Determination of the methane potential of blue mussels

Determination of the methane potential of

blue mussels

1

Content list

4. Materials and Methods……….. 6

4.1 Two-stage dry digestion system……… … 6

4.2 Experimental setup……… 8

4.3 Analytical methods……… 9

4.4 Calculations………... 10

5. Result and discussion second run……… 13

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1.

Summary

The aim of this study was to evaluate the methane potential of 35kg of blue mussels in a batch anaerobic two-stage dry digestion system (pilot-scale), which consists of a leach bed reactor (LB) and an up-flow anaerobic sludge blanket reactor (UASB).

We monitored the process daily by measuring temperature, pH, COD, VFA, NH4+, gas flow rate and gas content. The temperature was approximately 30 oC in the LB and 36 oC in the UASB reactor. The applied OLR was 1.5 g COD/l*d.

After 37 days process run, we obtained a total methane yield of 0.64 Nm3 respectively 0.29 Nm3/kg VS, of that 70% in the LB reactor and 30% in the UASB reactor.

2.

Introduction

The global energy demand and pressure on world energy markets will increase steadily in the coming years. A central challenge in the future is to ensure the increasing inquiry for energy. The energy supply is thereby closely related to climate protection and the securing of raw material supply.

The keyword in order to reduce greenhouse gas and to reduce the dependence on fossil fuels is renewable energy. In the future the variegated energy mix shall consist more and more out of renewable energy. For instance solar energy, wind energy and bioenergy.

The sustainable utilization of biomass (bioenergy) shall provide a larger contribution to the production of electric current, heat and fuels and will establish more eco-friendly production. To obtain the energy yield of biomass, it can be combusted or treated microbiologically under anaerobic conditions. The anaerobic treatment aims to win e.g.biogas, which can be

upgraded.

Biogas can be produced of different organic materials like maize, manure or varying substrates from the sea, for instance blue mussels as a new biomass type.

Blue mussels are widely distributed and living in intertidal areas where they can strong adhesive on every kind of solid surface with their byssal threads.

They are filter feeders. They filter water to get oxygen for breathing and organic material, mainly plankton and important nutrients. Hence, they prefer polluted environment with a high eutrophication level.

Carbon dioxide and indigestible components were delivered with the effluent filtered water. Due to the combination that they filter nutrients, organic substances and up to two liters water per hour makes them useful for water purification.

Eutrophication is a serious problem in the Baltic Sea (Nkemka and Murto, 2012). The Eutrophication level increases constantly due to fertilizer, which is used in agriculture and is spread on the land. The fertilizer seeps into the ground water and it will be transported from the land into the sea, causing algal blooms. Sunlight cannot reach the lower part of the water anymore and the algae die. Bacteria decompose the algae now by using oxygen in the water. This leads to oxygen-depleted zones and dying organisms.

3 work more difficult. This causes plants shutdown and high costs of maintenance (Yong-Giak Lim, 2007).

Actual solutions are a bride range of measures with focus primarily on prevention, removal and control. But afterwards thereby incurred costs for disposal. For instance for incineration (not economically since a high energy amount is necessary) and burying (strong odour development) (Yong-Giak Lim, 2007).

An adequate solution for disposal is the use for biogas production.

This means the expensive and disturbing disposal of blue mussels could be positive used. They don’t collide with the also increasing demand for food like maize, because they are in this region (Kalmar) too small for eating.

Meanwhile the digested residue from the biogas process contains recovered nutrients, which can be in turn recycled to the farmland as a fertilizer.

And one further benefit is also that harvesting is the most efficient way to remove nutrients in the sea and thus means to reduce eutrophication.

The aim of this study was to evaluate the methane potential of blue mussels and to verify if the process is working from a technical point of view. The experiments were performed in a pilot-scale dry digestion process which is under development.

3.

