Project Bt4ff19

2019
Phosphorous extraction from wastewater
Cynthia Nyawira Thomsen
Jonas Lisby Wesseltoft
Marianne Fuglsang
Peter Nørgaard Mortensen
Simon Ehlers Hove
Aalborg University
31-05-2019
BT4FF19 Biotechnological Analysis 31/05/2019
Aalborg University
Title: Phosphorous extraction from wastewater
Theme: Bioplastic and P-release
Project span: 01/02 2019- 31/05 2019

Study group: Bt4ff19
Supervisor: Reinhard Wimmer and Giulia Dottorini
Page total: 69 Abstract

Appendix total: 15 During the last century, there has been a large
increase in global population. This has led to an
Participants: increase in the need for Phosphorous (P) in
fertilizer to maintain food security. The
Simon Ehlers Hove depletion of natural sources of P and the
negative results of P in aquatic environments
has increased interest in recovering P from
wastewater. This is done with a process called
enhanced biological phosphorous removal, that
Peter Nørgaard Mortensen utilizes phosphorous accumulating organisms
(PAO) to fixate the P in polyphosphate
structures. This removes the P from the
wastewater before it is let out of the treatment
plant and enables the collection of the P-rich
Jonas Lisby Wesseltoft PAO for use in fertilizer. During this process, the
PAO also produce polyhydroxyalkanoates (PHA),
which can function as an alternative to plastics.
The initial process of this method was examined
in the experiment in this report. It was examined
Marianne Fuglsang
if acetate was a useful substrate for PAO. The
results were analyzed using Colorimetry, HPIC,
______________ NMR, TLC and GC-MS. The results of these tests
suggested a minor activity of the PAOs and no
Cynthia Nyawira Thomsen
discernable production of PHA.
The content of the report is freely accessible, but publication (with reference) may only happen in
agreement with the authors.
Side 1 af 69
Aalborg University
Indhold
Abbreviations .................................................................................................................................... 3
Introduction ...................................................................................................................................... 4
Experimental Methods Theory ........................................................................................................ 14
Applied Methods ............................................................................................................................. 18
Results ............................................................................................................................................ 23
Discussion ....................................................................................................................................... 45
Sludge incubation ........................................................................................................................ 45
Colorimetry ................................................................................................................................. 45
NMR - acetate ............................................................................................................................. 46
HPIC – Acetate ............................................................................................................................. 46
Thin Layer Chromatography - TLC ................................................................................................ 46
NMR-lipids................................................................................................................................... 46
GC-MS Lipid ................................................................................................................................. 47
GC-MS - PHA................................................................................................................................ 48
Conclusion....................................................................................................................................... 49
Bibliography .................................................................................................................................... 49
Appendix ......................................................................................................................................... 54
Side 2 af 69
Aalborg University
Abbreviations
ATP: Adenosine triphosphate
CoA: Coenzyme A
COD: Chemical oxygen demand
DNA: Deoxyribonucleic acid
DMPC: Dimyristoyl Phosphatidylcholine
DMPE: Dimyristoyl Phosphatidyl ethanolamine
DMPG: Dimyristol Phosphatidylglycerol
EIC: Extracted ion chromatogram
GC: Gas Chromatography
GCMS: Gas Chromatography Mass Spectroscopy
HB: Hydroxybutyrate
HPIC: High Pressure Ion Chromatography
HV: Hydroxyvalerate
MS: Mass spectroscopy
NAD(H): Nicotinamide adenine dinucleotide
NMR: Nuclear Magnetic Resonance
P: Phosphorous
PAO: Phosphorous accumulating organisms-
PE: Phosphatidyl-ethanolamine
PHA: Polyhydroxyalkanoates
RNA: Ribonucleic acid
TCA cycle: Citric acid cycle
TLC: Thin Layer Chromatography
VFA: Volatile fatty acids
Side 3 af 69
Aalborg University
Introduction
P is an important nutrient in agriculture globally. It is impossible for plants to grow without it
and therefore, P is essential for global food production. P have been in short supply since
mass production of food started, due to increased demand and increased global population.
This leads to a rapid consumption of natural P, which is a huge problem all over the world.
Without the natural resources of P, a new source needs to be discovered, to continue the
required production of food. In opposition to the need for P in agriculture, it can be disrupting
in natural aquatic environments such as rivers and lakes. Large amounts of P causes algae to
grow rapidly in the water leading to eutrophication.
There has been a raised interest around wastewater as an alternative source of P. Wastewater
has been found to contain a large amount of P. At wastewater treatment plants PAO can be
used to remove the P from the water. Treatment plants utilizes the PAOs ability to absorb or
release P under aerobic or anaerobic conditions respectively. PHA is a biproduct of the
anaerobic process and could be beneficial as bioplastic. The PAOs accumulate the P in an
aerobic environment and can then be collected, which can be used in industries to produce
fertilizer in agriculture.
In this project, acetate was used as a nutrient source for PAOs in order to determine their
ability to produce PHA under anaerobic environments. This was done by measuring the
uptake of acetate and the release of P which are conjugated reactions. In addition to this, the
lipids of the sample were analyzed, and their structure determined.
Side 4 of 69
Aalborg University
Statistics and source of phosphorus

P is one of the essential nutrients required by every living cell. P has always been in short
supply in agricultural production. The increasing demand on P has caused a depletion of P in
most of the reserves. The reserve that remains are in just a few countries, for example
Morocco, which have a phosphate rock reserve that amount to 50% of the world total
resources. This make P increasingly rare and expensive. The price for phosphate has, over the
years, risen since 2008 (figure 1). Therefore, it is uncertain whether the world’s main source
of P will be available and accessible in the future. There is no proved substitute for P and
therefore there is a need to continue mining the phosphate rock to maintain a sustainable
supply of P to meet global demand(D. Cordell & Neset, 2014; Dana Cordell, Drangert, & White,
2009).
Figure 1: World price of phosphate rock from year 2001 to 2020. On the y-axis is the price in USD and on the x-axis is the
time in years. A Peak is seen from around year 2007-2008 (Blanco, 2011 modified).
As previously mentioned, there is a limited amount of P and one of the candidates as a

potential source for P is the sludge from wastewater. A new way for recovering P from
wastewater with reduced environmental pollution is by using PAO. This is a practical method,
which is being practiced in many of the world’s developed countries. The method could be a
conventional solution for phosphate source since there is presently no substitute for P
(Herzel, Krüger, Hermann, & Adam, 2016).
Side 5 of 69
Aalborg University
Mining and impact

Phosphate mines produce toxic waste from the mining areas. In essential P areas waste can
dominant the landscape for miles. A good example is Florida, the world’s largest mining place.
Here, mining of phosphate has led to tons of mining waste, piling up at dump sites. Phosphate
mining has caused rivers to dry up and settling ponds have leaked which can lead pollution of
the surrounding environment. The phosphate rock contains hazardous chemical elements
that damage the soil, when used in agricultural fertilizers (Craig Pittman, 2017; Reta et al.,
2018).
Some of the risks involved in global P production and use, include loss of a source of irrigation
water, widespread water pollution from farm nutrient runoff, energy cost of producing and
transporting fertilizers, damage to fish spawning ground, reductions in tourism, as well as loss
of the essential natural value of ecosystems and nature. These risks are the negative impact
on the ecosystem, that are not considered in the direct market transactions in supply P from
industries worldwide (D. Cordell & Neset, 2014).
The Increasing global demand on P is caused by the increasing global population and the
following food demand, especially in developing countries. Another factor is an increasing
global demand towards some specific food diets, which are significantly more P intensive for
their production. The main fundamental objective of the P supply is to ensure that the
agriculture sectors have enough access to P. Therefore, around 90% of the global mined P is
for food and the rest for other industrial use. To attain a sustainable and suitable development
towards reducing P demand, the world must change the mining ways, usage and equitably
distribution of P globally (D. Cordell & Neset, 2014).
Fertilizer in agriculture
Humans have relied on agriculture in the past 10.000 years, since plants were first
domesticated. A gradual increase in agricultural techniques eventually allowed humans to
gather in towns and focus on other things than collecting food. Following the food surplus,
the global population has seen a significant increase, which led to increasing agricultural
demands all over the world. This increase led the agricultural community to find new ways to
enhance the output of crops. It gave an increase in cultivated land and a more intense
cultivation, which resulted in a big decline in soil fertility leading to a decline in crop yield.
Since the industrial revolution there has been a significant increase in the use of chemical
fertilizer which has greatly reduced the issue of declining crops (Hignett, 1985).
In temperate climates in developed countries, fertilizer inputs are estimated to attribute

about 40-60% of the crop yield. This value is even higher in tropic climates. For this reason,
fertilizers are essential for the global food security and must be secured in the future
especially with the increasing global population (Stewart & Roberts, 2012).
Side 6 of 69
Aalborg University
Important mineral for plants

In addition to oxygen, carbon dioxide and water, plants require a minimum of fourteen
specific mineral elements to grow. These minerals are split up in two groups, macronutrients
and micronutrients. The first group is needed in significantly larger amounts than the latter.
The macronutrients include: N, P, K, Ca, Mg and S. The micronutrients include Cl, B, Fe, Mn,
Cu, Zn, Mo and Ni. They are all necessary for optimal growth and lack of any of these will lead
to inhibited growth and reduced crop yields (White & Brown, 2010).
Plants need P in relatively large amounts, and agricultural crops usually have a P
concentration of between 0.1% to 0.5%. The P uptake happens through the roots of the plant
allowing it to take up the P from the soil. In the plant, P is included in several essential
molecules including DNA, RNA, phospholipids, enzymes and ATP. These molecules are all
necessary for the cells of the plants to function as they are involved in energy transport,
genetic information and metabolism (Schachtman, Reid, Ayling, S, & A, 1998).
P is often added to the soil in fertilizers because only around 20% of P in the soil is available
to the plants. This is caused by P high affinity to bind to soil matrix and become immobile in
soils. Much of the P exists as organic P which the plants cannot take up. Organic P counts for
20-80% of all P in the soil. This leads to a slow uptake of P in the plants and lead to slow grow.
The P that is bound to the soil has a risk to be eroded with soil, which can cause significant
environmental damage (Schachtman et al., 1998).
Eutrophication
The presence of elevated P levels in an ecosystem is not always beneficial. It can have
significant and unwanted impacts on aquatic environments in the form of eutrophication
(figure 2). Eutrophication is the sum of the effects caused by a major increase in plankton
growth, disturbing the level of primary production in the lake or river which can cause it to
lose its function e.g. drinkable water. This is one of the more challenging environmental
problems faced today (Yang, Wu, Hao, & He, 2008).
The increase in plankton happens when the environment receives a significant increase in P
or nitrogen (N) levels (figure 2). This is because these two usually are the most limiting factors
in aquatic environments. This is seen in the algae's experienced chemical formula
C106H263O110N16P. P accounts for about 80% of the limitation of eutrophication, N accounts for
10% and the last 10% is accounted for by other small factors. This can however vary from
location and environment. It means that an increase in these limiting factors will allow the
plankton to cause algal bloom (figure 2), which is when the algae is saturated with the
nutrients it usually lacks and is therefore allowed to grow rapidly (Yang et al., 2008).
Runoff water from agricultural land is one of the main causes of eutrophication since the
runoff contains nutrients like P and N. When soil erosion occurs, it causes these nutrients to
reach the water bodies, and the nutrients are extracted into the water, and this causes
eutrophication. Nitrates is water soluble and therefore becomes dissolved in the water and
Side 7 of 69
Aalborg University
are readily transport through the surface- or groundwater. Phosphate is not water soluble
and therefore is bounded to soil components and moves when the soil gets eroded (Khan,
2014).
Figure 2: Representation of some of the eutrophication causes and effects (Biology, 2018).
A result of elevated P levels in water is that autotrophic algae build up their biomass through
increased photosynthetic activity. The increase in algae biomass will decrease visibility in
water and reduce the amount of light that can reach the plant life at the bottom of the water.
This will in turn decrease or even stop photosynthesis, killing the plants. It can also lead to
both supersaturation and a lack of oxygen in the water, both of which are fatal to animal life.
Some algae will form green scum on the water surface and can also release toxins that are
deadly to animals and degrade organic material into poisonous gases. When the algae die it
also releases a lot of these dangerous toxins, which can be extremely severe if it happens in
an area containing drinking water (Yang et al., 2008).
Phosphorus extraction at treatment plants

