being similar to toxic commercial products with 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 eutrophication and 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 an integrated effort, where harvesting of algal bodies and e.g. converting them into biogas is initiated. This would possibly become a profitable means both from economic and environmental aspects.

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Biogas production by the anaerobic digestion (AD) process is a promising means of achieving multiple environmental 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, hydrocarbons and 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 this will 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 anaerobic decomposition or landfilling. Also, large scale harvesting of algal biomass would contribute to curb eutrophication 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 eutrophied Baltic Sea. The experiments were carried out in laboratory scale stirred digesters [Section 3.1.2] and as three non-parallel batch experiments [Section 3.1.1]. Additionally, several methods, i.e. codigestion of 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 to study 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 renewable source of energy as well as to a sustainable management of the toxic red algae to eliminate it’s environmental 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 the Baltic 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 minutes}

increase 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 g VS algae L-1_D-1 _{and be codigested with primary sludge at a rate of 0.5 g VS L}-1_D-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 fed with algae?

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Chapter 2

Background

2.1 Biogas: A green energy

Biogas is a mixture of CO2and the inflammable gas CH4, which is produced by bacterial conversion

of organic matter under anaerobic (oxygen-free) conditions [11]. Several studies have showed that the 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 biogas

yields, 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 yield and 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 resource recovery [10]. After the rise of energy prices in 1970s, the process received renewed attention due to the need to find alternative energy sources to reduce the dependency on fossil fuels [9]. Due to the environmental advantages, the interest in biogas process still remained in spite of the fuels price decreased in early 1985. Recently this has become more evident due to the concern on the greenhouse gas emissions to the atmosphere. Plant biomass assimilate and store atmospheric CO2

through their photosynthetic activity. Therefore, when biomass is degraded in the AD process the recovered biogas may be burnt without the occurrence of any additional CO2 emission into the

atmosphere. In contrast, fossil fuels combustion increases the overall level of CO2 since it has been

sequestered in the earth since many millions of years.

The biogas, apart from being used for heat and electricity production and as a vehicle fuel, can also be distributed on the natural gas grid [13]. Biogas has to be upgraded at least to 96-97% CH4

content for the use as a motor vehicle fuel or before being injected into the natural gas grid. Biogas has a lower calorific value than natural gas and in specific applications such as automotive fuel [9]. Table 2.1 shows the upper and lower caloric value of biogas in comparison to natural gas.

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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

a_{STP (standard temperature and pressure), i.e. the volume at 0}o_{C and 1 bar pressure.}

Biogas has several advantages from an environmental and resource efficiency perspective compared to other biomass based vehicle fuels, which have so far been introduced [7]. Indirect environmental benefits occur, e.g. anaerobic digestion of crop residues and manure reduces the plant nutrient leaching from arable land, the use of digestates as a fertilizer reduces the need for chemical fertilizers leading to a more sustainable use of phosphorus and lower production of energy-demanding nitrogen fertilizers [13].

In the case of algal biomass, however, indirect environmental benefits are added to those of energy recovery. During the degradation of algal bodies on the beaches, considerable methane emissions to the atmosphere may take place. Furthermore, leaching of toxic red effluents into the water, decreases the quality of beaches which in turn affects the ecosystem and the use of beaches for recreation. Large scale harvest will balance N and P levels of the water body. Additionally, N, K and P in digestates after 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 as vehicle fuel [14]. In January 2007, the European Commission adopted new guidelines for an ambitious energy policy for Europe with a binding target of increasing the level of renewable energy in the EU from the current level of <7% to 20% by 2020 [7]. The biogas potential in Sweden is estimated to be some 50 PJ/year, which is ten times higher than the current production and corresponds to 3–4% of the current energy consumption in Sweden [15].

It is noteworthy that Sweden is the world leader in biogas system utilization. In Sweden, the present attention 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 is degraded to CO2 and CH4 by subsequent oxidations and reductions in absence of oxygen. This

process occurs in the environment (e.g. sediments, wetlands, swamps, paddy field etc.), in intestinal tracts 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 with aerobic degradation. In addition, energy conversion during the digestion process in form of CH4

makes the process economically profitable. Currently, the AD process is mainly utilized in four sectors 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.

