Thisis_Can Chu_z3329270

THE UNIVERSITY OF NEW SOUTH WALES

SCHOOL OF PHOTOVOLTAIC AND RENEWABLE ENERGY ENGINEERING

A net-negative carbon energy system via methanogenesis of coccolithophorid

algal biomass

Can Chu

Bachelor of Engineering in Renewable Energy Engineering

Course Code: SOLA 4911

Submission Date: 28th Oct 2014

Supervisor: Dr. Robert John Patterson

AssessorA/Prof. Gavin Conibeer

II

Abstract

In this project, is looking for a system that could support both methanogens and

algae living together. Cyanobacteria were used in this system due to their high

growth rate and also they could be easily digested by methanogens as they do not

have any lignin or cellulose. During the experiments, the methane content of the

sample bottle headspace was monitored once or twice a week using gas

chromatography (GC). From the result, this cyanobacteria and methanogens system

could live for about 40 days with methane being produced continuously. The

maximum methane yield among all samples is the one where made by f2 media with

only1/8 of the recommended vitamin concentration. This sample gives 1852.3

µmoles of methane and 45.4% methane content in the headspace volume.

Key words: Algae, coccolithphor, CO2 concentration, methanogenesis, CH4

production, cyanobacteria,

III

Acknowledgements

The author gratefully acknowledges to the guidance and support of post-doctoral

supervisors Dr. Robert Patterson, Dr. Sabrina Beckman, Dr. Valentina, A/Prof. Mike

Manefield and A/Prof. Gavin Conibeer. This project could not get to successes so

smoothly without any of their help. Also gratefully to the support of undergraduate

student Leo Zhan whom helps with the whole process during the experiments.

IV

Table of contents

Abstract ....................................................................................................................... I

Acknowledgements ................................................................................................... III

Table of contents ...................................................................................................... IV

Introduction ................................................................................................................. 1

Background ............................................................................................................. 1

Coccolithophor ..................................................................................................... 2

Previous researches ................................................................................................ 5

CO2 analysis ........................................................................................................ 5

Sea water media .................................................................................................. 8

Photosynthetic microorganisms............................................................................. 10

Methanogenesis .................................................................................................... 12

Proposed experiments .......................................................................................... 13

Preparation for all experiments ................................................................................. 15

Making f/2 media ................................................................................................... 15

Culturing methanogens ......................................................................................... 16

Lighting condition .................................................................................................. 19

Temperature control .............................................................................................. 21

Making CO2 concentration .................................................................................... 22

Pressure ................................................................................................................ 23

General sample containment ................................................................................ 24

Results and discussions ........................................................................................... 25

Varying initial CO2 concentration for the combined system ................................... 25

Varying CO2 concentration for blue-green algae only ........................................... 28

Varying the media vitamin concentration for the combined system ....................... 30

Varying lighting condition for the combined system .............................................. 33

Conclusion ................................................................................................................ 37

References ............................................................................................................... 38

Appendix................................................................................................................... 45

I. Sample preparation ............................................................................................ 45

II. CH4 analyses ..................................................................................................... 47

III.CH4 data for all experiments .............................................................................. 48

IV.Detailed figures of result ................................................................................... 51

V. Media recipes.................................................................................................... 55

VI. Sample calculations ......................................................................................... 57

1

Introduction

Background

With the development of countries leading to a higher quality of life, the average energy

consumption per person is increasing continuously. Traditional power plants are

causing significant issues with respect to global warming and general pollution, so

alternative clean energy sources need to be developed. This could be achieved by

looking for a kind of biomass feedstock that, as a part of its photosynthetic processes,

already contains carbon capture and storage. Figure 1.1.1 shows the carbon cycle of

existing types of power plants and industries. From which could be seen that the

difference in using nature resources as well as net emission. Algae cultivation for CO2

fixation have been suggested since 1955 [1] [2] with the possibility of producing

methane as an energy source through an anaerobic degradation process. Also, it has

other advantages such as does not have the problem of competing with what we

conventionally think of as food for human consumption.

Figure 1.1.1 net emission of power plant or industry.[3] The positive sign means there

are CO2 emission into atmosphere (1), zero means no extra CO2 emission (2&3), and

negative indicate that this project is capturing CO2 from atmosphere (4). For our

experiment, we are focusing to develop a system which could achieve net negative CO2

emission.

2

Coccolithophor

Figure 1.1.2 below shows the ideal carbon cycle of a net-negative energy system. This

cycle is one of the largest naturally carbon storage systems which takes the carbon

from atmosphere and sediment them on the ocean floor. Coccolithophorid algae drive

this cycle. It is has a high pressure in the atmosphere CO2 will slightly dissolve in the

sea water which forms bicarbonate and hydrogen ions. Then coccolithophorid algae

take these bicarbonates to form calcium carbonate on their cell wall during the process

of photosynthesis. As shells are forming on its cell wall, old shells will start to separate

from the cells and drop off to the ocean floor. [4] The formation of this calcium

carbonate is the key that makes this system net negative on carbon.

Figure 1.1.2 Carbon cycle of algae energy system. CO2 is initially existing in the

atmosphere, with the photosynthesis and calcification of algae (step 1) it will form

C6H12O6 (glucose or cellulose) and CaCO3. After bacterial digestion of these sugars,

they will become smaller organic molecules that will be easier for methanogens to

digest (step 2). Then the methane is formed by anaerobic digestion performed by

methanogens (step 3). With the combustion of methane, CH4 will go back to being CO2

3

in the atmosphere (step 4). The process of algae calcification will make this cycle net

negative on carbon.

Figure 1.1.3 Cell structure and calcification of coccolithophorid algae Emiliania Huxleyi.

[5] This figure illustrates the process of algae calcification. The figure on the left shows

only two of the extracellular coccoliths (EC). In E.hux, coccoliths are composed of about

30 units of radially arranged crystalline units and each of them could subdivided into a

connecting wall (a) between a lower element (b) and an upper element (c). Different

intracellular organelles are also shown in the figure including chromatin (Ch);

chloroplast (Chl) cover (Cov); mitochondrion (M); nucleus (N); nucleus envelope (NE);

reticular body (RB); cell vacuole (v); coccolith vesicle (CV). [4] [6]

4

Figure 1.1.4 schematic diagram of one possible photobioreactor (PBR) configuration

suitable for a marine net-negative carbon energy system. Solar energy (1) is incident on

the system, and the algae layer (2) produce calcium carbonate (3) and drop on the

bottom of the reactor taking biomass with them. Methanogens produce CH4 and biogas

(4). After combustion and energy extraction (5), the exhaust gases are recycled (6) back

into the system, filtering (8) step is to prevent CO2 going out of the system since

anaerobic conditions need to be maintained within the PBR. As nutrition is needed to

grow of algae, nutrients will be constantly added into the system (7).