Basic knowledgement

Biogas production is an anaerobic degradation. This is very useful because only 5% is used for cell growth and maintenance in compare with 60% in the aerobic degradation. On the one hand this has the advantage that there are no high disposal costs for the low digested residues and more substrate will be converted into the desired product biogas. On the other hand the microbes grow slowly which leads to a long time till the necessary amount of microbes is available and that the process is sensitive and must be close monitored to prevent break downs.

In the following text is explained the anaerobic degradation of organic material and production of biogas proceeds through four sequential steps with involving different microbiological groups.

First step Hydrolysis:

High molecular substances like proteins, fats and carbohydrates must be hydrolyzed into low molecular substances like amino acids, long-chain fatty acids and sugars.

Hydrolysis is the basis for degradation because the bacteria’s can only use small metabolites for metabolism. This is done by different enzymes produced by obligate or facultative anaerobes microorganism.

Second step Acidogenesis:

4 In stable conditions main degradation is via acetate or carbon dioxide and hydrogen. But also low molecular acids like lactate, ethanol, propionate, butyrate (VFAs) occur in this step, which cannot be directly used for the methane production.

Third step Acetogenesis:

In this step the electron sinks will be converted into acetate or carbon dioxide and hydrogen by very sensitive obligate hydrogen producing acetogens (Björnsson, 2000). On the one side an increasing hydrogen concentration over a certain level might causes inhibition of the sensitive acetogens. VFAs cannot be degraded in this case.

And on the other side an increasing hydrogen concentration, which still has to be kept in a certain limit, benefits the methane production in the last step (Björnsson, 2000).

This means hydrogen producing (Step 3) and hydrogen consuming (Step 4) organisms are connected to each other mean they have a syntrophic cooperation.

Last/Forth step Methanogenesis:

In the last step (Methanogenesis) archae will produce methane. About 70% of the methane will be generated from the metabolite acetate (acetoclastic reaction). The rest of the methane will be generated from the reduction of carbon dioxide into methane in the presence of hydrogen gas. Only a few methanogens can perform acetoclastic reaction. These acetoclastic methanogens are very sensitive about environmental changes in compare to the hydrogen-consuming methanogens. Therefore is this often times the rate limiting step (Björnsson, 2000).

3.1 Influencing Parameters

Monitoring is very important to prevent costly overload failure and to perform the whole process more efficient. In time generally the reactor is performed at very low capacity and thereby uneconomically. It’s important to choose a suitable organic loading rate (OLR). Connected is here the monitoring parameter COD, chemical oxygen demand. COD provides information about the organic matter in water phase and thereby indirect about how much substrate is dissolved and how much food is available for the microorganism. It’s a controlling parameter during the whole process.

The dissolved organic material (COD value) will be converted into biogas. A higher amount of generated biogas means a higher amount of a possible utilization of energy. But it’s not only the amount of produced biogas, which is important, also the gas composition. Biogas composed mainly of two gases methane and carbon dioxide. It might also contain small amounts of ammonia, nitrogen and hydrogen sulfide.

But the energy generating step is the combustion and therefore oxidation of methane. The aim is obviously to reach high methane content so that it can be upgraded and used in further processes.

5 To reach the aim of a high methane yield it’s necessary to have knowledge about the

requirements of the microorganism for instance especially about temperature and pH. In general will the process be operated at mesophilic (25-40°C) or thermophilic (>45°C) temperatures.

The optimum pH for methanogens and acetogens is about 7 and they are very sensitive against changes. This is to the detriment of the acidogens, which pH optimum is about 6. But they are not rate limiting and can also grow at pH 7 (Björnsson, 2000).

It’s difficult to measure the amount of living microorganism but a suitable way to monitor the process is to measure the metabolites which indicate indirect the activity of the microbes. For monitoring indirect the activity of methanogens is volatile fatty acids (VFA) a good parameter.

The syntrophic cooperation can be used in this case. It is important that this cooperation works, otherwise VFAs like butyrate and propionate cannot be degraded anymore, when the hydrogen pressure concentration gets too high.