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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 green energy 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 anaerobic degradation can be used as organic fertilizer [17]. Consequently, the digestates are expected to be free 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, laminaran and mannitol), with zero lignin and low cellulose content, which should make them an easy material to 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 knowledge concerning the difference microorganisms involved. It has been reported that only a few percent of bacteria and archaea have so far been isolated, and almost nothing is known about the dynamics and interactions between these and other microorganisms [22]. However, the anaerobic microbiological decomposition in AD process is a process in which micro-organisms derive energy and grow by metabolizing 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 required

its own characteristic group of micro-organisms [Figure 2.1].

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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 and fermentative bacterial groups ideally breaks down biopolymers (C100 -C10,000) into soluble organic

compound such as mono- and oligomers (C10-C100) e.g., sugars, amino acids, long chain fatty acids

etc. In case of more complex biopolymers, pretreatments of organic materials are often needed to accelerate this phase. The polymers are unavailable for intracellular metabolism because of their size 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 activity

may 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 fatty acids and alcohols are converted into acetate, CO2and H2by acetogenic bacteria [23]. This bacterial

group is termed as acetogens and are obligate hydrogen producer. Later, those products are used by methanogens to produce CH4.

H2 concentration is an important factor regulating the metabolic activities in both methanogenesis

and acetogenesis. Biogas formation from the fermentation products is thermodynamically possible only 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 a continuous 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 transfer or interspecies electron flow. Additionally, low acetate level (usually 10-4 and 10-1 M) is required for acetogens 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, the sensitive part of the process, where microorganisms show most sensitivity with the system’s chemical and physical environment. The two major pathways of methanogenesis are known as acetotrophic and hydrogenotrophic. Some 60-70% CH4 is produced via the acetotrophic pathway [24]. Methanogens

can 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 three metabolic pathways: acetotrophic (acetate metabolized), hydrogenotrophic (H2/CO2 metabolized)

and methylotrophic (methylated one-carbon compound metabolized). Often the hydrogenotrophic methanogens are able to use formate, while those using acetate for methane formation can only use acetate. However, Methanosarcina is metabolically and physiologically most versatile which possess all three known pathways for methanogenesis.

When acetate-utilizing methanogens are inhibited by e.g., ammonia, sulfides etc., bacteria will oxidize acetate to H2 and CO2 which is then the source of methane [33]. Thus, there is a syntrophic

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relationship between acetogens and methanogens and an interspecies hydrogen transfer or interspecies electron flow takes place [32, and references therein].

However, in methanogenesis, mainly acetate is converted to CH4 and CO2 as the end products in

anaerobic degradation of organic matter. Some other or even same methanogens use CO2 and H2

for 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 factors such as accumulation of VFAs, hydrogen pressure, buffering capacity, bicarbonate concentration in liquid phase and CO2 concentration in the gas phase, pH, ammonia concentrations and other toxic

substances, 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 factors are temperature, pH, substrate composition and toxins [9]. Variation in those parameters affects the process 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 can grow in a specific pH range [9]. For methanogenic and acetogenic organisms, an optimum pH range is between 6.5 and 7.5, while acidogenesis and hydrolysis has their optima around 6. However, the pH of an anaerobic digester and its optimization largely depends on the characteristics of the substrates used. It is noteworthy that, the optimum ranges given for anaerobic digestion processes in different studies may be contradictory. The favorable conditions for microbial growth of a process and its stability are often related to the characteristics of the inoculum used. This means, if the microbes grow 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 the bacterial growth and conversion rate of organic materials. Most anaerobic reactors are carried out within the mesophilic and thermophilic ranges [9]. The microbial growth within the psychrophilic temperature range is slow resulting in long retention time. Although the reactor operational energy input 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 comparatively small digester volumes, high hydrolysis rate of particular matter, efficient destruction of pathogens etc.

Both pH and temperature factors influence methanogenesis heavily. In addition, toxic compounds concentration (e.g., ammonia, sulfide) are also influenced by pH and temperature. For instance, in high temperature and higher pH, ammonia concentration is higher and more toxic to the methanogens than optimum conditions.