Wastewater is polluted water that contains waste products from daily sources. This is purified
at treatment plants where all the pollutants are removed. However, the pollutants have a lot
of potential elements such as P, making wastewater an ideal source for recovering P (Yuan,
Pratt, & Batstone, 2012). This is beneficial since the treatment plant also needs to extract the
P to prevent it from contaminating the water at the outlet of the treatment plant (Nielsen,
Mcilroy, Albertsen, & Nierychlo, 2018). The extraction of P from wastewater can be done by
PAO. PAO are a group of bacteria often used in treatment plants to remove P from
wastewater by binding P in chemical structures such as ATP and Poly-P inside the cells.
Side 8 of 69
Aalborg University
However, depending on the environmental conditions PAO can either release or accumulate
P. Under aerobic conditions P is accumulated and under anaerobic environments P is
released. This process is called enhanced biological phosphorous removal (Barnard, Barnard,
Dunlap, & Steichen, 2017).
Figure 3: A process diagram of the purification process on a treatment plant. The triangles represent anaerobic
tank (to the left) and an aerobic (to the right) tan. The squares show different processes and products and the
octagon is a harvest tank for extracting the P (Kasper Reitzel1 et al., 2015).
To make these two separated environments, two tanks are used. The first tank is anaerobic
where PAO do the process that releases all the P currently in the cell. This is effective because
the PAO cells can take up P in the second tank. When the P is released it sinks to the bottom,
where it is collected and transferred to a storage container. The water, still filled with bacteria
and P, is transferred to the second tank (Barnard et al., 2017).
The second tank is aerobic where PAO bacteria take up P. When the bacteria take up P it sinks
to the bottom where it is collected. The water will then go through the process again a few
times to make sure all P is extracted and then released. The bacteria now containing P, can
be harvested. After harvesting the bacteria can be retreated to the anaerobic tank and be
reused for the process (figure 3)(Barnard et al., 2017).
Anaerobic and aerobic process

Under the anaerobic conditions the cells synthesize PHA and in the process P is released. PAO
uses PHA as chemical energy storage synthesized from organic carbon mainly short-chain
volatile fatty acids (VFA), poly-P and glycogen. In an environment containing VFA, like acetate,
PAO will absorb the acetate from its surroundings and then store it in the form of PHA. The
PHA can be used in the TCA cycle later to create energy in the form of NADH and ATP (figure
4) (Nielsen et al., 2018).
Side 9 of 69
Aalborg University
Figure 4: Pathway of acetate in a PAO bacteria. The figure 4 Shows processes in anaerobic environments (Moritz
Haigermoser, 2017).
To transport acetate through the membrane, a difference in pH is used and H+ will enter the
cell. PAO uses the energy generated from hydrolyzing polyphosphate to syntheses ATP, which
is used to remove the H+ and bind a CoA to the acetate and there by losing one of its P atoms.
The P then moves through a membrane protein out of the cell to balance the concentration
contrast between the outer environment and the cytoplasm and because of the gradient do
not require energy (figure 4) (Barnard et al., 2017).
Figure 5: Pathways of PAO processes in aerobic environments

modified (Moritz Haigermoser, 2017).
Side 10 of 69
Aalborg University
In aerobic conditions, PAO uses glycogen and the stored up PHA to generate Acetyl-CoA which
is then used in the TCA cycle. This process absorbs P and H + from the environment and uses
ATPase to generate ATP. The generated ATP is then used to synthesis poly-P. The ATP is used
in the metabolism to break down glycogen and PHA. The H+ And NADH2 react with O2 from
the environment in the electron transport chain (Figure 5) (Seviour, Mino, & Onuki, 2003).
Production of bioplastics
PHA is a byproduct of the anaerobic processes in a treatment plant. These macromolecules
are mono-polymers or co-polymers in the family bio polyesters with a high diversity in
structure. They contain different types of alkyl groups (figure 6) and can be synthesized by
microorganisms (Chen, 2010). The PHA producing cells uses PHA as an intercellular storage
for energy and carbon. Because of its diverse structure, chemical properties and being
complete biodegradable PHA’s have found applications in industrial, medical and daily use
e.g. bioplastic. Based on this, PHA could become beneficial in limiting the consumption of
fossil fuels(Chen, 2010; Marang, van Loosdrecht, & Kleerebezem, 2015).
Figure 6: General structure of PHA chains and PHA monomer: (3HB) 3-hydroxybutyrate, (3HV) 3-
hydroxyvalerate, (3HHx) 3-hydroxyhexanoate, (3HO) 3-hydroxyoctanoate, (3HD) 3-
hydroxydecanoate, (3HDD) 3-hydroxydodecanoate (Chen, 2010 modified)
It is possible to produce PHA by either a chemical approached or biosynthesis. However,

through biosynthesis it is impossible to obtain a complete control of the structure of
monomers in the PHA produced. The PHA-producing microorganisms can synthesize PHA
monomers together to create polymers (Chen, 2010). Studies have shown that single fatty
acids as substrates enables microbes to have superior storage capacity of PHA at 90 wt %.
However, glucose, starch and wastewater can be used as substrates, but will yield a lower
PHA storage capacity (Gerardi, 1979; Marang et al., 2015). Several pure cultures of bacteria
Side 11 of 69
Aalborg University
in controlled environments have been used to produce PHA. However, active sludge from
treatment plants have the potential to be more cost efficient and environmental beneficial.
A study of PHA producing in active sludge over 5 months, have showed the major contributors
to PHA production, by determining the genes involved with PHA production, Bacteroidetes
19-28%, Proteobacteria 17-26% and Actinobacteria 11-15% (Antonio Mendez-Vilas, 2007).
Figure 7: Biosynthetic pathways where fatty acids is transformed into PHA with enzymes and
genes (Chen, 2010 modified).
Several PHA synthesis pathway have been researched and the pathway II (figure 7) produces
PHA by using fatty acids such as acetate as substrate. Several reactions are involved in the
PHA syntheses from fatty acids.
First step of the PHA synthesis, the fatty acids are cleaved to acyl-CoA through beta-oxidation.
Several reaction ways lead to R-3-Hydroxyacyl-CoA from acyl-CoA which PHA synthase (PhaC)
can synthesize. The enzymes used to synthesize R-3-Hydroxyacyl-CoA involves 3-ketoacyl-CoA
reductase, epimerase, R-enoyl-CoA hydratase/enoyl-CoA hydratase, acyl-CoA oxidase and
Side 12 of 69
Aalborg University
enoyl-CoA hydratase I (figure 6) (Chen, 2010). Step 4 in the beta-oxidation 3-Ketoacyl-CoA is

cleaved into acyl-CoA and acetyl-CoA, but if acetate is used as the substrate acetyl-CoA can
be synthesized by acetyl-CoA syntheses. Pathway I then synthesizes 3-hydroxybutyryl-CoA
out of acetyl-CoA with the enzymes PhaA and PhaB, PHA is then synthesize by PhaC (figure
5). PHA can present a possibility for production of a more environmentally beneficial
alternative for regular plastics because of its biodegradable nature (Chen, 2010).
Plastic as a problem
Plastic is an important resource that is used for many purposes since it is cheap, versatile and
light weight. After (Ritchie & Roser, 2018). The plastic that ends up in the ocean has a big
impact on the wildlife. The animals occasionally consume the plastic by mistake, which can
be lethal if the plastic blocks their digestive system causing death by starvation. Some of the
plastic in the ocean are from old fishing net that has been dumped or lost. The animals can
get tangled up in the nets which can result in its death since they will be unable to breathe or
move (Gregory, 2009). A solution to this problem could be the use of bioplastic, which is
produced from renewable materials, and is degradable in the nature. There exist three
fundamental types of bioplastic/biopolymer groups. Degradable petrochemical-based
bioplastic, degradable (primarily) bio-based bioplastic and Non-degradable bio-based plastic.
Degradable bioplastic can be based on petrochemicals and renewable raw materials as its
base (Endres, 2017). The PHA produced by the PAO is an example of the degradable bio-based
bioplastic.
Bioplastic have a similar structure to normal plastic and is produced by petrochemicals. The
similarities result in the same processing methods and usages. The minor differences in
structure creates an easily accessible energy source for bacteria and other microorganisms to
degrade, making bioplastic biodegradable. The reactions depend on different environmental
conditions such as water, different microorganisms, humidity, temperature, oxygen and pH.
(Endres, 2017)(Gregory, 2009).
Biodiesel fuels from Sludge lipids

The use of sludge from wastewater is a promising method for the producing biodiesels
because sludge contains an important amount of lipids. Biodiesel is composed of lower alkyl
fatty acid with chain length C14 – C20, and esters of shorts chain alcohols mostly methanol.
Biodiesel is an ideal alternative to fossil fuels/diesel fuels since it is renewable, sustainable
and less toxic. It is produced from lipids by the process of transesterification with simple
alcohols and the cost of production varies depending on the source, the basic materials
needed, geographical areas among other factors. Oils produced from the sludge are available
and relatively cheaper compared to other types since the sludge is readily available. Biodiesel
fuels are environmental beneficial because of the combustion is CO 2 neutral (Demirbas,
Bamufleh, Edris, & Al-Sasi, 2017).
Side 13 of 69
Aalborg University
Experimental Methods Theory

Sludge incubation.
The sludge will be incubated with acetate as substrate to measure the substrate uptake and
P release for four hours. During incubation period, sludge would be subjected to an anoxic
environment to sustain the reaction that takes up the VFA and store them in PHAs. This allows
the PAOs to synthesize PHA energy reserves and release orthophosphate into the reactor
(Kristiansen et al., 2013). The reactors should be sealed with a lid containing three syringe
needles. Two of these syringes are for gas inlet and outlet, and the third syringe is for sample
collection every thirty minutes. Every inlet syringe should be connected to N gas to allow
smooth flow of N gas into the reactor. Every outlet needle is checked every 30 minutes to
make sure that the gas is getting out, to prevent the reactor bottle from exploding. N gas inert
properties enables the reaction to occur in anoxic environment. The substrate is added to
only two sludge reactors and water is used for the third reactor which is the control reactor.
Immediately after this, the first samples should be taken, time and type marked on the
collecting tube, and the sample should be frozen. This is repeated every thirty minutes for
four hours (Maekawa & Elert, 2003).
Colorimetry
Accurate determination of the content of P is important in measuring the efficiency of P
removal in wastewater treatment. A commonly used method is the Molybdenum blue
method(Gerardi, 1979; He & Honeycutt, 2005). In this method, P concentration is measured
by P reacting with acid ammonium molybdate, which forms a phosphor-molybdenum
complex. This is further reduced by ascorbic acid, forming molybdenum blue. Molybdenum
blue absorbs light at wavelength 880 nm, which a spectro-colorimeter can read. Since there
is a linear relationship between absorbance and concentration as seen in Lambert Beers law,
it is possible to determine P concentration by comparing the sample with standard curve. The
release of orthophosphate in the sample can indicate the production of PHA (Gerardi, 1979).
High Pressure ion chromatography (HPIC)

HPIC is used to identify the amount of acetate in different samples. In this experiment the
AS11-HC column will be used. This column is used to identify many organic anions and
inorganic acid in complex matrices, in this case it is in wastewater. The column uses a small
resin particle called microbeads, to efficiently separate the organic anions and inorganic acid.
Therefore, giving good results of organic acids and inorganic anions in complex samples. This
gives a more accurate peak integration and more reliable results (ThermoFisher, 2019).
To run the sample though the HPIC it is necessary to make standard solutions with a known
concentration of acetate. This must be done before sample testing, so it will be possible to
identify the acetate concentration by comparing with the standards. The sample is diluted
before putting it into the machine, to make sure that the concentration is not to high and will
not affect the peak and the results. After the sample have been injected into the machine, it
Side 14 of 69
Aalborg University
will have a run time, where the sample runs through the column resulting in various spectral
diagrams. These peaks show the concentration of acetate in the samples (ThermoFisher, 2019).
Thin Layer Chromatography (TLC)