A photobioreactor is a more complex system for culturing algae that will be relevant in

future work. The scope of the research performed in this report is for the purpose of

getting more knowledge about the integrated system of photosynthetic microorganisms

and a methanogenic consortia to determine the best approach to the overall net

negative carbon energy system.

The purpose of this report is to determine the maximum CO2 tolerance level and the

best initial CO2 concentration for culturing algae alone and also with methanogens. The

difference in performance when changing the vitamin concentration is also investigated.

Appropriate lighting is important for optimal algal culture, so an experiment on this

aspect has also been performed.

5

Previous researches

CO2 analysis

With its high lipid content and high sunlight conversion efficiency, algae haves been

considered to be a source of biodiesel during past oil crisis. However, the cost of

dewatering and harvesting makes the operational cost not competitive with other energy

sources. [7] [8] Therefore, growing algae for removing CO2 from industrial flue gases to

produce energy is likely to be a good solution [9]. From the previous research, Brennan

& Owende states that the oil content of most algae is between 20%~30% of their dry

weight and algae typically has an exponential growth rate which could get doubling

rates as short as 3.5 hours [10]. There are many various kinds of algae available in the

natural environment, but for lab conditions, Moheimani suggests that Pleurochrysis

Carterae is more resilient, with growth rate of 3.5x 104 cells/day, and a potential yield of

8.9x 10-13 grams of CH4/cell. [11]

Since 1970, algae have been used for waste-water treatment, a source of biomass and

many energy-related purposes. Growing algae for methane production as an energy

source has been proposed almost 50 years ago.[12] As algae has not being focused for

a long time, no one has been researched on the effect on growth rate when changing

the CO2 concentration, and the difference of lab media and ocean. Previous research

shows that, Euglena gracilis could grow under 5% to 45% of CO2 concentration, and it

will stop growing when the concentration is over 45%.[13] The best growth of this kind

of algae is when the CO2 concentration is at 5%. [13] Another type of algae, Chlorella

sp. has a CO2 tolerance of 40%. [14] Furthermore, a strain of Chlorella sp. T-1 was

found by Maeda et al, which could grow under 100% initial CO2 concentration, but the

maximum growth rate happens fewer than 10% CO2 concentration,[15] Scenedesmus

sp. has a CO2 tolerance of over 80%, and best performance happens between 10%-

20%.[14] Some CO2 tolerance of other types of algae is shown in the table below.

6

Table 1.2.1 CO2 tolerances of some high lipid content algae. [25]

Species Known Maximum

CO2 tolerance

Concentration

References

Cyanidium Caldarium 100% [16]

Scenedesmus sp. 80% [14]

Chlorococcum littorale 60% [17]

Synechococcus

elongatus

60% [18]

Euglena gracilis 45% [13]

Chlorella sp 40% [19]

Eudorina spp. 20% [20]

Dunaliella tertiolecta 15% [21]

Nannochloris sp. 15% [22]

Chlamydomonas sp. 15% [23]

Tetraselmis sp. 14% [24]

Although the types of algae are with high lipid content not coccolithophorid, but it still

suffices take these values as a reference or indicator of typical algae tolerances. This

table clearly shows the CO2 tolerance for different types of algae is varying over a large

7

range, so for our experiment the CO2 tolerance for our experimental photosynthetic

microorganisms will find out. After this, the CO2 concentration will vary to looking for the

concentration with the best performance of them.

Figure 1.2.2 Effect of CO2 concentrations in the atmosphere on E. hux (circles) and

G.oceanica (squares) [26]This figure is from a research on cocolithophorid algae, under

a prediction of atmosphere condition. With the CO2 concentration increasing, more CO2

will dissolved and lead to increase on pH level. Figure a) shows the trend of particulate

organic carbon (POC) formation rate and figure b) is the trend of calcification rate.

Therefore from the graph, with the increasing concentration of CO2 in atmosphere, the

trend is to form more POC but less calcification, which means that the cells will getting

sequester less carbon with a high CO2 concentration.

8

Sea water media

There is currently very little research on growing algae using just pure sea water, as the

sea water may contain a large amount of other elements that will effect on the growing

of algae and methanogens, such as sulphate ions. Therefore, for lab research, the

media we are using are always made up by the chemicals that algae and methanogens

both required. Also, vitamins are known to be necessary for culturing algae and there is

currently little research on the effects of varying their concentration in the growth media.

In these experiments, pure sea water will be investigated as a medium to culture algae.

Also how the vitamin concentration in the media affects the growth rate will be explored.

Table 1.3.1 Chemical composition of sea water [27]

9

Table 1.3.2 Recipe for f/2 media

Compare between table 1.3.1 and table 1.3.2, the presence of trace metals are missing

for the composition of sea water, also iron ions, vitamins, phosphate, and EDTA are all

required for culturing of the algae but not contained in sea water.

In the lab, we are using f/2 media for culturing the algae. Trace metals are present in

this media assist cellular formation therefore the concentration of them could affect the

growth of algae significantly. From the research on E. Hux before, the concentration of

[Fe] ion will affect the cell growth, and [Zn] ion is related to coccolithophore growth. [28]

Thus, the composition and concentration of media for culturing microorganisms could

influence on resulting growth rate significantly.

After completing these experiments, the result of the best CO2 concentration and the

CO2 tolerance when culturing cyanobacteria will be obtained. Also, algae are initially

living in the ocean, but for lab research, we are always making the media to culture

algae. Therefore, this project will investigate the difference in performance between lab

media and sea water, and how vitamin concentration effects the culturing of the algae.

10

Photosynthetic microorganisms

The photosynthetic microorganisms used in these experiments were bought from a local

aquarium shop. These were expected to grow faster and are cheaper than P.Cart. After

microscopy and researches online, the type of photosynthetic microorganism we were

used was found to be cyanobacteria (blue-green algae).

Figure 2.1.1 The algae used in these experiments imaged under a microscope. From

the image, shows that this microalgae is about 60µm in size, strip in structure, lack of a

visible cell wall and lack of the ability to see a nucleus, which indicate the specie of

these microorganisms to be cyanobacteria.