On the other side VFA decreases pH, which can lead to the inhibiting protonated molecule form of VFAs (Björnsson, 2000).

Therefore, pH needs to be monitored but connected with the pH is also the alkalinity (buffering capacity) of the system. This can be buffering compounds like ammonium

bicarbonate combined through ammonium from the fermentation of the amino acids in mussel tissues with carbon dioxide and water and also calcium carbonate made from the mussel shells (Yong-Giak Lim, 2007).

It’s important to have sufficient buffering capacity to keep the pH in an almost constant range. Furthermore the pH is sometimes not a reliable parameter to measure, because if the alkalinity of the system is really high, the pH will not change although there is an accumulation of ammonium or VFA.

The last significant parameter is Ammonium NH4+, which leads in high concentration to inhibition.

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4.

Materials and Methods

4.1 Two-stage dry digestion system

The anaerobic digestion of blue mussels was performed in a batch anaerobic two-stage dry digestion system, which consists of a leach bed reactor (LB) and an up-flow anaerobic sludge blanket reactor (UASB), figure 1.

This shown pilot scale dry digestion process was used in our experiments and under development.

7 Leach bed reactor:

The LB reactor was filled with 350l of water and 35kg of blue mussels, figure 2. The mussels were collected from the coast in Kalmar, Sweden and the characteristics are shown in Table 1. We digested the mussels without removing shells or crashing before.

A heating element had kept the liquid content at a mesophilic temperature (30°C) and a pump ensured the mixing and an adequate mass transfer in the LB reactor.

UASB reactor:

The UASB reactor was filled with 30l of water and about 10l of granules. A pump circulated the fluid with 30 Hz (approx. 1.8 l/min) through a heating system in order to keep the fluid at a mesophilic temperature (36°C) and to ensure a high mixing level. We measured the

resistance with a pT100 element and converted the resistance to temperature by use of a Table.

The leachate of the LB reactor was manually transferred daily into the UASB reactor using a pump operated at 25Hz (1.5 l/min). The applied OLR was 1.5 g COD/l*d (see analysis). The same volume was transferred back from the UASB to the LB reactor.

Figure 2: Three bins in the LB reactor filled with blue mussels

Biogas Biogas Leach bed reactor UASB reactor

8 The produced gas in both reactors flew through a continuous gas flow measurement (Alicat Scientific). After that we collected the gas in a tight bag in order to measure the gas content by a gas meter (GSM 410, Gasdata Ltd Coventry UK), figure 3.

To ensure that dry gas entered the continuous gas flow measurements, we led the biogas flow first through bottles filled with silica gel.

4.2 Experimental setup

While analyzing the results from the first experiment in February / March 2013, we

determined-based on the biogas values in the leach bed reactor-, that the reactor was not tight. To obtain reliable results, we decided in April 2013 to repeat the experiment, after fixing the two holes in the leach bed reactor.

The UASB reactor was working in both experiments.

In addition, we have also improved the sampling. The first improvement was only to do the sampling from the UASB reactor during the run of the recirculation pump, in order to prevent the settling of the granules.

The second improvement was to add another division valve in the connection tube between both reactors (feeding tube). This reduces the risk of mixing the samples and ensures better control of the fluid exchange.

The rest of the experimental setup remained unchanged to the first experiment (see construction chapter).

We started the second process run on the 8th of April. We filled the LB reactor with 35 kg of mussels (Table 1) and approx. 350 l of water. The UASB reactor was filled with 10 l of granules and approx. 30 l of water.

The reactors were maintained at a mesophilic temperature, around 30 oC for the LB reactor and 36 oC for the UASB reactor.

Daily samples were taken and after the evaluation (pH, COD, VFA, NH4+, gas amount,gas content), we calculated according to the measured results the daily feeding time. The applied OLR was 1.5 g COD/l*d (see analysis).

The first fluid manual transfer from the LB to the UASB was started at the second day of the experiment. Thereafter, the leachate was transferred daily into the UASB reactor. The same volume was transferred back from the UASB to the LB reactor. After 16 days the liquid transfer was stopped.