TLC is used to identify different types of lipid groups in a sample since the method can
separate lipids like cholesterol, triacylglycerols and hydrocarbons (Cyberlipid, 2019).
TLC consists of a polar solid phase (Silica gel) attached to an inert plate (aluminum plate) and
a nonpolar mobile phase (diethyl ether/methanol/acetic acid (90/1/1, v/v)). The method
functions on the same principles for chromatography in that a compound will have different
affinities for the stationary and mobile phases. This determines the speed at which the
compound will migrate. The method works by applying a sample onto the stationary phase
and inserting the plate into the mobile phase. The mobile phase proceeds to carry the sample
when it moves across the stationary phase resulting in spots along a vertical line after the
separation is completed. The measure of how well the compounds, match the two phases is
determined by the retention factor (Rf) with the following formula:
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑎𝑚𝑝𝑙𝑒

𝑅𝑓 =
𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
When the multiple results are obtained on the same plate, the compound with the lowest Rf
is the most polar, because it will tend to bind with the stationary phase. In the same vein, the
one with the highest Rf will be the less polar molecules. When implementing this along with
lipid standards, the different types of lipids in sample will be able to be determined. To do
this, the plates will first need to be treated with a substance that makes them visible, because
they are colorless. This is done in an iodine chamber. It works by adding iodine crystals to a
closed glass chamber and heating them up until a violet vapor is created. The iodine vapors
then create colored complexes with many organic molecules, including the lipids in the
sample. Once the lipids have been visualized, they can be analyzed (Chemistry Libretexts, 2019).
Nuclear Magnetic Resonance spectroscopy (NMR)

NMR is an analytical chemistry technique used in different research for determining the
content and purity of a sample as well as determining its molecular structure. NMR can
quantitatively analyze mixtures containing both known compounds and unknown
compounds. NMR can either be used to match against spectral libraries or to infer the basic
structure directly. Once the basic structure is known, NMR can be used to determine
molecular conformation in solution and enable the study physical properties at the molecular
level (Günther, 2013).
Side 15 of 69
Aalborg University
Several methods of NMR exist (see appendix A) however, the principle behind NMR is that
many nuclei have spin and all nuclei are electrically charged. If an external magnetic field is
applied, an energy transfer is possible between the base energy to a higher energy level. The
energy transfer has a specific wavelength which corresponds to a radio frequency and when
the energy level returns to its base level, energy is emitted at the same frequency. This signal
is measured processed to yield an NMR spectrum (Günther, 2013).
Gas chromatography mass spectroscopy (GC-MS)

This method is used to identify the lipids and PHA left in the reactors. The fatty acids and
monomers can then be identified by using the GC and MS. Lipids identification is done on the
(FAME) extracted after transesterification. PHA identification is done with the monomers of
PHA, which are an alkyne with a methyl ester, after transesterification (O. David Sparkman,
Zelda Penton, 2011).
GC for fatty acids and PHA
To run the fatty acids and PHA monomers through the GC, they are injected into the column
where they will be heated until they evaporate. This will cause them to move through the
column because of the gas flow, usually from helium (O. David Sparkman, Zelda Penton,
2011).In the column, the evaporates will shift between binding to the stationary phase or
moving with the mobile phase in the column (O. David Sparkman, Zelda Penton, 2011). A
detector is placed at the end of the GC column to detects all the vapors, when they exit. The
result of this analysis is recorded as spectral diagram showing spiked lines with time over
vapor detected. In the case of analyzing the fatty acids, the smaller they are the faster they
run through the column. This is also the case with the PHA monomers but based on the length
of the alkynes (O. David Sparkman, Zelda Penton, 2011).
In case of PHA monomers, they are analyzed by subjecting the dry cell samples to
methanolysis by reacting it with solvent methanol and chloroform in screw capped test tubes.
This is done to precipitate the PHA materials. After cooling, water is added to separate the
organic and aqueous phases. The organic phase containing methyl ester derivatives is then
analyzed by using GC system in the total-ion scan mode and the spectrum is measured as
described above (Huang, Okoshi, Mizuno, Hiroe, & Tsuge, 2018). Therefore, the fatty acids
and PHA monomers will be identified from the running time through the GC.
MS - Mass spectroscopy
When the vapors from the GC exits the column it enters the MS, where it is ionized. By doing
this they are separated into subunits. Breaking down a molecule like this will always provide
the same subunits. (O. David Sparkman, Zelda Penton, 2011).They are separated within the
mass-to-charge spectrum by acceleration and either electric or magnetic field. In this process
the same ions will have the same deflection. A detector then reveals the mass from the charge
rating of each group of ions. The results of this analysis are a diagram over the mass of
different charges from groups of ions (O. David Sparkman, Zelda Penton, 2011).
Side 16 of 69
Aalborg University
Collective results for GC and MS
The chromatogram provided by the GC shows several peaks with varying intensity,
corresponding with the different molecules exiting the GC. The integrals are calculated, and
the spectrums are extracted from the peaks. These provide insight into the mass distribution
of the fatty acids and alkyne monomer present in the samples (O. David Sparkman, Zelda
Penton, 2011).
Side 17 of 69
Aalborg University
Applied Methods
A flowchart (figure 8) was made to show all the protocols used during the experiments. Data
on all materials, machines and chemicals used is listed in appendix B.
Figure 8: Flowchart of applied methods in the sludge experiment.
Side 18 of 69
Aalborg University
Sludge incubation and sample collection

The sludge was aerated for one hour on arrival with an aquarium stone. After an hour the
aquarium stone was removed, and the sludge was moved under the fume hood. All bottles
had a magnet to enable the use of a magnetic stirrer. 200 mL sample were transferred to
three different bottles and they were sealed to create an enclosed environment. A long
hypodermic needle was inserted through the bottle cap of each bottle. This needle was used
to collect all samples from the reactor during the experiment. Two small needles were
inserted as an inlet and an outlet to enable a constant flow of N to create an anaerobic
environment. During the four hours incubation, the reactors were placed on a magnetic
stirrer to ensure homogenization. Before the experiment started, the reactors were circulated
with nitrogen for 30 minutes to ensure no oxygen was left in the reactor.
10 mL of acetate solution was added to reactor A and B. 10 mL water was added to control
reactor, to gain the same volume in all reactors. At the time 0, (t=0), 6 mL sample was
collected from each reactor, divided into three Eppendorf tube with 2 mL sample for each
purpose and frozen to stop any reaction. The 2mL for the colorimetry were filtered
immediately before being frozen. This was repeated every 30 minutes for four hours. The
remaining sludge in the reactors was transferred to two 50 mL tubes and frozen.
Determination of concentration of P by Colorimeter

It was estimated that concentration of P in the experiment would be between 20-60 mg /L
(Danish Standards Association” 2004). The sample was diluted by a factor of 1:100 to avoid
the shadow effect and increase accuracy. The standard was diluted by a factor of 1:100 to be
comparable. First, a P stock solution of 50 mg P/L (1.6 mM) was made by dissolving 0.022 g of
KH2PO4 in ELGA-water, 1 mL 4.5 M Sulphuric acid was added and the stock diluted to a final
volume of 100 mL. The 2 mg P/L Phosphorus stock solution (0.064 mM) was made by diluting
the 50 mg/mL stock 1:25 (Appendix C).
To the working standard and samples two reagents were made. An acid molybdate solution
consisting of 60 mL 4.5 M sulfuric acid, 20 mL 0.11 M molybdate and 20 mL 7.44 mM antimony
potassium tartrate hemihydrate. The second reagent was 100 g/L (0.57M) ascorbic acid and
it was made by dissolving 5 g ascorbic powder in ELGA-water to get 50 mL. Each working
standard consisted of 100 µL of ascorbic acid solution and 200 µL of acid molybdate solution
and a fixed ratio between the 2 mg P/L Phosphorus stock solution and ELGA-water for a final
volume of 5 mL to acquire the specific P concentrations for the standard curve, 0.04 mg P/L
(0.0013 mM), 0.08 mg P/L (0.0026 mM), 0.16 mg P/L (0.0052 mM), 0.24 mg P/L (0.0077 mM),
0.32 mg P/L (0.0103 mM), 0.4 mg P/L (0.0129 mM), and 0.6 mg P/L (0.0194 mM).
For each sample tested, 100 µL of ascorbic acid solution and 200 µL of acid molybdate solution
was mixed with the sample for a final volume of 5 mL. The standard curve was made from the
working standards and absorbance was measured at 880 nm wavelength. Absorbance for all
Side 19 of 69
Aalborg University
samples were tested and compared with the standard curve to predict the concentration of
P.
Identification of concentration of acetate by HPIC

The samples where centrifuged to separate the pellet from the supernatant. The supernatant
was moved to a new Eppendorf tubes, centrifuged again and the pellets were discarded. Then
the samples were diluted to 1:20. 2 mL of each sample was pushed through a syringe with a
0.2 µm filter into HPIC bottles. The samples were frozen until needed. All the samples were
vortexed after they were thawed and taken down to the HPIC machine.
Substrate degradation – PHA

The 50 mL tubes from reactor A was taken out of the freezer and submerged into a water
bath to accelerate the melting. Then all the tubes were put in the centrifuge for 15 minutes,
at 20 °C and 10,000 rpm. The supernatant was removed and discarded. The pellet was
lyophilized.
The lyophilized pellets were resuspended in 1 mL acidic methanol and 1 mL chloroform and
kept at a 100 °C overnight. After the heating to 100 °C the samples were put on ice for 30 min.
500 µL water was added to all the samples and the samples was vortexed. Then 500 µL more
water was added. The tubes were left to separate into two phases. Since the results was not
good, a saturated NaCl solution was made. 2 mL sodium chloride solution and 2 mL
chloroform were added to all samples. The samples were vortexed and after a while the
samples were clearly separated into two phases. The bottom layer containing the PHA was
moved to small glass tubes, sealed and frozen. The samples were later transferred to a GC vial
and analyzed by GC-MS.
Lipid extraction
The tubes for reactor B was taken out the day before and put in the fridge to slowly defrost
to the next day. Once the sludge was completely liquid, it was centrifuged for 15 min at 6000
g and 4 °C. The supernatant was then removed, and the remaining pellet was weighed and
recorded. Because the sludge was divided into 3 tubes previously, they were collected in one
tube and 10 mL chloroform and 5 mL methanol was added. To homogenize the sample, it was
vortexed for 4 minutes. The mixture was then added to a separatory funnel and mixed with 5
mL chloroform and 5 mL saline solution. The separatory funnel was then shaken vigorously,
with occasional gas release. It was then allowed to settle but did not separate into two phases
as it should. Additional 5 mL of saline solution and chloroform respectively was added, and
the shaking was repeated. It was left to settle but did not separate. The samples were
centrifuged for 15 minutes at 8000 g and 4 °C. After this, there were 2 phases, separated by
a layer of biomass. The bottom layer, which contained the chloroform and lipids, was
removed with a glass pipette into a round flask. This flask was transferred to a rotary
evaporator where the chloroform was removed. This left the dry isolated lipids in the round
flask.
Side 20 of 69
Aalborg University
NMR-Lipids and acetate