11

Figure 2.1.2 Image of Cyanobacteria (blue green algae) under microscope [29]

Marine biotechnology has been the focus of much attention due to the fact that marine

microorganisms are suitably involved in the biosynthesis of marine natural products. [30]

[31] The culturing of actinomycetes and fungi are well known to be culturable marine

microorganisms producing some kinds of bioactive metabolites. [32] Cyanobacteria

(blue green algae) are also one of the most promising groups of organisms that have

biochemically active natural products. [33] [34 [35] [36] Studies show that marine

cyanobacteria have the ability to produce secondary metabolites unlike those found in

any terrestrial species. [37]

During the past few decades, cyanobacteria have already been used in the biofuel area

to produce a number of different biofuel related compounds. [38] From previous

research, the nutrients needed for culturing cyanobacteria are simple, just nitrogen and

phosphorus similar to plants, which makes it ideal for large scale culturing of biofuel

production. [39]

12

Methanogenesis

Methane was first identified by Italian physicist Alessandro Volta as „combustible air‟ in

1776 [40], and further work by Bechamp, Popoff, Tappeneiner, Hoppe-Seyler, Sohngen,

and Omelianski revealed that this kind of „air‟ is methane gas. [41] During the past

decade, methane-producing microorganisms (methanogenesis) by anaerobic digestion

have been found to have a widespread and fundamental role in nature. [42] [43] [44] [45]

[46] These microorganisms haves been performing methanogenesis for centuries by

anaerobic digestion of biomass but there has been insufficient knowledge about what

species they are and how they work. [47]

With more research on methanogenesis and also the improvement in biological

characterization technology, there are 3 different broad types of methanogenesis known

at present: hydrogenotrophic (hydrogen consuming), acetotropic (acetate consuming),

and methylotrophic (consuming compounds with methyl groups). [48] The pathway of

hydrogenotrophic methanogensis is consists of the reduction of CO2 by H2, [equation 1]

This kind of methanogenesis is performed by up to 77% of known methanogens. [49]

The acetotrophic type of methanogenesis [equation 2] is used for approximately two-

thirds of biologically produced methane. [50] Finally, for the methylotrophic pathway, the

equations of the reaction are given by equation 3. [50]:

CO2 + 4 H2 → CH4 + 2 H2O (1)

CH3COO- + H+ → CO2 + CH4 (2)

CH3–A +H2O → CH4 + CO2 + A−H (3)

The rate and amount of methane produced by methanogens are always depended on

the conditions in which the methanogens are growing. As their metabolic processes are

anaerobic, oxygen must be prevented from entering their environment when culturing

them. Also, the methanogens are sensitive to the concentration of nutrients, organic

substrates, and lighting conditions.

13

Proposed experiments

For the variation initial CO2 concentration of combined cyanobacteria and

methanogenesis system, the CO2 concentration considered are selected with respect to

the research before as well as the living condition of methanogenesis. The performance

of this system is predict to have better performance at initial CO2 concentration between

60% and 40% which is the reason why more samples prepared within this region.

The initial concentration selected for the experiment on cyanobacteria only aim to find

out the CO2 tolerance level and best-performing CO2 concentration. Thus, samples are

made with high distribution between 100% and 5% initial CO2 concentration. With the

research previously, the best performance initial CO2 concentration for most of high lipid

content algae is in between 20% to 5% which result to have more samples within this

range for the experiments working with cyanobacteria.

The purpose of the experiment on varying the media vitamin concentration is to find out

the minimum vitamin required for culturing algae with methanogensis as methane

produced system, which could reduce the capital cost for industrialise cultivation.

For organisms living depends on the process of photosynthesis, the appropriate lighting

condition is highly demanded in general culturing. In which lack of brightness will

decrease the rate of photosynthesis and intensive light could bring the occur of

photoinhibition. Therefore, during the experiments, the optimal lighting condition for

experimental cyanobacteria will be found out.

14

Table 1.5.1 Samples considered for each experiment (detailed experiment management

see Appendix I)

Experiment Samples going to prepared

Varying initial CO2 concentration for the

combine system

100%, 80%, 60%, 55%, 50%, 45%,

40%, 30%

Varying CO2 concentration for blue-green

algae only

100%, 80%, 60%, 40%, 20%, 15%,

10%, 5%

Varying the media vitamin concentration for

the combined system

1/8 vitamin, 1/4 vitamin, 1/2 vitamin, f/2

vitamin (normal), pure sea water

Varying the lighting condition for the combined

system

Completely dark, normal light, LED light

with paper covers 12:12, completely

LED 12:12

15

Preparation for all experiments

Making f/2 media

According to the table 1.3.2, stock solutions need to be prepared before making the

media. Having these stock solutions will make it much more convenient to make the

media in future experiments. When making the f/2 media, a 2 liter schott is used, a 2L of

water from which the water filtered through a reverse osmosis system. 2ml of each

stock solutions 1~4 need to be added into the bottle before autoclaving After this, the

vitamin and NaH2PO4 as a phosphate provider are added to the media which vitamin

will get damaged and NaH2PO4 will form precipitate that auto-clave separately and

added in the end.

With this media made, the algae can be cultured, to make enough to use in the

experiments.

For the experiment on varying vitamin concentration, as the algae culturing for all the

experiments grow in f/2 media with the full vitamin concentration, to make the samples

for this topic could only change the amount of vitamin slightly with adding in a small

amount of new f/2 made with no vitamin presented. To start with, the f/2 media were

autoclaved with no vitamin added. Then a specific vitamin concentration from the lowest

concentration was added. For example, we make the 1/8 vitamin concentration first.

The normal vitamin concentration for f/2 media is 2ml per litre, so we put 0.25ml of

vitamin for this sample. Then 20ml of sand on the bottom and 10ml of this f/2 media and

48ml algae as well as 2ml methanogens was added, as before. Thus, to make other

samples with higher vitamin concentration, we just keep adding the vitamin. The CO2

concentration for this system is 80%. Also, a sample with only sea water adding in is

made with the sea water taken from Manly beach, and after autoclave, 10ml of that is

added as other samples before adding in the 48ml algae.

16

Culturing methanogens

All samples of methanogens for the experiments come from the beach and cultured by

black sea media with 0.2 ml of methanol adding-in as a food source. After the bottles

are sealed, CO2 was used to flush out the headspace of these samples as

methanogenesis need anaerobic condition for living. After three weeks culturing of

those methanogenesis, it is supposed to have enough methanogensis for the

experiments with checking the CH4 concentration of those samples by Gas

Chromatography (GC).

Figure 2.3.1 Schematic diagram of GC. The injector introduces the gas from head

space of a sample into a continuous flow of carrier gas, and this flows along the entire

column. The various components are separated inside the column section within the

column oven. The detector measures the quantity of the components that exit the

column. [51] The GC for these experiments are using flame ionization detector (FID)

which is based on the detection of ions formed during combustion of organic

compounds in a hydrogen flame. The generation of these ions is proportional to the

concentration of organic species in the sample gas stream.