On the 25th of April, after 18 days, we switched off the UASB reactor and on the 14th of May, after 37 days, also the LB reactor.

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Table 1

Some characteristics of the collected blue mussels

Mussels [kg] 35

TS [% of ww] 30

VS [% of TS] 21

Improvement:

- A better way of drying the gas so that the silica bottles do not have to be changed so frequently. One solution could be to cool the gas so that the water condenses and can be removed.

- A way to have a look in the reactor / knowledge about a good circulation

4.3 Analytical methods

The total solid (TS) and volatile suspended solid (VS) content were analyzed according to APHA (1985) standard methods.

Total solids (TS):

The samples were weighed and dried in 105°C over night and weighed again. The total solid is calculated according to the following equation (1).

(1)

Wvessel =Weight of vessel

Wsample =Weight of wet sample and vessel [mg] Wtotal =Weight of dried residue and vessel [mg]

Our calculated TS value is 30%. Volatile suspended solids (VS):

To calculate VS a dry sample need to be glowed in a furnace at 550°C for 2 hours. After this you can use the equation (2):

(2)

Wvolatile = Weight of residue and vessel after ignition [mg] Wtotal =Weight of dried residue and vessel [mg]

Wvessel=Weight of vessel [mg]

10 Daily measured parameters:

1) Volatile fatty acids (VFAs) according to Hach Lamge ( LCK 365)

2) Chemical oxygen demand (COD) according to Hach Lange (LCK 014) and (LCK 114)

3) Ammonium (NH4+) according to Hach Lange (LCK 303)

4) Alkalinity (KS 4.3) according to Hach Lange (LCK 362)

5) pH according to standard methods (pH-meter) 6) Temperature measured by amperemeter

7) Gas content by a gasmeter (GSM 410, Gasdata Ltd Coventry UK)

8) Biogas measured by a continuous flow measurement (Alicat Scientific, Tucson, USA)

4.4 Calculations

How much mussel should be put in?

The calculation was done with the values of the succeeded process run in Lund (Nkemka and Murto, 2012).

X = mussel [ ] = 200

TS = 41.2 % (Nkemka and Murto 2012) VS = 18.8% (Nkemka and Murto 2012)

(3)

11 Calculating with our values for TS=30% and VS=21% gives:

245.9 0.25 87.44 kg (350 l)

We filled the reactor only with 35kg (respectively in the first process run with 45kg) mussels since we had not enough space.

Daily feeding:

The determined COD values in connection with the VFA and ammonium concentration in the LB and UASB reactor is important for calculating the feeding time, equation (4). We took almost the same value from the succeeded process run in Lund. They were performing with OLR of 1 g COD / l*d.

Calculating for instance:

X = COD value in the LB reactor Y= Feeding time

(4)

We calculated and applied about 1.5 g COD / l*d.

1.5 l/min flow rate from the pump feeding the reactor.

Temperature:

We measured the resistance with a pT100 element and converted the resistance to temperature by use of a Table.

Interpolation between 115.54 Ω = 40°C and 111.67Ω = 30°C, equation (5)

(5)

12 Biogas production/methane content:

Methane and gas volumes were normalized to 0°C, assuming constant pressure 1 atm and expressed as Nm3.

Biogas production UASB: 0.28 Nm3 Average methane content UASB: 69% Methane production UASB: 0.19 Nm3

Total VS (what we put in): 2.205 kg VS The methane value calculated per kg VS.

(6)

The value from Lund for the total methane production was 0.33 Nm3/kg VS.

(7)

Only in the UASB reactor they had a value

(68%) and we had .

The same calculation for the LB reactor gives: Biogas production LB: 0.85 Nm3 Average methane content LB: 53% Methane production LB: 0.45 Nm3

We yielded 0.20 Nm3 methane/kg VS.

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5.