The NMR used were a BRUKER AVIII-600 MHz NMR spectrometer equipped with a CPP-TCI
probe. All spectra were recorded at 298.1 K. All the NMR samples, from t = 0 to t = 240, were
centrifuged at 7800 rpm for 5 minutes to sediment the particles in the samples. Acetate
samples were prepared by adding 5 μL of D20 with TSP-d4 (56.7 mM) to 0.5 mL supernatant
of each sample, followed by 20 μL D2O. The mixture was homogenized by vortexing. All the
NMR tubes were analyzed by NMR except t = 210, since it had too little liquid.
The dry isolated lipids were collected from the rotary evaporator and resuspended in 600 μL
Deuterated Chloroform and 600 μL Methanol. This solution was analyzed by NMR.
TLC
The eluent was prepared by mixing diethyl ether, methanol and acetic acid with volumes of
45 mL, 0.5 mL and 0.5 mL respectively. A beaker was then covered on the inside with a filter
paper both on the bottom and on the sides. A small gap was left uncovered on the sides for
following the process. Half of the eluent was then added to the beaker and immediately it
was covered with an aluminum foil to create a gas chamber and prevent the evaporates from
escaping the beaker. TLC plates were then marked with four points each with a height of 2
cm from the bottom and 1 cm apart. About 2 μL of the sample in the NMR solution and each
of the three types of lipid standard were transferred to the marked points with a thin glass
capillary. The lipids standards were Triglyceride (Rapeseed oil) (TG), Dimyristoyl
phosphatidylcholine (DMPC) and Dimyristoyl phosphatidylglycerol (DMPG). The plate was left
to rest for few minutes until the solvent was evaporated, then placed inside the beaker
carefully so that the samples did not get into contact with the eluent. The plate stayed inside
the beaker until the eluent reached about 1 cm from the top and then the plate was removed
and marked where the eluent reached. After the solvent was dried from the plate, it was
ready to use in the iodine chamber for lipid observation.
The iodine chamber was prepared in the hood by adding a spatula full of iodine crystal to a
beaker, which was then covered with an aluminum foil to create iodine gas chamber. The
beaker was placed on a heater at a maximum of 50-70 °C until a deep violet vapors was visible
in the chamber. The plate was then transferred to the chamber using a pair of forceps and
left inside until all or most spots were visible. After this it was removed from the chamber and
all the spots were marked and the plate photographed.
GC-MS
The GC were equipped with an automatic liquid autosampler and used an Agilent column 122-
5532UI-INT: US18460252. The temperature of the GC inlet was 250 °C to prevent the sample
from condensing in the inlet. GC had a constant flow of helium on 1.2 mL. The column’s
temperature was 50 °C to make sure the sample condensate. After 2 minutes, the
temperature was raised to 200 °C with 10 °C /min. After 5 minutes at 200 °C, the temperature
was further raised to 250 °C with 5 °C/min. After 5 minutes at 250 °C, the temperature was
Side 21 of 69
Aalborg University
raised to 300 °C to clean the column of the remaining sample. The MS began the
measurement after 3.5 minutes after the first substance entered the MS. The MS was
programed to measure the substance mass between MS1 start mass at 50 and MS1 end mass
at 400. The scan time was 100 ms. The electron volt (eV) used by MS was 70 eV.
Transesterification of lipids
For the GC-MS to be able to measure the fatty acids of the lipid sample they had to be trans-
esterified. An NMR was run before putting it into the GC-MS to check if it had worked. This
was done by comparing the spectrum of FAMEs to the original lipid spectrum.
0.5 M sodium methoxide solution was prepared by adding 0.14 g sodium hydroxide to 5 mL
anhydrous methanol and mixing until all the crystals were dissolved. Glass pipette was used
to transfer the remaining NMR lipid solution after the TLC to 50 mL tube. 2 mL of 0.5 M sodium
methoxide was added, and the mixture was heated at 50 °C for 30 minutes. 4 mL was then
removed and left to cool in an ice bath. 10 mL of hexane was then added, and the mixture
was left for few minutes to create two layers. The top layer, hexane, was transferred to a new
tube and a small scoop of anhydrous magnesium sulphate crystals were added to dry the
hexane. The mixture was then hand centrifuged to sediment the particles and supernatant
transferred to a new clean tube and dried under nitrogen gas for one hour. 600 μL deuterated
chloroform were added and the sample was analyzed by NMR
Side 22 of 69
Aalborg University
Results
NMR – acetate
To obtain usable results, the TPS-d4 signal was calibrated to 0 ppm on the shift-axis. The
baseline was corrected. The phases of all the peaks except the water phase was adjusted
and made symmetrical. Following these steps, the integral tool was used to find the area
under each peak. Using the formula below, the unknown concentration of acetate could be
calculated by using the integral of the acetate and TSP-d4 peaks, and TSP-d4 concentration.
𝐿𝑥 𝐶𝑥 ∗ 𝑁𝑦
=
𝐿𝑦 𝐶𝑦 ∗ 𝑁𝑥
L is the intensity, the relation measured with the integrals. C is the concentration of the
respective compounds and N is the number of nuclei per molecule that contributes to the
integrated peak. H atoms are significant factor in this calculation, TSP-d4 contained 9 and 3
acetate. The concentration of TSP-d4 was known to be 56.7 mM in the original solution and
the final concentration can be calculated using the formula below:
C1 * V1 = C2 * V2
The TSP-d4 final concentration was calculated to be 0.54 mM.
After adjustments and calculation of the integrals, the supernatant NMR spectra looks as
follows (figure 9). The other spectra are provided in (appendix D).
Figure 9: NMR spectrum for acetate sample t=0. The red numbers represent the integral of the TSP-d4 peak and the acetate
peak.
Side 23 of 69
Aalborg University
This spectrum shows three distinct peaks. The TSP-d4 peak at zero, an acetate peak around 2
ppm and a water peak just below 5 ppm represent the water in the supernatant and solvents.
The spectra also provide a ratio between the two peaks of interest, TSP-d4 and acetate.
When all the concentrations have been calculated (table 1 in Appendix E), these values were
plotted to show the concentration of acetate over the course of the experiment (Figure 10).
Figure 10: The graph shows the decline of acetate in the sample after specific amount of time .
The graph shows a slight decline of acetate concentration over the course of the experiment
(figure 10). It appears to be relatively stable around 6-7 mM. This is slightly lower than
expected of our starting concentration. The results would suggest, that there is little to no
acetate uptake over the course of the experiment. However, the results are not consistent at
any point, which means the results are not reliable.
Side 24 of 69
Aalborg University
NMR – Lipid
After the NMR spectrum from the measurements of the lipid samples have been calibrated,
it is compared to 4 lipid standards, Triglyceride (TG), Dimyristoyl Phosphatidylcholine (DMPC),
Dimyristoyl Phosphatidylglycerol (DMPG) and Phosphatidyl ethanolamine (PE) to determine
the composition of the lipid sample.
Lipid:
Figure 11: NMR spectrum for lipid sample on the range from 0 to 3.5 ppm.
Side 25 of 69
Aalborg University
Figure 12: NMR spectrum for lipid sample on the range from 3 to 7.5 ppm.
The NMR spectrum of the lipid sample shows defined peaks throughout the spectrum. The
first spectrum shows a peak at 0.8 ppm, which indicates presence of a CH 3 group present in
the terminal end of the fatty acids. The second peak at 1.2 ppm represent presence of CH 2 in
the sample. In lipids, CH2 is present as the main component in unsaturated fatty acids and
especially in the saturated fatty acids. The peak at 1.6 ppm shows a measure of the hydrogen
bonded to the C-2. The peak at around 2.3 ppm represents the C-1. The signal at 2 ppm
indicates double bonds and stems from the hydrogen of a carbon bonded to one other carbon
which is involved in a double bond. A peak is also seen at 2.8 ppm which indicates the
presence of conjugated double bonds (figure 11-12).
The large peak at 4.7 ppm represent the water that remained in the sample or in the solvents.
The peak at 5.2 ppm indicates the hydrogen bound to the central carbon in a glycerol
backbone while the peak at 5.3 ppm indicates the double bonds in the fatty acids. The
remaining glycerol peaks should be between 3-4.5 ppm which shows only moderate peaks
(figure 12).
Side 26 of 69
Aalborg University
Lipid - Triglyceride:
Figure 13: NMR Spectrum for Lipid sample (Red graph) compared with triglyceride standard (Blue graph) in the range from 0
to 3 ppm.
Figure 14: NMR Spectrum for Lipid sample (Red graph) compared with triglyceride standard (Blue graph) in the range from 3
to 5.5 ppm.
Side 27 of 69
Aalborg University
Comparing the triglyceride standard lipid with the lipid sample, there is a significant
overlapping of peaks. There is a significant overlap at 5.3 ppm which indicates the double
bonds. Most importantly there is a significant overlap of the peaks that describe the glycerol
backbone, since that’s the defining feature in glycerides. This is the peak at 5.2 ppm which
indicates the central carbon atom on the glycerol. The remaining peaks between 3 and 4.5
ppm indicates the other carbons in glycerol, is however not as defined as expected for
triglycerides. The good peak-overlapping between the lipid sample and this lipid standard
suggest that the lipid sample contains triglycerides.
Lipid - Dimyristoyl Phosphatidylcholine (DMPC):

The peak at 0.9 ppm indicates the presence of CH 3 groups, the peak at 1.3 ppm indicates the
amount of CH2, the peak at 1.6 ppm indicates -CH2-C-CO2 and the peak at 2.3 ppm indicates
CCH2CC. These peaks all indicate fatty acids in the sample. The peak found in the sample but
not in the DPMC standard is the one at 2 ppm, which indicates CH 2CO2.
Figure 15: NMR Spectrum for Lipid sample (Blue graph) compared with Dimyristoyl Phosphatidylcholine standard (Red
graph) in the range from 0 to 3 ppm.
Side 28 of 69
Aalborg University
Figure 16: NMR Spectrum for Lipid sample (Blue graph) compared with Dimyristol Phosphatidylcholine standard (Red
graph) in the range from 3 to 5.5 ppm.
On the chemical shift between 3 ppm and 7 ppm, there is no overlapping by most of the peaks
(figure 16). These peaks from the DMPC lipid standard are the main parts of the backbone
normally holding the fatty acids in structure. Since these peaks are missing from the samples,
none of these components should be considered present in the lipid samples spectrum.
From DPMC comparison results, the DPMC is not confirmed to be present in the lipid sample.
This is because the peaks identifying the presence of the backbone of the lipid DMPC are not
overlapping with the lipid sample spectrum. This could indicate that the structure of DMPC is
not present in the sample, because only the fatty acid part of the spectrum overlaps, and
these could be from any other type of lipid.
Side 29 of 69
Aalborg University
Lipid - Dimyristol Phosphatidylglycerol (DMPG):
Figure 17: NMR Spectrum for Lipid sample (Blue graph) compared with Dimyristol Phosphatidylglycerol standard (Red
graph) in the range from 0 to 3 ppm.
Figure 18: NMR Spectrum for Lipid sample (Blue graph) compared with Dimyristoyl Phosphatidylcerol standard (Red graph)
in the range from 3 to 5.5 ppm.
Side 30 of 69
Aalborg University
The spectrum from the sample (blue) have clear overlaps with significant peaks for fatty acid
at 0.8, 1.2, 1.6, 2 and 2.3 ppm in the Dimyristol Phosphatidylglycerol NMR spectrum(red)
(figure 17). The samples significant peaks in the spectrum 3.5-5 ppm do have 1-2
overlapping peaks with the DMPG template however, this is insufficient to the overall
DMPG spectra profile to determine the presents of DMPG in the sample (figure 18).
Lipid - phosphatidyl-ethanolamine (PE)
Figure 19: NMR Spectrum for Lipid sample (Blue graph) compared with phosphatidyl-ethanolamine standard (Red graph) in
the range from 0 to 5.5 ppm.
The spectrum from the sample (blue) have clear overlaps with significant peaks for fatty acid
at 0.8, 1.2 and 1.6 ppm in the PE NMR spectrum(red) (figure 19). The samples significant
peaks in the spectrum 3.5-5 ppm do have 1 overlapping peak with the template. This is at
4.6 and it is insufficient to the overall PE spectra profile to determine the presents of PE in
the sample. The other significant peak at 3.3 ppm is not overlapping with the sample.
Side 31 of 69
Aalborg University
FAME.
Figure 20: NMR spectrum for FAME sample on the range from 0 to 3 ppm.
Figure 21: NMR spectrum for FAME sample on the range from 3 to 7.5 ppm.
Side 32 of 69
Aalborg University
FAME - Lipid
Figure 22: NMR Spectrum for Lipid sample (Blue graph) compared with FAME (Red graph) in the range from 0 to 3 ppm.
Figure 23: NMR Spectrum for Lipid sample (Blue graph) compared with FAME (Red graph) in the range from 3 to 5 ppm.
Side 33 of 69
Aalborg University
The results of the comparison between the lipid sample and the FAME sample is shown in the
spectrums above. The NMR spectrums show a solid overlap in the range from 0 to 3 ppm
which is the area describing most of the fatty acid structure which was expected to be intact.
However, the peaks describing the glycerol backbone, specifically at 5.2 and 4.2 is gone,
replaced by a peak at 3.8 which fits to be the newly formed methyl esters groups on the fatty
acids.
Side 34 of 69
Aalborg University
Thin Layer Chromatography analysis (TLC)