In this project, 17 sample bottles are obtained in total all of which need methanogenesis.

In order to have enough methanogens solution for all samples, and also considering the

ratio of methanogenic organisms with the algae, 2ml of methanogenesis is considered

to add in each sample with 48ml of algae, which makes up to 50ml in total liquid area.

The reason why 2ml of methanogenesis is selected to obtain in each sample is due to

the consideration of toughness and growth rate of methanogenesis together with

cyanobacteria.

17

From the GC software, the amount of CH4 contained within the sample injected is

shown as in the area under result graph (figure 2.3.2). In order to convert the data into

concentration of CH4, samples with known CH4 concentration are tested using this GC

then calibrated to get a coefficient for calculate area into µmol of CH4.

Figure 2.3.2 Result shown in GC software

18

Figure 2.3.3 Calibrated graph of getting the coefficient for GC result, thus to calculate

the amount of CH4 in the samples, the process is to divide the resulting area by a

coefficient of 6029.3 to convert it into molar amount of CH4. (table see Appendix II)

19

Lighting condition

In order to get sufficient light for algae to photosynthesize, two lighting bulbs are bought

from an aquarium shop. One of these is a LED light, and the other one is a florescent

lighting tube. Also switches which are enable to switch off automatically after several

hours are bought for the lights. During the experiments, the lighting hours are set to be

12:12 which means 12 hours photosynthesis of algae and 12 hours dark condition for

them to reproduce.

Figure 2.4.1 lighting condition for the experiments

From figure 2.4.1, it shows the pure algae samples are cultured in the front four

containers. The samples for experiments are placed behind which are in small sample

bottles.

20

Figure 2.4.2 Fluorescent lighting bulb using for the experiments lighting. From the

information of the lighting bulb shown in the instruction, and compare with the light

spectrum, this lighting source slightly contains ultraviolet light, and it might affect the

growth of the experimental cyanobacteria.

Figure 2.4.3 LED light for the experiment, this additional light is done on purpose to

provide sufficient lighting for the growth of cyanobacteria.

With the restriction in space, the samples with varying CO2 concentration for blue-green

algae only is placed under the light in the orbital shaker that is only an orange colored

fluorescent lighting bulb. The other samples are all placed within the tank under both

LED and Fluorescent except the one under normal light for the lighting experiment.

21

Temperature control

To keep the temperature in constant, a container tank is used and a temperature control

thermal-meter, which could heat up the water in the tank (shown as figure 2.4.1) when

the temperature is below the set point which is 20oC. Therefore, samples placing in the

tank are supposed to culturing under same and constant temperature.

Figure 2.5.1 Heater used in the tank to maintain a constant temperature

22

Making CO2 concentration

As CO2 cylinder is available in the lab, pure CO2 is going to use for preparing all of

those samples. The procedure of making CO2 concentration is to flush the head space

with pure CO2 for about 2 minutes, and it is assumed to have pure CO2 concentration

within its headspace. For other concentrations, as the headspace left for all samples are

all 100ml, pure CO2 of headspace are made first and then use a needle to inject certain

calculated amount of air to obtain specific concentration of CO2 as the molar volume of

all gasses are the same.

Figure 2.6.1 Flushing head space with CO2

The presence of two needles on the top is for the purpose to enable gas flowing out of

the sample. The one with a pipe connected is for CO2 injection where the pipe is

connected with CO2 cylinder. If gas is flowing into the system without outlet, pressure

inside the sample will keep increasing which is not recommended for all those

experiments, the present of second needle is for the reason to release the redundant

gas replaced by the injection of CO2.

23

Pressure

As the pressure in the sample bottles will influence the amount of CO2 dissolved in the

liquid part, thus change the pH level. To balance the pressure for each sample is kind of

an important issue that needs to be considered. To do this, after finishing making the

CO2 concentration, a needle is used to balance the pressure of each sample with

atmosphere.

Picture 2.7.1 Pressure balancing, the amount of gas taking out depends on the amount

of inject. For example, if a sample of 50% initial CO2 concentration is going to prepared,

initially the headspace is flushed with pure CO2, and another 50ml of air will be injected

into the system, which means 100ml of CO2 and 50ml of air are presented, then

balancing the pressure by using a needle tube to flush the head space several times in

order to finely mix the gas, then 50ml of mixture gas will be taken out of the system.

Thus, the pressure inside this sample will be the same as atmosphere pressure and

which is made as 50% in CO2 concentration (which is 66.67% actually which realised

after the entire experiments, initial concentrations mentions below are all prepared in

this method).

24

General sample containment

After culturing enough algae for making all samples, the experiments could start. For

the combined system of methanogenesis and cyanobacteria, sand should be placed at

bottom of the sample bottle as to enable the methanogens to „hide‟ away from the light,

also the O2 produced by algae. Thus, in total there will be 20ml of sand with 10ml of

new f/2 media, 2ml of methanogens, 48ml of algae with media and 100ml head space

with different concentration of CO2 for each sample of varying initial CO2 concentration

experiment but 83.33% for the other combined system experiments.

For the sample varying initial CO2 concentration of cyanobacteria only, in order to get

the CO2 concentration more accurate, the samples are prepared by firstly fill all the

sample bottles full with algae, and placing two needles on the top, one is for CO2

injecting, the other one for algae outlet. Therefore, if the sample is going to have 70%

concentration in CO2, 70mL of CO2 in volume will be injected with eject of 70mL of

cyanobacteria. Same method is using for injecting air thus 30mL of air is injected into

the bottle and eject 30mL of cyanobacteria to get 70% initial CO2 concentration. The

volumes of headspace for all samples are in 100ml.

25

Results and discussions

Varying initial CO2 concentration for the combined system

The purpose of this experiment is aim to find out the influence of initial CO2

concentration within the headspace on growth of algae and the amount of CH4

produced.

Figure 3.1.1 Samples of varying CO2 concentration for cyanobacteria-methanogens

combine system

Before checking the methane content of the headspace gases using the GC, a

prediction of result is that the samples with higher CO2 concentration might produce

more CH4 as the methanogens need an anaerobic environment to live. This statement

has been overturned after finish this experiment.

To check the result, GC is used to measure the CH4 concentrations, which will show the

production rate under each CO2 concentration of this algae-methanogens combined

system. Measurements are taken every week or twice a week, data is included in

Appendix III.

26

Figure 3.1.2 final CH4 concentration (day 40th checking account first GC checking as

day 1) in the head space of all samples

From figure 3.1.2, it shows the final CH4 output for all samples. In the final checking, it

clearly shows that for the overall CH4 yield, sample with 55% initial CO2 concentration

gives the lowest performance which has only 29.2% CH4 in volume of headspace, and

50% CO2 gives the maximum of 38.8%

.