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2.2.2.2 Bicarbonate alkalinity

The pH of a reactor system is primarily controlled by the bicarbonate concentration. CO2 produced

during the biological conversion of biopolymers reacts with ammonia released from the degradation of nitrogen-containing organic matter and, thus, produce bicarbonate alkalinity. In this case ammonia acts as a strong base to react with the CO2. It is a better idea to control a system’s pH by measuring

bicarbonate alkalinity instead of measuring only pH. When bicarbonate concentration is low, pH may decrease quickly because of the low buffering capacity. The buffering mechanism is shown below:

CO

2-3+ 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 the}

alkalinity 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 even temporary or complete cessation of methane formation. However, VFAs are important substrates that are readily used by methanogenic microorganisms [29, 30]though high VFAs accumulation affects the 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 will

decrease. This may result in further accumulation of VFAs and decrease of pH. Accumulation of higher molecular weight VFAs may lead to complete cessation of methanogenic activities. Usually feeding 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 or formed during its degradation will lead to instability of the process. Although anaerobic microor-ganisms are less sensitive to toxic compounds than are aerobic microormicroor-ganisms, the growth rate of the anaerobes makes the re-establishment of a microbial community more time consuming. Toxicity of 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 the temperature and pH, the higher the toxicity [17, 31]. The adaptation to an inhibitory compound is, 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].

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2.2.2.5 Nutrients

Nutrients for anaerobes are grouped as macronutrients and micronutrients. Two macronutrients essential 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 possess

several unique enzyme systems leading to the need for some micronutrients, e.g. cobalt, iron, nickel and sulfide. Out of these micronutrients, methanogens need some other obligatory micronutrients such as selenium, tungsten, molybdenum as additional trace elements to complete the metabolism. Additional micronutrients of concern are calcium, magnesium, barium and sodium. The shortage of macro- 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 of algae and enhance the biogas production. The suggested optimum C/N ratio for anaerobic digestion is in the range of 20:1 to 30:1 [34, 56, and references therein]. The unbalanced nutrient composition of the algal sludge (low C/N ratio) was regarded as a limiting factor for its use in an anaerobic digestion process [56]. However, the C/N ratio was about 5.3/1 in algal sludge reported in previous studies [56]. The analyses reports show that algae (new and old) has a C/N ratio varying from 13/1 and 20/1. Therefore, the addition of primary sludge from a sewage treatment plant may provide the nitrogen 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 rate limiting as a result of the extent at which the substrate is possible to hydrolyze [9]. To enhance the overall degradability of substrate, different pretreatment techniques are being introduced [69]. The core function of different pretreatments is to break down the complex biopolymers, disrupt cell walls and bring out the chemical substances from polymers. In this study, autoclavation and ultrasound pretreatments were applied.

2.2.4.1 Pretreatment with autoclave

Steam and heat pretreatments have been applied in several studies to open up cellular structure making cell components accessible to hydrolytic enzymes. This is one of the proven mechanisms to accelerate the AD process.

2.2.4.2 Pretreatment with ultrasound

Numerous studies reveal that ultrasonic pretreatment affects anaerobic digestion [51, 52, 53], and for some substrates the biogas yields increase substantially. In other cases the effect is an increase of the process rate occur as a result of pretreatment with ultrasound. This means that a shorter retention time (RT) may be applied [17]. Ultrasound basically acts on particular material by decreasing particles 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 of bio-solids [69]. Increased microbial activities take place due to this disintegration resulting in higher biogas yields and higher organic solid reduction.

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2.3 The Baltic Sea eutrophication