Two TLC plates were used for this analysis. The first plate (figure 24, left) showed defined
spots for the sample and the triglyceride standard only. The procedure of labelling the plate
and immersing it in iodine chambers was repeated on a new plate and the amount was
changed according to former results (figure 24, right).
Both TLC plates shows a clear spot on both the TG standard and the sample. These spots have
travelled the same distance up the plate which could suggest that they contained the same
lipids. Both the DMPC and DMPG, however, did not show any indication of moving up the
column once they were colored in the iodine chamber. This could have been because of the
eluent that was used. It is possible that it could not dissolve DMPC and for this reason did not
carry it up the column. The DMPG did not appear on the plates after the staining, even with
the increased volume, which could indicate that it needs an even larger volume to show on
the plate. For these reasons it is not possible to determine whether the sample contains
DMPC and DMPG but the fact that the spot for the sample and the spot for TG line up, is a
good indicator that the sample contains triglycerides. However, it is also possible that the
solvent was not able to dissolve the DMPC and DMPG. Resulting in no DMPC or DMPG in the
TLC.
Table 1: Labels and amount used in the two TLC trial
Label Samples 1st 2nd amount

amount
1 Lipid sample 2 ul 0.5 ul
2 DMPC 2 ul 10 ul
3 TG 2 ul 3 ul
4 DMPG 2 ul 10 ul
Side 35 of 69
Aalborg University
Figure 24: The two TLC trials, with first plate on the left and the second on the right.
Colorimetry
The absorbance measured for the different P stock concentrations are plotted as a graph to
produce the standard curve. The standard curve is linear as expected (figure 25). This
standard curve can be used to calculate the concentration of P in sample A, B and N with the
measured absorbance (figure 26).
Figure 25: A graph of absorbance over concentration of the acetate stock solution. The y-axis is measured absorbance and
the x axis is concentration in the unit mM. The formula above the graph is of the linear tendency linear regression.
Side 36 of 69
Aalborg University
Figure 26: A graph of concentration over time for all samples. On the y-axis is the time. On the x-axis is the concentration. The
two formula corresponds with the tendency line of the same color.
From the calculated concentrations, a graph showing concentration over time is made. The
tendency lines can be used to estimate the progress in the P-concentration. However, a few
outlying values is seen in the graph. These numbers are removed to get a more precise result
(figure 26). Graphs with these outliers removed is made (figure 27-29).
Figure 27: Graph of concentration over time of the sample A. The formula above the graph is of
the linear tendency line. 240 has been removed.
Side 37 of 69
Aalborg University
Figure 28: Graph of concentration over time of the sample B. The formula above the graph is of the
linear tendency line. 240 has been removed.
Figure 29: A graph of concentration over time of the sample N. The formula above the graph is of
the linear tendency line. 60 and 120 have been removed.
Side 38 of 69
Aalborg University
HPIC
HPIC is used to estimate the decrease of acetate concentration during the experiment. By
measuring the concentration every 30 min to make a tendency line (figure 30 is one of the
results). The rest of the measurements are in appendix F.
Figure 30: Measurement for reactor B at time 30. Notice that the amount is noted as M. This is a mistake and it should be
mM.
The amount of acetic A for each reactor was written down corresponding with the time it
was measured. The amount tells how much of the substance is in the sample and the
specific time.
Side 39 of 69
Aalborg University
Table 2: Concentration over time for each reactor
Reactor B Reactor A Reactor N

Time Concentr Time Concentr Time Concentr
ation ation ation
(mM) (mM) (mM)
t:0 0.0106 t:0 0.1296 t:0 0.4105
t:30 0.0121 t:30 7.7198 t:30 0.2652
t:60 0.0149 t:60 7.5967 t:60 0.2626
t:90 7.5329 t:90 7.1567 t:90 0.3129
t:120 3.858 t:120 7.8745 t:120 0.2803
t:150 7.5586 t:150 7.1602 t:150 0.2192
t:180 7.3519 t:180 7.3585 t:180 0.2907
t:210 5.275 t:210 8.0675 t:210 0.2796
t:240 7.408 t:240 7.2676 t:240 0.2654
Figure 31: A graph of concentration for acetate over time. Time is shown in minutes.
Side 40 of 69
Aalborg University
The concentration seems to be relatively stable except for the initial measurement which is
extremely low in comparison. The remaining points shows no indication of either increasing
or decreasing, even though a steady decrease would be expected (figure 31).
Figure 27: A graph of concentration for acetate over time. Time is showed in minutes
It is impossible to tell which measurements could be accurate because reactor B have no

consistent measurements except for the three initial which is zero (figure 32).
Figure 28: A graph of concentration for acetate over time in reactor N. Time is shown in minutes.
Side 41 of 69
Aalborg University
The results for acetate concentration in the negative sample reactor shows a similar
inconsistent graph over the duration of the experiment. It can be seen on the y-axis that the
concentration is significantly lower than that in the other results, which is to be expected
since this reactor did not contain added acetate. On this graph however, it could be argued
that there is a slight decrease in concentration over the course of the experiment (figure 33).
GCMS - Lipid
The FAME - lipid sample was analyzed with the gas chromatograph Agilent Intuvo with an
automatic liquid autosampler and the Mass Spectrometer Agilent 7010B triple qudrupole.
The resulting chromatogram was then integrated and the average MS spectrums from each
peak was extracted. The standard chromatogram was compared with the chromatogram of
the sample to find any potential overlaps. The standard contained 10 different FAMEs (table
3)
Table 3: All the FAMEs in the standard provided, along with their structure and respective concentrations.
Myristic acid, methyl ester (C14:0) 4%

Palmitic acid, methyl ester (C16:0) 10%
Stearic acid, methyl ester (C18:0) 6%
Oleic acid, methyl ester (C18:1, cis-9) 25%
Elaidic acid, methyl ester (C18:1, trans-9) 10%
Linoleic acid, methyl ester (C18:2, cis-9,12), 34%
Linolelaidic acid, methyl ester (C18:2, trans-9,12) 2%
Linolenic acid, methyl ester (C18:3, cis-9,12,15) 5%
Arachidic acid, methyl ester (C20:0) 2%
Behenic acid, methyl ester (C22:0) 2%
In order to understand the results, the monoisomeric masses were found for some of the
most common fatty acids in addition to those found in the standard. These were then
compared to the masses found in the mass spectrum corresponding to each peak in retention
time (Rt) in the standard. The results are recorded (table 4). In the standard, the two peaks at
Rt 23,7-23,8 and 23,9-24 minutes were found to have the same mass. This mass was a fit for
both oleic acid and elaidic acid because they are isomers. However, the integral for the peak
at 23,9-24 was smaller. With the knowledge that oleic acid was present in the standard in a
higher concentration (table 3), it had to correspond to the peak with the highest integral.
The FAME sample was compared to the same values along with the Rt of the peaks, of the
standard in order to determine the structure of the FAMEs. If the mass and Rt was a fit, it was
determined to be the same fatty acid. The FAMEs that had the same Rt as the standard but
different maximum masses, were checked if they had a significant peak in the expected
location in the mass spectrum. The fatty acids that were found to be present in both standard
and the sample are palmitic acid, stearic acid, myristic acid, oleic acid and linoleic acid. Two
Side 42 of 69
Aalborg University
of the fatty acids found in the standard was not found in the sample. These were elaidic acid
and behenic acid. In addition to the identified peaks, 9 other peaks in the sample were not
identified, and 4 of these had the same mass at 206,8 and a similar Rt between 32,2 and 33
minutes (table 5).
Both palmitic acid and stearic acid were found to have the same Rt and mass as in the
standard and were therefore determined to be present in the sample. Myristic acid had two
peaks that had the same Rt. It was identified by investigating if the mass spectrum of the peak
in the sample matched the peak at the mass expected for myristic acid. This was also the case
for oleic acid and linoleic acid except that their mass spectra did not contain the expected
peak. Despite this, it was suspected to be the same substance based on Rt. A peak was
identified at a slightly lower Rt than palmitic acid with a mass that was 2 units lower than that
of palmitic acid. This corresponded with a similar structure only containing a double bond and
was determined to be palmitoleic acid.
Table 4: Results for FAME-standards presenting Rt and substance in the standard
Retention time (Rt) Name of the ubstance

17,3 Myristic acid, methyl ester C14:0
20 Palmitic acid, metyl ester C16:0
23,6-23,7 Linoleic acid, methyl ester C18:2 (9Z,12Z)
23,7-23,8 Oleic acid, methyl ester C18:1 (9Z)
23,9-24 Elaidic acid, methyl ester C18:1 (9E)
24,3-24,4 Stearic acid, methyl ester C18:0
32,460 Behenic acid, methyl ester C22:0
Table 5: Results for GC-MS for FAME sample presenting Rt and the expected substance in the sample
Retention time (Rt) Name of the substance

14,8-14,9 N/A
17,3 Myristic acid, methyl ester C14:0
17,3-17,4 N/A
17,7 N/A
19,6-19,7 Palmitoleic acid, methyl ester C16:1 (9Z)
20 Palmitic acid, methyl ester C16:0
23,5-23,7 Linoleic acid, methyl ester C18:2 (9Z,12Z)
23,7-23,8 Oleic acid, methyl ester C18:1 (9Z)
24,3-24,4 Stearic acid, methyl ester C18:0
28,5-28,6 N/A
30,4-30,5 N/A
32,2-3,23 N/A
32,5-32,6 N/A
32,6-32,7 N/A
32,9-33 N/A
Side 43 of 69
Aalborg University
GCMS - PHA
The GC-MS analysis of the PHA sample results were treated similarly to the results of the
lipids. The Rt and the mass from the MS for the substances in the lipid sample were recorded
(Appendix G). The HB and HV were the expected monomer in the sample. The monoisotopic
masses of these monomer were calculated, recorded (table 6) and compared with spectra
and mass from the MS. In order to make a comparison, their masses were used to create an
extracted ion chromatogram (EIC), which provided a spectrum that showed the intensity of
the chosen mass at every Rt. This made it possible to find an area that contains a high
concentration of mass spectra with measurements at that mass. This gave a possible Rt for
the substance with that mass thereby enabling the investigation of both HV and HB most
significant peaks. For HV, the EIC provided one defined peak. However, once the average mass
spectrum of the specific peak was investigated, it became clear that a higher mass was more
prevalent, which suggests that the peak did not represent HV. In the case of HB, the EIC did
not provide a clear peak anywhere on the spectrum. The most significant peaks' mass
spectrum was investigated for the expected mass, but none of them showed any sign of this.
Because of this the probability of HV and HB being present in the sample were low.
Table 6: The expected monomer and their masses
Name of the substance Mass