27

Figure 3.1.3 CH4 production under different CO2 concentration

Figure 3.1.3 combined all data together, and it could clearly show the difference in CH4

concentration of those samples. From the graph, the sample with 50% CO2 always have

higher CH4 concentration than others, and it has higher production rate at the beginning,

also the sample with 100% CO2 is producing the least CH4 for the first 7 checking, but it

nearly catch up with the 50% sample in the final checking. This might due to the

concentration of CO2 is changing during experiment period and also the algae are

constantly dying. Thus methanogens will have more „food‟ to consume. Therefore, from

that result, shows that the best initial CO2 concentration for algae and methanogens

combined system are at 50% (which is 66.67% in the actual calculation of CO2).

The reason why 50% initial CO2 concentration gives the best result might because the

cyanobacteria lives better under that concentration and the methanogens will have

more cell to consume, also the pH level under that concentration (amount of CO2

dissolved) could affect the activeness of them both. And the CH4 production stops after

50 days culturing due to the lack of nutrition.

28

Varying CO2 concentration for blue-green algae only

Refer to table 1.2.1, different types of algae can have extremely different CO2

tolerances. Since cyanobacteria and similar organisms are found to produce most of the

oxygen presented in earth‟s atmosphere, experiments on several kinds of cyanobacteria

could live under pure CO2 condition is done previously. [52] So for our experimental

cyanobacteria, it is supposed to have relatively high CO2 tolerance and the best

performing CO2 concentration should also be high.

For this experiment, as the growth of cyanobacteria could be seen clearly by difference

in color, the gas concentration of headspace is not needed in this experiment.

Figure 3.2.1 visual checking on 25/08/2014 (4 days after made). As it shown in the

picture, the samples with 100%, 80% and 20% are already almost dead at this time.

Figure 3.2.2 visual checking on 04/09/2014 (14 days after made). In this checking, only

the samples which still alive are shown, from this figure, it is still hard to tell the

difference in between each concentration.

29

Figure 3.2.3 visual checking on 16/09/2014 (26 days after made). This time, the result is

more obvious, only 15%, 40% and 60% are still alive, and it seems like 60% performs

better than others.

Figure 3.2.4 visual checking on 26/09/2014 (36 days after made). In this checking, the

sample with 20% CO2 concentration surprisingly turns back to green but other than that,

the sample with 60% still perform the best whereas others seems already died. Thus for

most of the samples under high CO2 concentration (compare with the atmosphere, 5%

pure CO2 concentration account as “high”) this type of cyanobacteria could survive for

long as 26 days of living.

From the pictures and researches performed in this work, this kind of cyanobacteria is

determined to have CO2 tolerance between 80% and 60%, and its best performance

concentration is at about 60%. Therefore, a suggestion for future experiments with

different photosynthesizing microorganisms is to find out the CO2 tolerance and also

best performing CO2 concentration should be found out first before making other

samples. For cyanobacteria, samples with headspace CO2 concentrations around 60%

are recommended.

30

Varying the media vitamin concentration for the combined system

Due to the fact that the sea water might not have enough nutrients for the growth of

algae, we are thinking that the result of that sample will not be optimistic, and the

samples with more vitamins should perform better than others.

As methanogens are used in this experiment, GC could be used for checking the

performance of each sample.

Figure 3.3.1 Final CH4 concentration in the headspace of each sample.

From figure 3.3.1, it clearly shows that the sample with 1/8 vitamin gives the most CH4

produced at the end which is 1852umol, and pure sea water gives the lowest of

939umol. The growth rates for f/2 media but different vitamin concentration are not

obvious, but it burst up after the check on the 14th day.

31

Figure 3.3.2 CH4 production under different vitamin concentration

This figure shows the data for all samples, and from the graph, the sample with pure

sea water clearly have higher CH4 growth rate than others, but it stops growing after the

14th day of the experiment. This might due to the methanogens and algae having used

up all the nutrition in the media and then died after that. For the other samples, the

reason why CH4 production rate burst up might due to the methanogenesis is more

likely to living under low vitamin concentration. However, why the sample with less

vitamin performs better- is probably because of the vitamin using in our lab is already

being used for a long time which means it might already been oxidized and the

methanogenesis is not much activated under those oxidized vitamin contained media. If

the vitamins are considered to be not oxidized, the result here will consider being-

methanogenesis are more likely to produce more CH4 under low vitamin concentration.

The reason why the sample with pure sea water produces much more CH4 at the

beginning is probably because the type of methanogens used in the experiments are

taken from the beach which is living under nature sea water condition. Thus, for this

experiment, the sea water using is after autoclave, which means the microorganisms

32

contained are all being killed, therefore, more suitable for the growth of methanogens.

With the bloom of methanogenesis at the beginning, the cyanobacteria will be

consumed up in a short term, which indicates that the concentration of CH4 grows up

quickly after the sample was prepared, and then stops due to the lack of food for

methanogens to consume.

33

Varying lighting condition for the combined system

There is a LED light in the sample tank, which might be too bright for algae growing,

and one of the lights slightly contains ultraviolet and might harmful to the algae and

methanogens. Also, the methanogens are more likely to survive in dark conditions. So

the purpose of this experiment is to find out the appropriate lighting condition for this

combined system. In order to make the lighting condition different, one of the samples is

in the tank with others, one with a translucent paper cover in the tank as well, another

one just place on the table so with normal lighting condition, and the last one is covered

entirely with aluminium foil.

Figure 3.4.1 Lighting sample with a translucent paper cover

As we already know, the methanogens are more likely to live in dark conditions, but

algae need lights to photosynthesis. Therefore, for this experiment, the one with dark

condition is expected to produce the most CH4 of all those samples. For the sample

with lighting, as mentioned in the lighting section, the direct light in the tank might not

appropriate for both algae and methanogens, so the result of that sample might not be

optimistic. Thus, as the sample of methanogens using is coming from the beach were

tide takes away the sand and puts it back every day, before using the GC to check the

34

methane content of the headspace gases, the sample with normal light is predicted to

give the best result.

To check the performance of methanogens under these different lighting conditions, GC

is still used to measure the amount of CH4 production.

Figure 3.4.2 Final CH4 concentration in the headspace of all samples.

Figure 3.4.2 shows the data collected from each measurement and surprisingly that the

samples under dark condition and normal light performance much better than the

samples under LED lighting. Therefore, it clearly means that our LED lighting system

using is not suitable for this kind of algae and methanogen combination. Also the algae

under dark condition appear to be still alive after all the data taken, which indicate that

these cyanobacteria could be living under dark condition, but the density is visibly less

than the one under normal light.