The Baltic Sea is almost enclosed by land with a narrow and shallow straits connected with North Sea around Denmark and Sweden. More than 200 large rivers characterized by cold temperature bring fresh water into the Baltic Sea, which makes it the world’s biggest brackish sea. Exchange of water with the open sea is very limited. It typically takes about 25-30 years for all the water in the Baltic Sea to be replaced [44]. The Baltic Sea habitats and species are threatened by eutrophication and elevated amounts of toxic substances from agriculture and industrial waste stream regulated by human activities. At present, sixteen million people live in nine countries along the coast of the Baltic 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 waters during 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 higher forms of plant life to produce an undesirable disturbance to the balance of organisms present in the water and to the quality of the water concerned’’ [8]. Numerous papers explained and discussed the causes, consequences and definitions of eutrophication [35, 36, 37, 38]. Industrialization, intensifying agricultural 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 the mid-19th century [40] as a result of over-fertilization with phosphorus (P) and nitrogen (N) [39, and references therein]. Discharges of both N and P from sewage treatment plants are also significant contributors to eutrophication [41]. Interestingly, scientists have demonstrated that eutrophication is mainly regulated by the P level in the water body. Because, when the P level is high in the water, 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 by phosphorus [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 of nitrogen fixers may cause severe problem as they supply so much nitrogen that they aggravate local or regional eutrophication. Excessive nitrogen fixers generate undesirable excess of biomass in the water which exceeds the ecosystem’s ability to assimilate. According to MARE (Marine Research on Eutrophication) [42], Aphanizomenon, Nodularia and Anabaena are the most nitrogen producing genera 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 important species of nitrogen-fixing cyanobacteria in the Baltic Sea. Both species are heterocystous, filamentous and colonial. Surface blooms, which are patchy and episodic, are generally dominated by Nodularia but 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 the summer period though they occur the year round in the water. Toxic blue-green algal blooms can represent a considerable health risk for people and animals, and people are advised not to swim in bloomy water [43].

The sources of nutrients causing eutrophication in the Baltic Sea are often classified into point sources (settlements, industrial plants or fish farms), diffuse sources (agriculture, forestry, dispersed settlements, storm water), or airborne pollution (emitted from traffic or fossil fuels combustion for power 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 fauna and flora, additional undesired organic matter production, increase in oxygen consumption, oxygen depletion 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

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quantities of algae eventually end up on the seabed where their decomposition uses up oxygen. This can lead to anaerobic condition near the seabed. Moreover, subsequent decay of high plant biomass causes an increase in oxygen consumption which may lead to anoxic conditions in bottom waters and sediments, since the biological oxygen consumption exceeds the supply of oxygen by diffusion by orders of magnitude [45]. When the uppermost sediments on the seabed become anaerobic in this way, they release nutrients, particularly phosphorus, back into the water through a phenomenon known as internal loading [43]. Today roughly one-third of the bottom of the Baltic is practically dead, and the deepest basins mostly contain hydrogen sulphide instead of oxygen [46].

Consequently, filamentous macroalgae (red, brown and green) are reported abundant in the Baltic Sea and proliferated as a result of eutrophication. At the end of the summer, filamentous algae produce thick, loose mats covering shallow bottoms close to the sea shore. Huge masses of algae are accumulated on the beaches by winds and currents movements. Malm et al. [3, 4] recorded extended banks at the shores of south eastern Sweden, amounting up to five tonnes of algae per meter beach. The quality of beaches are declined, affecting tourism and severe environmental degradation is 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 most of 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 of red colored effluents leak and gradually mixed into the water. Several studies revealed that marine macroalgae produce a number of organohalogen compounds and many of these compounds are similar to toxic commercial products like pesticides. Some of them are claimed to have acute effects on the central nervous system and chronic endocrine effects [5], carcinogenic and nerve toxic effects [3, and references therein]. The extracts from accumulated filamentous red algae (Polysiphonia, Rhodomela and Ceramium) increase mortality in crustaceans, fish and fish larvae in the Baltic Sea [47, 4, 3, and references therein]. The red macroalgae under the family Rhodomelacea is dominating in the central Baltic Sea.

2.4.2 Characteristics and productivity

The term algae refer to a large and diverse assembly of eukaryotic organisms that contain chlorophyll and 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 easy material to convert to methane by anaerobic digestion processes [16]. Typically, algae are unicellular and microscopic, but assembled to multicellular organisms they constitute seaweeds. The most common groups of macroalgae are red algae (Rhodophyta), brown algae (Phaeophyta) and green algae (Cholorophyta). Polysiphonia, a genus of red algae under the division Rhodophyta, with more than 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 and heavily overgrown on the sea shore. Filamentous and typically well branched with a length up to 30 cm and polysiphonous construction are basic characteristics of Polysiphonia. The life cycle of triphasic (three phases) red algae Polysiphonia, consists of a sequence of a gametangial (gametes producing), carposporangial (carpospores producing) and tetrasporangial (tetraspores are produced from meiosis) phases.