3- hydroxybutyrate methyl ester. 118,1
3-/4- hydroxy valerate methyl ester 132,1
Side 44 of 69
Aalborg University
Discussion
Sludge incubation
When the sludge was transferred to an incubation tanks, the sludge sedimented leaving a
solid sludge in the bottom of the liquid water. After vortexing, the sludge did not stay
homogenized long enough to transfer a sample to each incubation tank before
sedimentation. Due to this, it is not certain that the amount of sludge and water in the sample
transferred was equal for all tanks. To ensure that an equal amount of the sample is taken, it
could be helpful to use a method where a sample is taken while the sludge is being
homogenized under a magnetic stirrer. It could also be helpful to be fast in taking out a sample
before sedimentation begins.
Another error was that the first sample, t=0, was frozen after 22 minutes such the reaction
continued for longer than it was expected. This could influence the results for the rest of the
procedures done, especially because the 22 minutes of continued incubation would be
aerobic. This would explain the results of the HPIC showing low concentrations of acetate at
time 0. However, this conclusion would be contrary to the results provided by both the NMR
and colorimetry.
Colorimetry
The standard stock solution prepared did not show signs of any reactions. Therefore, no
absorbance could be measured. The reaction of the standard and reagents used was
supposed to give a blue color with an increasing intensity correlating to an increase in P
concentration. To measure the absorbance, reagents from another group were borrowed and
used together with the standard stock solution. Lack of the blue color could have been caused
by low concentration of the reagent´s reactive components. This would verify why the t=240
sample had a reaction and turned slightly blue. This loss of components could be explained
by exposure to light or other errors.
The standard curve made from the acetate stock solution was expected to have a linear
formula with a positive slope. In the results from tank A and B the measurements from t=240
were both outliers resulting in a higher slope. Presence of small particles in the sample or the
sample being unhomogenized could have been a reason for these outliers. Apart from the
outliers, reactor A and B gave resulted as expected, indicating that there was an increase in
concentration of P.
Since the samples from reactor A and B came from the same treatment plant, both reactors
were expected to show similar results, which they did with a difference in the slope of 0.0002.
The slope of A was 0.0074 and the slope of b was 0.0072. The results from reactor N were
expected, as they did not give an increasing curve. The expected results were a tiny P release
due to a small amount of P in the cells from the sludge and a small amount of nutrients in the
tank. In conclusion, the results suggest a slight release of P.
Side 45 of 69
Aalborg University
NMR - acetate
The results used in NMR-acetate analysis were provided by the supervisor, since there was
inconsistency in the results obtained by the group. All the spectra graphs from t=0 to t=240
shows spectrum between 0 ppm and 3 ppm. In all graphs, acetate spectrum is appearing
around 2 ppm. This shows there is a good relation between the acetate peak and TSP-d4. The
third spectrum peak represent the water remaining in the acetate-sample and in the NMR
solvent used. By reading the integration of the acetate peaks, it was possible to calculate the
concentration of acetate during the four hours incubation. These concentrations were used
to plot a graph for visualizing the acetate consumption by the bacteria during incubation. The
plotted graphs were expected to start by a concentration of 8 mM but instead they start by
around 7 mM. This can be due to the acid and base solution used to create a neutral pH
environment. It could have led to a lower start concentration than the expected. The sample
at t=210 was not measured because there was less solution than the required in the NMR
tubes. The results provided by the acetate NMR graph are highly inconsistent to a point where
it becomes unreliable. However, it does suggest a relatively stable concentration with a minor
decline in concentration.
HPIC – Acetate
The graphs results provided by the HPIC could not be used for further determination of
acetate consumption by bacteria in the sludge, since the graphs do not show any consistent
results. The concentration of acetate during the incubation time was expected to decrease
gradually over the course of the incubation, because it was being used up by bacteria. The
results do, however vary immensely from reading to reading, which could indicate that
something went wrong either in the production of the samples or in the process of making
the measurements.
The assumption that the results were caused by mistakes during the experiment is further
supported by the fact, that the HPIC machine broke down before and during the experiment.
This could indicate that the machine was still not fully functional, while it was used doing the
experiment. Another explanation could be that the sample either completely or partially
evaporated before the sample was analyzed.
Thin Layer Chromatography - TLC

The results provided by the TLC suggested the presence of triglycerides in the samples, but
inconclusive results regarding DMPC and DMPG, since these didn’t move on the TLC plate.
This could be because the two lipid standards had no affinity for the eluent and for this reason
did not move with it up the TLC plate. Therefore, it cannot be entirely concluded that they
were absent. If the experiment were to be attempted again, the eluent would be tested for
each of the lipids.
NMR-lipids
The NMR spectrum of the lipid sample contains peaks corresponding with what would be
expected of a lipid sample. It contains the peaks that indicates all the major components of
Side 46 of 69
Aalborg University
fatty acids. In addition to these, there was other peaks identified which corresponds with the
peaks of a glycerol backbone. This is a suggestion that the sample contained glycerolipids.
This analysis was further supported by the comparison between the NMR spectrum of the
sample and that of a triglyceride standard.
All the fatty acid peaks were similar and so was the glycerol peaks even though some were
quite small. These results would indicate that the sample did contain triglycerides. This
conclusion is further supported by the conducted TLC analysis which also indicate the
presence of triglycerides in the sample. A similar NMR comparison was conducted on both
DMPC, DMPG and DMPE, however these did not provide the same definitive results. In the
comparison it was clear that the peaks provided by the fatty acids were similar, but the peaks
for backbones were not overlapping.
The signal in the Lipid NMR that indicated double bonds is likely caused by the unsaturated
fatty acid found in the sample. These were identified in the GC-MS as oleic acid, linoleic acid
and palmitoleic acid, which are all unsaturated fatty acids. The presence of linoleic acid is
likely responsible for the peak that indicates conjugated double bonds. Considering these
results, it can be concluded that the sample probably contained only triglycerides with regards
to lipids. The large, wide peak around 7 is not an indicator for any group present in lipids and
is therefore likely to be caused by contaminants in the sample.
GC-MS Lipid
The results from the GC-MS analysis of the FAME sample was analyzed and compared to a
FAME standard in order to determine the composition of the fatty acid methyl esters in the
FAME sample. A few of the most common fatty acids and those from the standard were
recorded and the monoisomeric mass of each fatty acid's methyl esters was calculated. These
masses along with the Rt were used to determine the presence of specific fatty acids methyl
ester in the sample.
The results from the standard indicated the presence of 7 FAMEs when compared to the
known masses of common FAMEs. These 7 are myristic acid methyl ester, palmitic acid methyl
ester, linoleic acid methyl ester, elaidic acid methyl ester, oleic acid methyl ester, stearic acid
methyl ester and behenic acid methyl acid. The FAME standard was expected to contain ten
different FAMEs. However, this is not the case because the results show only 7, but all the
FAMEs identified in the GC-MS was expected to be present in the standard. This difference
could be caused by a degeneration of the fatty acids in the standard. A more likely reason is
that those that were not found were simply lost in the disturbance of other signals because
of low concentrations. This seems to fit with the fact that those not found in the standard are
some of those with the lowest concentration.
The results from the FAME sample were first compared to the FAMEs in the standard. Then
the remaining peaks were compared to the most common fatty acids. The results analysis
from the FAME sample showed the presence of 6 different FAMEs namely, palmitic acid
methyl ester, palmitoleic acid methyl ester, linoleic acid methyl ester, stearic acid methyl
Side 47 of 69
Aalborg University
ester, myristic acid methyl ester and oleic acid methyl ester. In addition to the FAMEs in the
standard, an extra FAME was identified, namely palmitoleic acid. Palmitoleic acid has two
lower mass per charge and lower Rt than that of palmitic acid. This is caused by the fact that
palmitoleic acid contains a cis-double bond. All the FAMEs found in the standard were found
in the sample except for elaidic acid and behenic acid. This is expected since it is a trans fatty
acid that is rarely found in organic organisms. Trans fatty acids are usually a result of a reaction
of the cis-isomer of that fatty acid.
All these FAMEs follow the expected pattern in which longer fatty acids will have a higher Rt,
since they have a higher boiling point. In addition, it is seen that more cis double bonds in the
fatty acids leads to a lower Rt, which is caused by their three-dimensional structure that
lowers its boiling point. The bends of the fatty acid chains reduce the cohesion compared to
saturated fatty acids with the same amount of carbon. This is also apparent in the standard,
which shows that the trans fatty acid have a higher Rt than its cis-isomer. The cis-isomer
retains its straight structure allowing a higher cohesion between the fatty acid chains.
Among the peaks found in the sample, nine of them were left unidentified. These were most
likely contaminants since their masses does not fit the pattern of the FAME’s increasing Rt
along with its increasing mass. In addition to this, they do not fit any Rt of the FAMEs found
in the standard or any of the masses of the most common fatty acids. This conclusion is also
supported from the NMR results of the lipid sample, which also showed a significant amount
of contamination. This contamination is likely caused by the method of which the lipids were
isolated as it is not a particularly selective method. If this experiment was to be repeated this
would be a point of interest for improvements. In addition to this, the standard would have
increased concentration in order to make it easier to identify every FAME.
GC-MS - PHA
The GC-MS analysis PHA did not provide any usable results, since there was no indication of
the presence of HB or HV in the sample. The lack of these monomers could be due to the PAO
not utilizing the acetate added to the sludge, hence being unable to produce the PHAs. This
is supported by the NMR acetate results as it shows no or low consumption of acetate.
However, the colorimetry results show a steady increase of P in the reactors A and B indicating
some P were released during the incubation.
The contents of the sludge are unknown and therefore, it can only be assumed that either the
sludge had a low concentration of PAOs, or they were inactive compared to what was
expected which would lead to the low uptake of acetate and an insignificant production of
PHA. There was no standard used for the PHA analysis with GC-MS. If the experiment was to
be repeated it would be beneficial to add this to the analysis. This will be helpful in obtaining
the Rt of some PHA monomers that could be used in identifying the substances found in the
sample. Another method to improve the results, could be to increase the amount of activated
sludge, since there will be a larger number of bacteria and thereby increasing the production
of PHAs.
Side 48 of 69
Aalborg University
Conclusion
The NMR for lipids spectra provided insight of the different groups of lipids in the sample once
compared with lipid standards. It was shown that only triglyceride was present in the sample
of the four lipid standards. This conclusion was supported by the TLC results and it confirms
that lipids can be extracted using this method. The lipid GC-MS indicated that the sample did
contain lipids and that the transesterification was successful. With these results it was also
possible to determine the structure of the fatty acids in the sample of which there were six
different.
The colorimetry results showed that the concentration of P increases over time, which
confirms that P is released during the incubation. This does not correspond with the acetate
NMR spectrum which only shows a subtle decrease of acetate over time during the
incubation. This could indicate that the P is released by other means than the reaction which
was expected. The HPIC results of acetate were inconsistence to point where it was
unreliable.
Since the results of the GC-MS did not provide any indication of PHAs in the samples and the
acetate NMR only indicated a minor uptake of acetate, it cannot be confirmed that acetate is
an effective substrate for PAO in the anaerobic reaction examined in this report.
Bibliography
Antonio Mendez-Vilas. (2007). Current Research Topics in Applied Microbiology and
Microbial Biotechnology. Retrieved March 12, 2019, from
https://books.google.dk/books?id=wrBpDQAAQBAJ&pg=PA320&lpg=PA320&dq=Chlor
oflexi+pha&source=bl&ots=KnYcK2hIRq&sig=ACfU3U2OcoBXk-XdKXV-
MfLW9Q9DIrXnPA&hl=da&sa=X&ved=2ahUKEwiBu4K_wf3gAhUFMuwKHeXsBpcQ6AE
wAXoECAkQAQ#v=snippet&q= Chloroflexi pha&f=false
Side 49 of 69
Aalborg University
Barnard, J., Barnard, J. L., Dunlap, P., & Steichen, M. (2017). Rethinking the Mechanisms of
Biological Phosphorus Removal, (November).
https://doi.org/10.2175/106143017X15051465919010
Biology, B. (2018). Eutrophication: Causes, Effects and Controlling measures - Online Science
Notes. Retrieved May 22, 2019, from https://onlinesciencenotes.com/eutrophication-
causes-effects-and-controlling-measures/
Blanco, M. (2011). Supply of and access to key nutrients NPK for fertilizers for feeding the
world in 2050. UPM, Madrit.
Chemistry Libretexts. (2019). Thin Layer Chromatography - Chemistry LibreTexts. Retrieved
May 14, 2019, from
https://chem.libretexts.org/Ancillary_Materials/Demos%2C_Techniques%2C_and_Exp
eriments/General_Lab_Techniques/Thin_Layer_Chromatography
Chen, G. Q. (2010). Plastics from Bacteria. Microbiology monographs (Vol. 14).
Cordell, D., Drangert, J.-O., & White, S. (2009). The story of phosphorus: Global food security
and food for thought. Global Environmental Change, 19(2), 292–305.
https://doi.org/10.1016/j.gloenvcha.2008.10.009
Cordell, D., & Neset, T.-S. S. (2014). Phosphorus vulnerability: A qualitative framework for
assessing the vulnerability of national and regional food systems to the multi-
dimensional stressors of phosphorus scarcity. Global Environmental Change, 24, 108–
122. https://doi.org/10.1016/j.gloenvcha.2013.11.005
Craig Pittman. (2017). The Clock is Ticking on Florida’s Mountains of Hazardous Phosphate
Waste | Sarasota Magazine. Retrieved from
https://www.sarasotamagazine.com/articles/2017/4/26/florida-phosphate
Cyberlipid. (2019). TLC of acylglycerols | Cyberlipid. Retrieved May 14, 2019, from
http://cyberlipid.gerli.com/techniques-of-analysis/analysis-of-simple-
lipids/acylglycerols/tlc-of-acylglycerols/
Demirbas, A., Bamufleh, H. S., Edris, G., & Al-Sasi, B. O. (2017). Biodiesel production from
lipids of municipal sewage sludge by direct methanol transesterification. Energy
Sources, Part A: Recovery, Utilization and Environmental Effects, 39(8), 800–805.
https://doi.org/10.1080/15567036.2016.1263259
Endres, H.-J. (2017). Bioplastics (pp. 427–468). Springer, Cham.
https://doi.org/10.1007/10_2016_75
Gerardi, M. H. (1979). Wastewater Bacteria. Retrieved from
http://ssu.ac.ir/cms/fileadmin/user_upload/Daneshkadaha/dbehdasht/markaz_tahghi
ghat_olom_va_fanavarihaye_zist_mohiti/e_book/Wastewater_Bacteria.pdf
GEROTHANASSIS, I. P., TROGANIS, A., EXARCHOU, V., & BARBAROSSOU, K. (2002). Nuclear
Magnetic Resonance (Nmr) Spectroscopy: Basic Principles and Phenomena, and Their
Applications To Chemistry, Biology and Medicine. Chem. Educ. Res. Pract., 3(2), 229–
252. https://doi.org/10.1039/b2rp90018a
Gregory, M. R. (2009). Environmental implications of plastic debris in marine settings-
Side 50 of 69
Aalborg University
entanglement, ingestion, smothering, hangers-on, hitch-hiking and alien invasions.