35

Figure 3.4.3 CH4 production under different lighting condition

This figure combined all data in one graph, and it shows that the sample under dark

condition produce a large amount of CH4 in the beginning and then stops. The reason

why more CH4 produced under normal light might because the algae are growing faster

with that condition. Thus methanogens could have more food to consume. However if

the algae are growing too fast, it will produce more oxygen which will lead death to the

methanogens, and that could be the solution why the CH4 concentration stops growing

after the GC measurement on the 24th day.

In addition, photoinhibition could be a reason to explain why the samples under LED

lighting condition are all in an „unhappy‟ stage which seems to have died- having a

yellow colour. There are three different time scales for the dynamic characteristics of

photosynthesis: rapid photoresponse (minutes), photoinhibition (hours) and photo

adaptation (days). Photoinhibition mostly happens at the process of electron transfer

chain located at photosystem II (PSII); photo adaptation is the mainly process to change

in physiological and biochemical composition for the light-harvesting system. Significant

36

progress in modelling photo adaptation has been achieved. [53] [54] [55] [56] [57] [58]

[59] [60] [61] The phenomenon of photoinhibition is represented by a decrease in

photosynthesis exhibited by plants when they are exposed to high irradiance. High

irradiance could also cause photo-oxidation of plants and other consequences, but

photoinhibition is usually used to describe the reduction of photosynthetic capacity,

independent of gross change in pigment concentration, induced by exposure to high

irradiance [62] [63]

37

Conclusion

Culturing algae as a bioenergy source has become a pretty mature technology at

present, but the technology for coccolithophorid algae cultivation still needs to be

developed. During this project, the importance of CO2 concentration and lighting

conditions could be observed in the cultivation of algae and the influence on methane

production with varying vitamin concentration.

From the result, this type of cyanobacteria and methanogens system could live for

about 40 days with methane being produced continuously. The maximum methane yield

among all samples is the one where made by f2 media with only1/8 of the

recommended vitamin concentration. This sample gives 1852.3 µmoles of methane and

45.4% methane content in the headspace volume

Therefore, for future experiments on coccolithophorid algae (Eg: E.hux, P.cart, etc.) it

will be better to find out the most suitable CO2 concentration, and the appropriate

lighting condition before other variations to the experiments are undertaken.

38

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45

Appendix I. Sample preparation

Sample Name/Number

Sample Owner (Can/Leo)

Date Made

Total Liquid Volume [mL]

Amount Algae [% or mL]ml

Amount Methanogens [% or mL]ml

Amount Sand [% or mL or g]ml

Volume of Headspace [mL]

Lighting Conditions [aquarium, natural, dark]

Measurement Technique [GC, microscope, visual inspection/picture]

Varying CO2 concentration A&M

100% CO2 with methanogen

Can 18/08/2014 50 48 2 20 100 aquarium GC measure the concentration of CH4


Can 18/08/2014 50 48 2 20 100 aquarium GC


Can 18/08/2014 50 48 2 20 100 aquarium GC


Can 18/08/2014 50 48 2 20 100 aquarium GC


Can 18/08/2014 50 48 2 20 100 aquarium GC


Can 18/08/2014 50 48 2 20 100 aquarium GC


Can 18/08/2014 50 48 2 20 100 aquarium GC


Can 18/08/2014 50 48 2 20 100 aquarium GC

Varying CO2 concentration Algae only

100% CO2 Can 21/08/2014 70 70 0 0 100 orbital shaker visual and pressure

46








Varying lighting

LED lighting 12:12

Can 21/08/2014 50 48 2 20 100 aquarium GC

normal roomlight 24 hrs

Can 21/08/2014 50 48 2 20 100 normal GC

LED lighting with paper cover 12:12

Can 21/08/2014 50 48 2 20 100 aquarium with paper cover

GC

no lighting Can 21/08/2014 50 48 2 20 100 dark GC

Varying vitamin concentration

1/8 vitamin concentration

Can 18/08/2014 50 48 2 20 100 aquarium GC & visual or microscope





f2 Can 18/08/2014 50 48 2 20 100 aquarium GC & visual or microscope

47

II. CH4 analyses

volume of gas (uL)

amount of gas (umol)

volume of bottle (mL)

peak area 01August

2013 CH4 (umol/L) Peak Area

1 mol of gas or hydrogen is 24.5 L at 21 oC

0 0 120 0 0 0

50 2.040816327 120 91133.2 17 91133

200 8.163265306 120 360286.1 68 360286

1000 40.81632653 120 3302443.9 340 3302444

2000 81.63265306 120 5078531.4 680 5078531

3000 122.4489796 120 5994208.6 1020 5994209

5000 204.0816327 120 10598098.2 1701 10598098

20000 816.3265306 120 40793339.7 6803 40793340

48

III. CH4 data for all experiments

1 6 14

29/08/2014 4/09/2014 12/09/2014

time area height CH4 time area height CH4 time area height CH4

100 1.894 39492 21871.7 6.550014 1.894 115063 64290.8 19.08397 1.897 402554.5 228881.6 66.76637

30 1.881 34415.5 18531.5 5.708042 1.892 164294.3 90104.8 27.24932 1.902 908532.5 511997.2 150.6862

40 1.873 29154.4 15779.6 4.835454 1.886 132243.3 72955.6 21.93344 1.902 585332.7 329011 97.08137

45 1.883 33985.9 18336.5 5.63679 1.9 153218.1 84569 25.41225 1.903 592234.5 334232 98.22608

50 1.89 74999.2 40916 12.43912 1.899 366571.6 200578.4 60.79837 1.902 2798021.1 1559820 464.0706

55 1.893 10149.9 5183.8 1.683429 1.888 19006.9 9879.4 3.152422 1.902 321355 179586.3 53.29889

60 1.888 22568.1 11968.4 3.743071 1.891 111690.4 61516.5 18.5246 1.9 473984 265692.4 78.61344

80 1.898 24609.9 13371.6 4.081718 1.897 97117.9 54129.7 16.10766 1.895 395151.5 221773.9 65.53854

lighting dark 1.897 123094.4 68035.8 20.41604 1.894 171418.5 96832.4 28.43091 1.906 2735829.9 1525817 453.7558

lighting LED 12v12 1.885 92245.2 50966.7 15.29949 1.887 114668.1 62234.4 19.01848 1.893 431573.1 241169.1 71.5793

lighting LED paper cover 1.899 91905 51433.8 15.24306 1.895 109571.3 59316.2 18.17314 1.893 859305.2 481076.8 142.5216

lighting normal light 24 1.896 124223.7 68676.9 20.60334 1.895 147799.7 80774.8 24.51358 1.901 716757.9 401725.1 118.8791

pure sea water 1.879 1092126.8 604922 181.1366 1.891 2876572 1556979 477.0988 1.899 4789730.9 2586841 794.4091