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(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 with other seaweeds, but less than at 10%. Newly produced new algae (NA) appeared to be darker and with a shorter thallus than older sample (OA).

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Chapter 3

Materials and methods

3.1 Experimental overview

The anaerobic digestion of collected red algal biomass Polysiphonia were carried out in three separate batch experiments in 330 mL glass bottles [see 3.1.1] with a working volume of 100 mL and small stirred reactors [see 3.1.2] with an active volume of 4 L and once a day feeding. Batch experiments and reactor experiments were carried out during the period of November 2007 to July 2008 and April to 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 to incubate a small amount of material to be treated with an anaerobic inoculum and measure the methane 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 and Tiedje [49] with some modifications. Three individual series of experiments were carried out to determine 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 two

different 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, where inoculum, solutions and water were mixed with the substrate at oxygen free condition to produce a volume 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 in a climate room for the incubation at 37°C. Gas pressure in serum bottles was measured to calculate the biogas production. Gas samples were collected each time of pressure measurements to determine

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the methane concentration by gas chromatography (GC) [Section 3.4.6]. Pressure measurement and analysis of methane concentration were performed twice a week for the two first weeks, then once a week and finally less frequent. Gas production and methane concentration was followed over 35, 51 and 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 of algae and primary sludge in laboratory scale stirred tank reactors (STR). The effects of an increasing organic loading rate as well as of pretreatments of substrates were also applied to monitor reactor performance. In this experiment, Polysiphonia algal biomass was codigested with primary sludge collected from Himmerfjärdsverke water treatment plant, which is the fourth largest wastewater treatment 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 electronic regulators. Feeding occurred once-a-day. Two digesters were operated with the same working volume of 4 L [Section 3.3.2.1] under mesophilic conditions (37±2°C) in the dark.

The two digesters were labeled as D9and D10, D9was operated as control reactor; i.e., no significant

changes in digester operation conditions occurred. D10 was the test reactor, receiving increased

organic 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 4L

HRT Over the period 20 days 20 days

Mixing Level Over the period Intermittentb Intermittent

Digestion 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 PS

Day 105-120 1.5 g Algae + 0.5 g PS 3.0 g Algae (pretreated)d

+ 0.5 g PS

a_{From April 3 to July 28, 2008 (117 days).}

b_{Minimal and intermittent, designed stirring speed -400 rpm. 4 times a day, 15 minutes/time.}

c_{Organic Loading Rate, units g VS/L active volume/day.}

d_{The 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 sludge was performed five days a week (Monday-Friday). The liquid volume in the digesters was adjusted to 4L on every Monday. The following measurements were performed:

Biogas yields: The biogas production was determined daily by gas meters [Figure - 3.2] equipped

with 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 a

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Gas composition: Produced biogas from both reactors were captured in plastic balloons once a

week 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 VS

reduction [Section A].

Digestate nutrients: The final digestate from the reactors was also analyzed at the termination of

the experiment to evaluate the nutrients and heavy metals concentration. These analyses were done 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 Island of Öland in Sweden two times, early autumn (October 2007) and early spring (March 2008). Algae were packed in several layers in plastic air tight bags in a thick paper box and delivered withing one day to our laboratory, where it was stored . Once received, the collected seaweeds were kept in the refrigerator 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 were cut 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 kept in refrigerator at -20 °C in polythene bags for further use. Before using algae as feeding, TS and VS were 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 a higher length than that of New algae, greenish - brown color were main visual characteristics of Old algae. The organic matter content of these algae was lower than new algae (NA). However, the sand content 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 performed at same place on the Island of Öland and by the same person. Filamentous and branched but then shorter the OA. Their color was dark reddish with comparatively higher sand content as experience during preparation of samples. A comprehensive characteristic is given in the table 4.5.

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3.2.3.3 Primary sludge

The primary sludge was collected twice a month from Himmerfjärdsverket in southern Sweden and stored at 4-7°C for maximum one week before use. When received, the primary sludge container was shaken 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, if stored 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 substrate pretreated in autoclave at 121 °C for 30 minutes. About 100g homogenized OA was transferred to a serum bottle with an average volume of 330 mL and placed in the autoclave. The pretreatment was carried 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 and VS reduction rates in the batch Expt.-3. Upon a positive result the ultrasound pretreatment would be introduced for STR digestion.