Philosophical Transactions of the Royal Society B: Biological Sciences, 364(1526), 2013–
2025. https://doi.org/10.1098/rstb.2008.0265
Günther, H. (Harald). (2013). NMR spectroscopy : basic principles, concepts, and applications
in chemistry. Retrieved from
https://sfx.aub.aau.dk/sfxaub?ctx_enc=info%3Aofi%2Fenc%3AUTF-8&ctx_tim=2019-
05-22T12%3A24%3A41IST&ctx_ver=Z39.88-
2004&frbrversion=4&req.language=dan&rfr_id=info%3Asid%2Fprimo.exlibrisgroup.co
m%3Aprimo3-Article-maruzen&rft.artnum=&rft.atitle=&rft.au=Günthe
He, Z., & Honeycutt, C. W. (2005). A Modified Molybdenum Blue Method for
Orthophosphate Determination Suitable for Investigating Enzymatic Hydrolysis of
Organic Phosphates. https://doi.org/10.1081/CSS-200056954
Herzel, H., Krüger, O., Hermann, L., & Adam, C. (2016). Science of the Total Environment
Sewage sludge ash — A promising secondary phosphorus source for fertilizer
production, 542, 1136–1143. https://doi.org/10.1016/j.scitotenv.2015.08.059
Hignett, T. P. (1985). Role of Fertilizer in Agriculture. In Fertilizer Manual (pp. 18–31).
Dordrecht: Springer Netherlands. https://doi.org/10.1007/978-94-017-1538-6_3
Huang, P., Okoshi, T., Mizuno, S., Hiroe, A., & Tsuge, T. (2018). Gas chromatography-mass
spectrometry-based monomer composition analysis of medium-chain-length
polyhydroxyalkanoates biosynthesized by Pseudomonas spp. Bioscience, Biotechnology,
and Biochemistry, 82(9), 1615–1623. https://doi.org/10.1080/09168451.2018.1473027
John D. Roberts. (2006). Nuclear Magnetic Resonance, 133.
Kaseman, D., Davis, U. C., Srinivasan, R., & Iyer, G. (2003). Nuclear Magnetic Resonance : An
Introduction.
Kasper Reitzel1, Per Halkjær Nielsen, Morten Lykkegaard Christensen, Haiyan Qu, Reinhard
Wimmer, Marta Nierychlo, … Ulla Gro Nielsen. (2015). ReCoverP – genvinding af fosfor
fra spildevandsslam. Dansk Kemi, nr. 6/7, 96. Retrieved from www.retsch.dk
Keeler, J. (2002). Understanding NMR Spectroscopy.
Khan, M. (2014). Eutrophication: Challenges and Solutions, (March 2014).
https://doi.org/10.13140/2.1.3673.8884
Kristiansen, R., Nguyen, H. T. T., Saunders, A. M., Nielsen, J. L., Wimmer, R., Le, V. Q., …
Nielsen, P. H. (2013). A metabolic model for members of the genus Tetrasphaera
involved in enhanced biological phosphorus removal. The ISME Journal, 7(3), 543–54.
https://doi.org/10.1038/ismej.2012.136
Maekawa, S., & Elert, K. (2003). The Use of Oxygen-free Environments in the Control of
Museum Insect Pests - Shin Maekawa, Kerstin Elert - Google Bøger. Los Angeles,
California: Getty Publications. Retrieved from
https://books.google.dk/books?id=7rtvOQe3sMYC&pg=PA90&lpg=PA90&dq=creating+
anoxic+environment+with+nitrogen+flow&source=bl&ots=7Y1e6Hr-
FX&sig=ACfU3U17YP8ZEtRb-
qscpRngIoxiH7hlgw&hl=da&sa=X&ved=2ahUKEwjGrPOUw8fhAhWDLVAKHQxjCTMQ6A
Side 51 of 69
Aalborg University
EwA3oECAkQAQ#v=onepage&q=cre
Marang, L., van Loosdrecht, M. C. M., & Kleerebezem, R. (2015). Modeling the competition
between PHA-producing and non-PHA-producing bacteria in feast-famine SBR and
staged CSTR systems. Biotechnology and Bioengineering, 112(12), 2475–2484.
https://doi.org/10.1002/bit.25674
Moritz Haigermoser. (2017). Einsatzmöglichkeiten molekulargenetischer Verfahren zur
Optimierung der biologischen Phosphatentfernung und der Phosphatrückgewinnung in
Kläranlagen - PDF. Retrieved May 13, 2019, from https://docplayer.org/63490034-
Einsatzmoeglichkeiten-molekulargenetischer-verfahren-zur-optimierung-der-
biologischen-phosphatentfernung-und-der-phosphatrueckgewinnung-in-
klaeranlagen.html
Nielsen, H., Mcilroy, S. J., Albertsen, M., & Nierychlo, M. (2018). Re-evaluating the
microbiology of the enhanced biological phosphorus removal process 6 8 Per. Retrieved
from https://aaudk-
my.sharepoint.com/personal/pnmo17_student_aau_dk/Documents/P4-
bioplastic/Projekt/Kilder/Nielsen COBIOT- EBPR final.pdf
O. David Sparkman, Zelda Penton, and F. G. K. (2011). Gas Chromatography and Mass
Spectrometry : A Practical Guide (2. edition). Elsevier Science & Technology. Retrieved
from https://ebookcentral.proquest.com/lib/aalborguniv-
ebooks/reader.action?docID=670206
Reta, G., Dong, X., Li, Z., Su, B., Hu, X., Bo, H., … Xu, S. (2018). Environmental impact of
phosphate mining and beneficiation: review. International Journal of Hydrology, 2(4),
424–431. https://doi.org/10.15406/ijh.2018.02.00106
Ritchie, H., & Roser, M. (2018). Plastic Pollution. Retrieved from
https://dx.plos.org/10.1371/journal.pone.0111913
Schachtman, D. P., Reid, R. J., Ayling, S. M., S, D. B. D. P., & A, S. S. S. M. (1998). Update on
Phosphorus Uptake Phosphorus Uptake by Plants: From Soil to Cell. Plant Physiology,
116, 447–453. https://doi.org/10.1104/pp.116.2.447
Seviour, R. J., Mino, T., & Onuki, M. (2003). The microbiology of biological phosphorus
removal in activated sludge systems. FEMS Microbiology Reviews, 27(1), 99–127.
https://doi.org/10.1016/S0168-6445(03)00021-4
Stewart, W. M., & Roberts, T. L. (2012). Food Security and the Role of Fertilizer in Supporting
it. Procedia Engineering, 46, 76–82. https://doi.org/10.1016/J.PROENG.2012.09.448
ThermoFisher. (2019). DionexTM IonPacTM AS11-HC-4µm IC Columns. Retrieved May 14,
2019, from https://www.thermofisher.com/order/catalog/product/078031
White, P. J., & Brown, P. H. (2010). Plant nutrition for sustainable development and global
health. Annals of Botany, 105(7), 1073–80. https://doi.org/10.1093/aob/mcq085
Williams, D. H., & Fleming, I. (1995). Spectroscopic methods in organic chemistry. McGraw-
Hill. Retrieved from https://www.ebay.co.uk/itm/302855505051
Yang, X., Wu, X., Hao, H., & He, Z. (2008). Mechanisms and assessment of water
Side 52 of 69
Aalborg University
eutrophication. Journal of Zhejiang University SCIENCE B, 9(3), 197–209.