1/8 vitamin 1.882 53550.2 29050.9 8.881661 1.895 319277.3 176221.5 52.95429 1.881 3513381.4 1911927 582.718

1/4 vitamin 1.895 41245.9 22176.4 6.84091 1.899 204089.9 113055.8 33.84968 1.898 820224.9 455669.6 136.0398

1/2 vitamin 1.889 9625.6 3015.3 1.596471 1.885 214709.8 118527.3 35.61107 1.901 866527.1 476701.4 143.7194

f/2 1.92 41127.5 17015.8 6.821273 1.889 164686.4 91775.1 27.31435 1.901 602801.1 337992.1 99.97862

49

18 21 24

16/09/2014 19/09/2014 22/09/2014

time area height CH4 time area height CH4 time area height CH4

100 1.889 715122.2 404429.6 118.6078 1.882 2027959 1118067 336.3507 1.845 3011976 1666203 499.5565

30 1.897 2854183 1602859 473.3855 1.894 4598402 2547746 762.676 1.862 5696689 3111602 944.8342

40 1.897 1600862 885754.8 265.5137 1.896 4120704 2262317 683.4465 1.82 5218538 2857475 865.5296

45 1.898 1887423 1057296 313.0419 1.885 3353622 1841314 556.2208 1.85 4391088 2427313 728.2916

50 1.889 4463525 2459201 740.3057 1.894 7047968 3848281 1168.953 1.837 7938030 4308079 1316.576

55 1.896 1739214 952413.4 288.4604 1.897 4040281 2238885 670.1078 1.853 5277674 2898090 875.3378

60 1.897 1796476 1009936 297.9576 1.893 2597384 1448913 430.7936 1.849 4283326 2320841 710.4184

80 1.893 727264.1 413497.9 120.6216 1.889 2170537 1189927 359.9982 1.8 3399017 1875861 563.7499

lighting dark 1.896 2925618 1611509 485.2335 1.93 2989592 1657217 495.8439 1.838 3069638 1694319 509.1201

lighting LED 12v12 1.9 643371.7 355573 106.7075 1.895 853940.4 466591.7 141.6318 1.829 939953.5 519353.1 155.8976

lighting LED paper cover 1.894 1351775 754642.4 224.201 1.873 1676085 920644.3 277.99 1.843 2087928 1150766 346.2969

lighting normal light 24 1.894 1897954 1065003 314.7884 1.909 2140364 1179862 354.9937 1.835 2941357 1600060 487.8439

pure sea water 1.894 5129888 2813553 850.8264 1.874 5102103 2805389 846.2182 1.851 5877317 3191682 974.7925

1/8 vitamin 1.891 4161891 2277310 690.2776 1.888 7498037 4095242 1243.6 1.848 9461318 5169009 1569.223

1/4 vitamin 1.9 1291006 707009.5 214.122 1.899 4060414 2240788 673.447 1.842 5799935 3158415 961.9583

1/2 vitamin 1.9 1291006 707009.5 214.122 1.902 5489204 3031144 910.4214 1.86 7926242 4328738 1314.621

f/2 1.893 1776565 995579.1 294.6553 1.904 3412114 1874230 565.922 1.84 4773879 2619238 791.7799

50

28 40

26/09/2014 8/10/2014

time area height CH4 time area height CH4

100 1.907 4748052 2624475 787.4963 1.899 9027985.5 4982917 1497.352

30 1.899 7289178 3972709 1208.959 1.89 8463578.7 4645702 1403.742

40 1.903 7111378 3899557 1179.47 1.902 9135362.9 5070610 1515.161

45 1.905 5742162 3143953 952.3762 1.903 8571139.8 4730236 1421.581

50 1.908 8744579 4805086 1450.347 1.902 9538683.9 5269867 1582.055

55 1.902 6043142 3354370 1002.296 1.905 7186959.3 3911769 1192.006

60 1.905 6199682 3405582 1028.259 1.903 8604752.3 4796202 1427.156

80 1.914 4632261 2537882 768.2916 1.904 7651462.5 4164776 1269.047

lighting dark 1.908 2867399 1609304 475.5773 1.894 3077783 1719411 510.471

lighting LED 12v12 1.914 939649.9 513060.2 155.8473 1.905 939111.3 522016.9 155.7579

lighting LED paper cover 1.908 2054471 1146616 340.7478 1.907 2116618.6 1181337 351.0554

lighting normal light 24 1.91 2887823 1592321 478.9648 1.907 2933129.9 1654230 486.4793

pure sea water 1.9 5861376 3151138 972.1486 1.908 5660876 3067545 938.8944

1/8 vitamin 1.903 10437529 5693011 1731.134 1.902 11168176.6 6133081 1852.317

1/4 vitamin 1.906 7116780 3889620 1180.366 1.901 9401888.9 5100639 1559.367

1/2 vitamin 1.904 7422388 4099832 1231.053 1.907 8626319.1 4818823 1430.733

f/2 1.902 6316054 3498480 1047.56 1.905 8601391 4748275 1426.599

51

IV. Detailed figures of result Varying initial CO2 concentration for the combine system

Figure 4.1.1 CH4 concentration of algae with 100% CO2 (left) and 30% CO2 (right)



52


53

Varying the media vitamin concentration for the combined system

Figure 4.2.1 CH4 concentration of algae with pure seawater (left) and 1/8 vitamin (right)

Figure 4.2.2 CH4 concentration of algae with 1/4 vitamin (left) and 1/2 vitamin (right)

Figure 4.2.3 CH4 concentration of algae with f/2 media Varying the lighting condition for the combined system

54

Figure 4.3.1 CH4 production under dark condition (left) and LED lighting 12:12 (right)

Figure 4.3.2 CH4 production under LED lighting 12:12 with paper cover (left) and normal light condition (right)

55

V. Media recipes Medium f (f2 and fE) - CSIRO Modification Marine and Atmospheric Research

Reference: Guillard, R. R. L. and Ryther, J. H. (1962) Canad. J. Microbiol., 8: 229-239. Stock Solutions