Result and discussion second run

The methanogens are very sensitive and an anaerobic process is often limited due to the low specific growth rate. To protect the methanogens against environmental changes and to retain them in the process could be used a biofilm or granulated sludge. Common reactors to use are for instance an UASB reactor, an anaerobic packed bed or a fluidized bed reactor (Björnsson, 2000).

The continuously stirred tank reactor (CSTR) is in wide use. The reactor has advantages like a high mixing level and suits for most substrates. CSTRs have furthermore been closely studied.

Disadvantages are that the CSTR requires a long retention time to prevent washout of active biomass and another one is the problems of clogging, pumping and accumulation of inert material resulting from for instance the mussel shells and sand will occur. The solution of remove the shells and sand is very costly and thereby expensive.

The UASB reactor is one of the most efficient once for anaerobic processes, but would have the same disadvantage like the CSTR according to the clogging problems.

One answer to solve this kind of problem is a two stage-system, which might consist of a leach bed reactor (LB) and an UASB, as used in our experiment. In this process the mussels are hydolysed in the LB reactor and then the hydrolyzate is transferred into the UASB reactor.

After 37 days of treatment period with an OLR of 1.5 g COD/l and 35kg of mussels, the methane yield was 0.64 Nm3 respectively 0.29 Nm3/kg VS (Table 2).

Once the leachate was transferred from the LB into the UASB reactor, the production of biogas/ methane started rapidly in the UASB reactor,-already on the 4th day of the experiment. During the first days, the methanogens need a short adaptation time in their new environment. Furthermore some time is needed to reach an adequate hydrogen accumulation as a proper food source, so that the methane bacteria are able to start their metabolism.

The average VFA concentration in the UASB is about 200mgl-1. It’s good that the value is not too high, because a high concentration would indicate inhibition.

Since we started feeding on the 2nd day, the 4th day is a realistic time period for the start of production of biogas. This transfer was very successful, which is clearly shown by the COD concentration in figure 6.

Figure 6: Biogas and COD as a function of the time in the UASB reactor 0 500 1000 1500 2000 2500 0 5000 10000 15000 20000 25000 30000 35000 5 6 7 8 9 10 11 12 13 14 16 17 C O D [ m g O 2 /l ] B io g a s [m l/ d a y ] Time (days)

14 The feeding stream is one main advantage of the two stage-system, because the inflow stream from the Leach Bed into the UASB can be closely monitored and therefore ensure the

protection of the methanogens.

On day 18, we disconnected the UASB reactor, because the biogas production decreaseddue to the low food supply. The COD concentration almost had the same level as in the LB reactor, which would lead to the need of a large volume exchange between both reactors during feeding.

After 13 days of biogas production, the UASB reactor yielded 0.19 Nm3 of methane, respectively 0.28Nm3 total biogas with an average methane content of 69%. This makes up 30% of the total methane yield from the entire process run.

Despite some minor fluctuations in the VFA and ammonium concentration, the pH value was due to the buffer capacity of the system in an ideal range for methanogens, namely between 7.1 and 7.9.

The biogas production in the LB reactor started at the 8th day. The exchange of liquid between both reactors led to the transfer of microorganisms from the UASB to the LB and initiated the methanogenesis.

After 37 days of biogas production we disconnected the LB reactor, since the biogas production was so low that the recorded measurement values were no longer reliable. The low biogas production in the end of the experiment is due to the low VFA and COD concentrations, Figure 7.

Figure 7: Biogas, VFA and COD as a function of the time in the LB reactor 0 1000 2000 3000 4000 5000 6000 0 10000 20000 30000 40000 50000 60000 0 10 20 30 40 V F A [m g/l ], CO D[m g 0 2/l ] B iog as [m l{ da y] Time [days]

15 The previous assumption that blue mussels are hydrolysed fast and easily was confirmed and reflected by the COD/VFA concentrations in the leach bed reactor in the first 8 days.