A custom-made sonicator was provided by Scandinavian Biogas Fuels AB. This is a single transducer ultrasonic 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 output power). A combination of energy input and pumping speed gives the actual treatment energy.

Treatment energy (Wh/L) =U ltrasonic input load (W )

Sludge f low (L/h)

200g of homogenized NA with 100 mL tap water (to make the substrate pumpable) was ultrasonicated at 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 and following controls:

1. Positive controls with Whatman paper (Whatman filter paper, 18.5 cm, 3 qualitative, Kebo Lab AB),

2. Inoculum only

3. 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].

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T able 3.2: Exp erimen tal design for the batc h digestion exp erimen ts. P a rameter Expt.-1 Expt.-2 Expt.-3 Exp erimen tal p erio d No v ’07-Jan ’08 Apr-Jun ’08 Ma y-Jul ’08 Duration of incubation 35 da ys 51 da ys 61 da ys T otal n um b er of tak en measuremen ts 5 4 8 TEST SUBSTRA TE → O A A uto cla v ed O A Whatman P ap er O A NA Whatman P ap er NA Ultrasonicated NA Whatman P ap er Pretreatmen t → None A u t o cla v e a None None None None None Ultrasonic b None OLR (g VS L -1 ) 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 TS (%) 12.48 12.65 98.61 16.41 14.69 96.91 10.0 9.9 98.7 VS c (% TS) 60.89 59.93 99.46 58.01 68.39 99.60 75.2 74.2 99.5 Liquid v olume in serum b ottles (mL) 100 100 100 100 100 1 00 100 100 100 Substrate digested (g) 3.25 3.35 0.25 2.56 2.65 0.25 3.00 3.00 0.25 Note: Th r ee replicat ions w ere p erformed for eac h substrates in eac h and ev ery batc h exp erimen ts in order to minimize t h e exp erimen tal errors [i.e.-3 incubation b ottles for O A in Expt.-1]. A v erage TS and VS v a lues are gi v en here in the t able 3 .2. aA t 121 °C for 30 min utes bWith ultrasonic equipmen t at 19 kHz and energy input 1-5 Wh/L. cMeasured during preparation of b ottles

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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 appropriate time during the bottle

preparation-W3 Saline solution (MgCl2.6H2O, NH4Cl, NaCl, CaCl2.2H2O) i.e. a mineral nutrient solution, 2

mL 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 sewage sludge 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). Preparation of 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 in room temperature until the remaining water disappear from bottles. In every series of experiments the 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 for cooling 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 into

the bottles to adjust the amount to 100 mL. Then the bottles were immediately sealed with the stoppers and aluminum crimps.

4. The N2 gas phase was exchanged for N2/CO2 (80%/20%) by nine evacuations and fillings of

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(a) Substrates were inserted in bottles (b) Evacuation and filling with nitrogen and carbon dioxide.

Figure 3.1: Preparation of experimental bottles for incubation.

5. Over pressure inside the bottles was released by inserting needle. However, in the Expt.-1 and Expt.-2, the overpressure in the external methane standard bottles was not released after flushing to keep the over pressure inside the vials.

6. 0.3 mL of W7was 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 this substrate were prepared in the same manner as the other substrates. As the theoretical and practical gas production of Whatman paper is known, it is suitable for a validation and performance of the experiments. 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 carbohydrates

corresponds to 1/162 mol = 0.006173 mol. If C6H10O5 is assumed to be completely oxidized to CO2

and 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. CH4is formed from 1 mol C6H10O5, why the 0.006 mol C6H10O5gives 0.019 mol CH4

in 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 moles R = Gas constant (8.314)

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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 series of methane bottles was incubated in same manner with the test bottles. Inoculum was not added in this series of bottles. With the 100 mL of boiled MilliQ water, 10 mL of pure CH4(99.99%) was

injected 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 difficult

to maintain high pressure inside the bottles during the pressure measurement. In the Expt.-3, the pressure was released like other experimental bottles, but in that case 20 mL of CH4was injected in

each 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 make substrate 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 volume of 4L in this study. They were equipped with two openings of each: one for feeding and withdrawal of 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 incubation

room. (c) Gas meters, placed in incubationroom.