1. NaNO3 150.0 g L-1 H2O 2. Trace metals CuSO4.5H2O 19.6 mg (Weigh and add each chemical individually) ZnSO4.7H2O 44.0 mg (in quantities stated to 750 mL distilled) CoCl2.6H2O 22.0 mg (water, dissolving between each addition.) MnCl2.4H2O 360.0 mg (Make up to final volume of 1 litre.) Na2MoO4.2H2O 12.6 mg 3. Na2SiO3.5H2O 22.7 g L-1 H2O 4. Fe citrate: Ferric citrate 9.0 g Add both chemicals to 1 litre of Citric acid 9.0 g distilled water and autoclave to dissolve. 5. Vitamins Stock Solution to 100mL of distilled water, add the following: Biotin 1.0 mL Vitamin B12 1.0 mL Thiamine HCL 20.0 mg (fresh solution every 3 months) Primary Stocks Vitamin B12 10.0 mg 100 mL-1 H2O Biotin 10.0 mg 100 mL-1 H2O 6. NaH2PO4.2H2O 11.3 g L-1 H2O 7. Na2EDTA.2H2O 30.0 g L-1 H20 (used in fE and fE2 media only) All stock solutions are made up in distilled water. Store all stock solutions in the refrigerator. To reduce risk of contamination and for longer shelf life, stock solutions can be autoclaved or filter sterilised using a 0.22 um filter.

Australian National Algae Culture Collection Street Address Castray Esplanade, Hobart, Tasmania 7000, Australia Mail GPO Box 1538, Hobart, Tasmania 7001, Australia Telephone (03) 6232 5316 Int +61 3 6232 5316 Facsimile (03) 6232 5471 Int +61 3 6232 5471 Web Site http://www.csiro.au/ANACC Email [email protected]

http://www.csiro.au/ANACC

mailto:[email protected]

56

Medium f (f2 and fE) - CSIRO

Modification Marine and Atmospheric Research

Reference: Guillard, R. R. L. and Ryther, J. H. (1962) Canad. J. Microbiol., 8: 229-239.

Preparation Methods 1. To Prepare Medium f Add 1 mL of each stock solution (1 – 5) to 1 litre seawater. Dispense to flasks and autoclave at 121°C (15PSI, 15 mins). Phosphate must be sterilised separately from seawater to prevent precipitation. Dilute original phosphate stock with distilled water such that 1 mL added to each flask of sterile medium will give the required concentration of phosphate in the medium. Autoclave dilute phosphate stock at 121°C (15PSI, 15 mins). After cooling, dispense aseptically with sterilised automatic dispenser. For example: For 100 x 125 mL Erlenmeyer flasks, each containing 75 mL medium, prepare dilute phosphate stock as follows: f and fE media: Take 7.5 mL of original phosphate stock and make up to 100 mL with distilled water. Pour into a 250 mL Schott bottle and autoclave to sterilize. Dispense 1 mL per flask asepically. f2 and fE2 media: Take 3.75 mL of original phosphate stock and make up to 100 mL with distilled water. Pour into a 250 mL Schott bottle and autoclave to sterilize. Dispense 1 mL per flask asepically. Scale up in the same proportion for larger volumes. To Prepare Medium fE Prepare as medium f, add 1 mL of Na2EDTA.2H2O stock solution. To Prepare Medium f2 Prepare as medium f, but using 0.5 mL of each stock solution instead of 1.0 mL of each. To Prepare Medium fE2 Prepare as medium f2, add 0.5 mL of Na2EDTA.2H2O stock solution. 2. To Prepare Medium f2 concentrated nutrients Take 5 mL of each stock solution (1 – 6) and make up to 100 mL with distilled water. Pour into a 250 mL Schott bottle. Autoclave at 121°C (15PSI, 15 mins). Alternatively, filter sterilise using a 0.22 um filter into a sterile 250 mL Schott bottle. Use 1 mL/100 mL sterile seawater. Add correct amounts of nutrients aseptically.

Australian National Algae Culture Collection Street Address Castray Esplanade, Hobart, Tasmania 7000, Australia Mail GPO Box 1538, Hobart, Tasmania 7001, Australia Telephone (03) 6232 5316 Int +61 3 6232 5316 Facsimile (03) 6232 5471 Int +61 3 6232 5471 Web Site http://www.csiro.au/ANACC Email [email protected]

http://www.csiro.au/ANACC

mailto:[email protected]

57

VI. Sample calculations

1. Making f/2 media

For making the media recipes of f/2, the stock chemicals available within the lab are inadequate. Therefore, when making the stock

solutions, similar substitution are in used.

Table 6.1.1 Substitution recipe calculation

Original Chemical

Recipes

Molar

mass(g/mol)

Amount needed for

stock solution (g/L)

Target

ion

No. of moles

present

Substitution

Recipes

Molar

mass

Amount

needed (g/L)

NaNO3 85 150 N 1.764705882 KNO3 101 178.2352941

CuSO4.5H2O 249.5 0.0196 Cu 7.85571E-05 Cu(NO3)2.3H2O 241.5 0.018971543

ZnSO4.7H2O 287.38 0.044 Zn 0.000153107 Zn(NO3)2.6H2O 297.38 0.045531074

Na2MoO4.2H2O 242 0.0126 Mo 5.20661E-05 (NH4)6Mo7O24 1164 0.008657851

Na2SiO3.5H2O 212 22.7 Si 0.107075472 Na2SiO3 122 13.06320755

Fe citric C6H5FeO7 244.94 0.009 Fe 3.67437E-05 FeCl3.6H2O 270.35 0.009933657

NaH2PO4.2H2O 156 0.0113 P 7.24359E-05 NaH2PO4 120 0.008692308

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2. CH4 concentration calculation

The molar volume of gas is 24.5 L/mol under 25oC condition, and the headspaces are

known to be 100mL for all the samples. Thus the concentration of CH4 in percentage of

headspace could be calculated.

Table 6.2.1 Calculation of the final CH4 produced in percentage of headspace

Total mole of headspace (μmol) 4081.633

Final CH4 presented (μmol) % of headspace

30 1403.741512 34.4%

40 1515.161445 37.1%

45 1421.581245 34.8%

50 1582.054948 38.8%

55 1192.005589 29.2%

60 1427.156104 35.0%

80 1269.046573 31.1%

100 1497.35218 36.7%

lighting dark AandM CC 510.4710331 12.5%

lighting LED 12:12 AandM 155.7579321 3.8%

lighting LED 12:12 paper cover 351.0554459 8.6%

lighting normal light 24 486.4793425 11.9%

pure sea water 938.894399 23.0%

1/8 vitamin 1852.317284 45.4%

1/4 vitamin 1559.366577 38.2%

1/2 vitamin 1430.733103 35.1%

f/2 1426.59861 35.0%

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