The hydrolyzation rate of the solid organic material is a very good basis for the further degradation and the resulting biogas production. From now on the nutrients/soluble organics are available for microbial metabolism. This could be a limiting factor when using other substrates.

The COD increased from 1244 to 4913 mg O2 / l and VFA increased from 199 to 2560 mg/l, Figure 7. From the 8th day the COD and VFA subsequently decreased to 530 mg O2 / l and 88.4 mg / l. This is because of the fluid recirculation with the UASB reactor and the efficient conversion of soluble organic compounds into biogas.

In the prior mentioned 37 days of production, 70% of the total methane of the entire process could be yielded, thus means 0.45 Nm3 methane of 0.85Nm3 biogas. Very interesting is the increasing methane content shown in figure 8.

The methane production increased from 1.8% up to an almost stable value at day 19 of 74.2%.

Figure 8: Biogas and Methane as a function of the time in the LB reactor

From day 33 on, the biogas production decreased to a low level, approx. 5 l/day. Nevertheless we continued the process for another 4 days, because the longer digestion time is necessary to fully harness the biogas potential of our certain amount of mussels.

The pH value increased after the first week of 6.6 almost up to an ideal value for the methanogens to 7-8.

This is explained by the decreasing VFA concentration on day 8 from 2560 to 88.4 mg/l as well as the slight increase in ammonium concentration to an average value about 430 mg/l, which will be released by the hydrolysis of the bound ammonium in proteins, figure 9. Furthermore it is explained by the fluid exchange between the two reactors.

0 10 20 30 40 50 60 70 80 90 0 10000 20000 30000 40000 50000 60000 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 M e th a n e c o n te n t [% ] B io g a s [m l/ d a y ] Time [days]

16 Figure 9: Ammonium, VFA and pH as a function of the time in the LB

To determine how successful the process run was, a comparison was done with the results from the Lund experiment (Nkemka and Murto, 2012).

Lund calculated the daily feeding at an OLR of 1 g COD / l*d and we calculated with 1.5 g COD / l * d. Both OLRs are very low to ensure the safe operation of the process and prevent break downs. In our experiment we aim to find out the potential yield of methane and not the maximum possible loading rate. To evaluate the maximum capacity of the process a higher OLR up to 20 g COD l-1 d-1 is needed (Björnsson, 2000).

Lund had a 1 l reactor with a total methane production of 0,33 Nm3/kg VS, 68% in the UASB and 32% in LB reactor.

Our values are reversed with 30% in the UASB and 70% in the LB reactor. Overall, we received 88% of the total methane potential from Lund.

Table 2

Duration time, methane yield and methane content of a two-stage anaerobic digestion of blue mussels.

Reactor Duration time Methane yield [Nm3/kg VS] Methane content [%]

Both (two-stage process) 17 0.29 -

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5.

References

[1] Lim, Yong-Giak; Niwa, Chiaki; Nagao, Norio; Toda, Tatsuki (2007):

Solubilization and methanogenesis of blue mussels in saline mesophilic anaerobic

biodegradation. In: Elsevier Ltd.2007; International Biodeterioration & Biodegradation 61 (2008) 251-260

[2] Nkemka, V.N.; Murto, M. (2012):

Two-stage anaerobic dry digestion of blue mussel and reed. In: Elsevier Ltd. 2012; Renewable Energy 50 (2013) 359-364

[3] Björnsson, L. (2000):

Intensification of the biogas process by improved process monitoring and biomass retention. In: PhD thesis Lund University, Department of Biotechnology;

ISBN:91-7874-075-4 [4] http://www.mifratis.de/acidogenese.php [5] http://en.wikipedia.org/wiki/Blue_mussel [6] http://www.planet-wissen.de/natur_technik/weichtiere/muscheln/mies.jsp [7] http://weichtiere.at/Muscheln/index.html?/Muscheln/miesmuschel.html [8] http://www.wri.org/project/eutrophication/about

[9] Moestedt, Jan; Nilsson-Paledal, Sören; Schnürer, Anna (2013):

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