Figure 3.2: The laboratory-scale anaerobic digesters used for this study, equipped with steering equipments and gas meters.

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Stirrer was a 3 bladed propeller [Figure 3.2] powered bySwitchmode Power Supply PSU24-075, type-MACOO-R1, manufactured by JVL, Denmark with 24 VDC power supply. The propeller was set 5.5 cm up from the bottom of the reactor, which indicates one third of the liquid volume. The motors were run initially at 400 rpm. The speed was increased up to 600 rpm in the reactor D10 after 30

days 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 the digital 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 in Linköping and directly transported to the laboratorium and stored in the incubation room for about one hour prior to the inoculation reactors. After inoculation, the reactors placed in the incubation room 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 sludge according to designed OLR. Since the designed HRT was 20 days, and total active volume (V) of the reactors were 4L, the volume of exchanged sludge (R) per day was calculated following the equation

below-R = _HRTV = 4000 mL

20 Days = 200 mL/Day.

The volume of exchanged sludge represents the volume of feed for the reactors. The same amount of sludge was withdrawn from reactors to keep the active volume constant at 4.0L. The designed g VS algae and primary sludge was supplied with water up to 200g. Usually feed was prepared for four days 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 at the same time on April 02, 2008. First feeding was introduced on the following day (day 1) as given in the table 3.1. Since the start-up period is sensitive to microbial adaptation in the reactors, pH and VFAs were determined more often during the start-up period. However, the pH value 7.4 for both reactors D9and D10 and no VFAs found in the inoculum during the first retention period. The

stirring equipments were installed on day 4. Until then, manual mixing was performed by shaking the digesters for about two minutes by hand.

To evaluate the performance and role of the inoculum during the start-up, the digesters were operated at the same. Both digesters were fed with similar feed portions, mixture of 1.5 g VS of OA and 0.5 g VS primary sludge (PS) .

After 17 days of reactors operation, the feeding was changed to NA. OLR was increased as designed from the Day 18. A thin foam layer was observed in the digesters on Day 21, why the stirrer speed was increased to 400 rpm at the same stirring schedule as before. After 2 days, the layer was dissolved in the reactor D9, but in D10, the thin foam layer remained and increased later on.

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On Day 35, the OLR was increased further to 3.0 g VS , while the primary sludge loading was unchanged. In the period Day 35-54, the test reactor D10 showed slight deterioration (foaming

occurred and VFAs accumulated), while control reactor D9 was stable. In order to recover stable

conditions of D10, the stirring speed was increased to 500 rpm on Day 42. The aim of this modification

was to break down the thick layer and dissolve it in the sludge. On Day 54, the reactor D10

experienced 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 solids such as minerals. The analysis includes sample homogenization achieve representative subsamples and followed the Swedish Standard SS-EN 12880:2000 [54]. Sample is dried to constant mass in an oven at (105±5) °C for 20 hours. The difference in mass before and after the drying process is used to calculate the dry residue and the water content. Oven dried (105 °C) crucibles were used and placed in desiccator with an active drying agent silica gel. To determine TS of a substrate, 0.5-5.0g was taken depending on the type of substrate, so that the dry matter obtained has a minimum mass 0.5g, in each crucibles. Analytical balance has been used with an accuracy of 1 mg. The oven was thermostatically controlled with forced air ventilation and capable of maintaining the set temperature.

3.4.2 Determination of volatile solids (VS)

The organic fraction of a substrate is given as Volatile Solids (VS). The sampling and the analytical procedure followed in the European Standard EN 12879:2000 [55]. The principle was: ‘Samples of dried substrates are heated in a furnace at (550±25)°C. The difference in mass before and after the ignition is used to calculate the loss of ignition’. The dry samples (after 105°C) in the crucibles were ignited at (550±25)°C for two hours in muffle furnace.

3.4.3 pH measurement

The pH meter, used for this study, was manufactured byChristan Berner AB, model WTW Inolab pH 730. pH values of the digested sludge of the reactors were measured twice a week. The pH meter was 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 a

Biomethanation of Red Algae from the Eutrophied Baltic Sea