https://doi.org/10.1631/jzus.b0710626
Yuan, Z., Pratt, S., & Batstone, D. J. (2012). Phosphorus recovery from wastewater through
microbial processes. Current Opinion in Biotechnology.
https://doi.org/10.1016/j.copbio.2012.08.001
Side 53 of 69
Aalborg University
Appendix
Appendix A: NMR
The NMR applies an external magnetic field to enable an energy transfer is possible between
the base energy to a higher energy level usually a single energy gap. The energy transfer takes
place at a wavelength that corresponds to radio frequencies and when the spin returns to its
base level, energy is emitted at the same frequency. The signal that matches this transfer is
measured in many different ways and processed in order to give an NMR spectrum for the
nucleus concerned in the sample taken. (Günther, 2013).
The atomic nucleus rotates about an axis. Among the nuclei the proton has a nuclear or
intrinsic angular momentum P and the spin of a nucleus can be explained using quantum
number of nuclear spin (I) and magnetic field (m). m is the magnetic or directional quantum
number that characterizes the corresponding stationary of the nucleus. The atomic nuclei
with even numbers of protons and neutrons have zero spin while all the other atoms with
odd numbers have a non-zero spin. These atoms with a non-zero spin exhibit a magnetic
moment μ and the relationship between P and μ is given the equation below:
μ=γ * P
Where γ is the gyromagnetic ratio (a constant characteristic of the nucleus and it can be
negative or positive depending on the sense of nuclear rotation).
The magnetic moment of the nucleus forces the nucleus to behave as bar magnet, which is
randomly oriented in the absence of an external magnetic field. When a sample is placed on
the external magnetic field B0, the bar magnet is forced to align with the B0 or to be against
the B0. When the bar magnets line up with the B0 it causes low energy levels while when the
bar magnet line up against the B0 it causes high energy, see figure 34 (Günther, 2013;
Kaseman, Davis, Srinivasan, & Iyer, 2003).
Figure 34: Magnetic field of a randomly oriented magnetic bar showing the external magnetic field align or
against the field (Kaseman et al., 2003).
Side 54 of 69
Aalborg University
The magnetic quantum numbers are related to the spin number of the respective nucleus.
The total number of possible energy levels is (2I + 1) different values of m. The 1H and 13C
nuclei are the most important nuclei in NMR spectroscopy because they can take up a low
orientation lined up with the applied field and a high energy orientation opposed to the
applied field. These two nuclei have I = 1/2, which have two m-values (+1/2 and -1/2) (see
figure q). Thus, if these nuclei are immersed in an external magnetic field, they can only be
regarded as being effectively lined up with the field (m = +1/2) or against the field (m = -1/2).
The energy difference between the states (upper state minus lower state) ∆E is found to be
proportional to the strength of the applied field B at the nucleus and is given by the equation
stated below: (Kaseman et al., 2003; Williams & Fleming, 1995).
∆Ε= γħB0
Figure 35: representation of the nuclear spin energy levels of a spin -½ nucleus in a magnetic field (GEROTHANASSIS et al.,
2002).
NMR spectrometer
An NMR spectrometer consists of several components. The first component is a magnet which
consists of a coil that produces a magnetic field and a probe which enables the coils to excite
the nuclear spins and detect the signal when placed close to the sample. Secondly it contains
a high-power radio-frequency (RF) transmitter or oscillator capable of delivering short pulses
and a pulse programmer to produce precisely times pulses and delays. In order to pick up the
signals, a sensitive receiver is needed to amplify the NMR signals. In addition to these a
digitizer is necessary to convert the NMR signals into a form which can be stored in computer
memory(John D. Roberts, 2006; Keeler, 2002).
Side 55 of 69
Aalborg University
Figure 36: Principle of NMR spectrometer consisting of a magnet, a radio-frequency transmitter or oscillator, a sample, a RF
receiver and a detector (John D. Roberts, 2006).
The magnet
It consists of a coil of wire through which a current pass in order to generate a magnetic field.
The wire is superconducting and thus when the resistance goes to zero at low temperatures
(less than 6 K). When the current is set running in the coil it will continue nonstop, thereby
generating a magnetic field without the need for further electrical power. The wire is
immersed in a bath of liquid helium in order to maintain it in its superconducting state. The
surrounding is usually acting as a heat shield and is kept at 77 K by contact with a bath of
liquid nitrogen to reduces the amount of liquid helium which boils off due to heat flowing in
from the surroundings. This is also because the liquid helium is more expensive than the liquid
nitrogen, hence nitrogen is used. The whole assembly is constructed in a vacuum flask to
further reduce the heat flow (Keeler, 2002).
The probe.
It is a cylindrical metal tube which is inserted into the bore of the magnet. In the probe there
is a small coil used to both excite and detect the NMR 8signal (magnetization). To be able to
optimize the sensitivity, this coil needs to be as close as possible to the sample (Keeler, 2002).
Side 56 of 69
Aalborg University
The transmitter.
The RF transmitter generates the pulses. The RF source, frequency synthesizer, produces a
stable frequency which can be set precisely so that the transmitter to different parts of the
spectrum. The frequency synthesizer (RF source) is controlled by a computer interface and it
is also relatively easy to phase shift the output from such a synthesizer, in order to create
phase shifted pulses.
To create a pulse of RF energy, the RF need to be applied for a short time and the output of
the synthesizer has to be gated (channeled) under computer control so that the length and
timing of the pulse can be controlled. The RF source is usually at a low level and it will provide
a useful B1 field when applied to the probe. In the transmitter there is an RF amplifier which
boosts the small signal to a power of 100 W or more (Keeler, 2002).
The receiver
The NMR signal that comes from the probe is small and therefore the amplifiers is placed as
close to the probe as possible so that the weak signal is boosted before being sent down a
cable to the spectrometer console. The receiver coil is tuned to the oscillator frequency but
is oriented with its axis perpendicular to both the direction of the principal magnetic field and
the axis of the oscillator coil. This arrangement is used to minimize the overloading of the
necessarily sensitive receiver which would result from direct coupling between the oscillator
and receiver coils. Therefore, a nuclear resonance signal id formed from an indirect coupling
between the oscillator and receiver coils produced by the sample itself (Keeler, 2002).
The NMR of various spectrum
D- Chloroform - Met-OH 1:1 is used as the internal reference compound because it can easily
be removed from the sample by evaporation due to its volatile properties. TMS is at a lower
frequency than most other signals because its methyl protons are in a more electron dense
environment than most protons are because silicon is less electronegative than carbon. The
position of the signals depends on the chemical shift.
Chemical shift is a measure of how far the signal produced from the proton is from the
reference compound signal by means of dimensions parameter δ (lowercase delta symbol). If
the sample absorbs to high frequency of the reference absorption at constant field of δ with
positive values. The TMS value increases toward the left and is measured in ppm (parts per
million), thus the proton chemical shifts range from 0 ppm to about 15 ppm and the chemical
shift is identical for a specific proton regardless of the spectrometer used (GEROTHANASSIS
et al., 2002).
Side 57 of 69
Aalborg University
Figure 37: Characteristics chemical shifts for 1H resonances of a number of commonly occurring groups (GEROTHANASSIS et
al., 2002).
The magnetic moment of the 13C is smaller than he proton by factor 4 and this makes 13C less
sensitive to use in the NMR than the proton. The 13C chemical shifts are like that of 1H though
the δ scale of 13C range from 0 ppm to about 250 ppm, see figure 38 (GEROTHANASSIS et al., 2002).
Figure 38: Characteristics chemical shifts for 13C resonances of a number of commonly occurring groups(GEROTHANASSIS et
al., 2002).
Side 58 of 69
Aalborg University
.
Some other heteronuclear such as 14N, 15N, 17O, and 19F has a ranger δ scale range than the
1H and 13C. The 17O has a δ scale range > 2000 ppm and has a resonance of C-O functional
groups, which indicates a clear distinction between alcohol, ether and C=O groups (figure 39)
(GEROTHANASSIS et al., 2002).
Figure 39: Representation of 17O shielding ranges in ppm for different C-O bonds (GEROTHANASSIS et al., 2002).
Side 59 of 69
Aalborg University
Appendix B: Lot and machines numbers

• Terumo syringe without needle – 2.5 mL
o REF: SS*Q2SE1
• Terumo syringe without needle – 10 mL
o REF: SS+IOES1
• Q-Max RR Syringe filters
o Lot: 180314004
• Centrifuge 5430, eppendof, nionlab cordozasvinget 6, DK-2680 solrød strand
o 104479
• Colormeter: UVmini-1240, strimadzu shimadzu suzhou china
o AAU: 80345
• Centrifuge Ramcon
o AAU: 1015663
• Agilent Technologies GC/MS triple Quad
o AAU: 104460
• Vacuum pump: scanvac
o AAU:93917
• HPIC: ICS-5000
o AAU: 93772
o AAU: 84405
o AAU: 84406
o AAU: 84407
• Chloroform
o SZBE014MV
• Methanol
o Product code: 83967.290
o Batch: 13Z2556
• Acetic acid
o Product: 20102.292
o Batch: 13G080031
• L-Ascorbic acid
o Lot: SLBV2291
• Sodium chloride
o Lot: SLBS2340V
• Sodium hydroxide solution
o Lot: BCBN0619V
Side 60 of 69
Aalborg University
Appendix C: Colorimetry
Table: Colorimeter schema for working standards
Standard 2 mg P/l water Ascorbic acid Acid Molybdate Final

concentration stock volume volume [µl] [µl] [µl] volume
mg P/L (in the [µl] [µl]
final volume)
0.04 20 920 20 40 1000
0.08 40 900 20 40 1000
0.16 80 860 20 40 1000
0.24 120 820 20 40 1000
0.32 160 780 20 40 1000
0.4 200 740 20 40 1000
0.6 300 640 20 40 1000
0.8 400 540 20 40 1000
Side 61 of 69
Aalborg University
Appendix D: NMR results
Figurxx: NMR results for acetate concentration at time 30.
Side 62 of 69
Aalborg University
Side 63 of 69
Aalborg University
Side 64 of 69
Aalborg University
Side 65 of 69
Aalborg University
Appendix E: NMR acetate results

Table: NMR acetate calculation results
tid [min] acetate TSP Concentration [mM]

Integral Integral
0 4,18 1 6,77808
30 4,74 1 7,6788
60 3,89 1 6,30342
90 4,36 1 7,06482
120 4,01 1 6,50268
150 3,70 1 5,99238
180 3,91 1 6,33582
210
240 4,06 1 6,573462
Table: Values used in the acetate

concentration NMR
Side 66 of 69
Aalborg University
Appendix F: HPIC Results

Table: HPIC results from the reactors A#H and B#H, and control reactor N
B#H A#H N#H

t:0 0,0106 0 t:0 0,1296 t:0 0,4105
t:30 0,0121 30 t:30 7,7198 t:30 0,2652
t:60 0,0149 60 t:60 7,5967 t:60 0,2626
t:90 7,5329 90 t:90 7,1567 t:90 0,3129
t:120 3,858 120 t:120 7,8745 t:120 0,2803
t:150 7,5586 150 t:150 7,1602 t:150 0,2192
t:180 7,3519 180 t:180 7,3585 t:180 0,2907
t:210 5,275 210 t:210 8,0675 t:210 0,2796
t:240 7,408 240 t:240 7,2676 t:240 0,2654
Side 67 of 69
Aalborg University
Appendix G: GC-MS PHA

Table: GC-MS PHA results of all the substances found in the sample with their mass per change and Rt.
Retention time Maximum

(min) m/z
4.096-4.134 280,6
4.996-5,037 392,4
5.064-5.113 280,8
5.445-5.483 116,9
5.646-5.684 386,6
5.848-5.884 280,6
6.006-6.115 393,4
6.510-6.546 336
6.546-6.587 381,4
6.906-6.979 337,8
7.082-7.125 113,9
7.379-7.409 125,9
7.546-7.582 129,9
8.266-8.304 116
9.719-9.780 395
10.158-10.202 378,9
10.541-10.579 149,9
12.542-12.597 377,3
13.874-13.912 153,8
14.325-14.376 342,2
14.777-14.810 396,8
14.810-14.844 333,3
15.052-15.088 213,9
16.685-16.725 399,2
16.903-16.964 243,6
17.021-17.057 378.1
17.074-17.112 398,9
17.229-17.333 241,9
17.722-17.763 208,2
17.856-17.856 223
18.032-18.074 256
18.129-18.176 255,8
18.284-18.345 328,4
18.500-18.550 255,8
19.139-19.224 271,4
19.219-19.478 396,8
19.588-19.652 237
19.673-19.732 236,8
19.751-19.802 239,3
19.830-19.883 236,8
20.020-20.080 269,9
Side 68 of 69
Aalborg University
21.215-21.299 393,4
21.503-21.600 250,2
22.049-22.126 380,6
23.203-23.280 358,8
23.603.23.689 264,8
23.763-23.852 264,7
23.896-23.977 298
30.470-30.565 388,1
32.177-32.289 352,2
32.530-32.605 207,8
32.931-33.035 366,9
Side 69 of 69

Project Bt4ff19

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Project Bt4ff19

Uploaded by

Copyright:

Available Formats

2019

Phosphorous extraction from wastewater

Cynthia Nyawira Thomsen

Jonas Lisby Wesseltoft

Peter Nørgaard Mortensen

Simon Ehlers Hove

Title: Phosphorous extraction from wastewater

Theme: Bioplastic and P-release

Project span: 01/02 2019- 31/05 2019

Supervisor: Reinhard Wimmer and Giulia Dottorini

Page total: 69 Abstract

Statistics and source of phosphorus

As previously mentioned, there is a limited amount of P and one of the candidates as a

Mining and impact

In temperate climates in developed countries, fertilizer inputs are estimated to attribute

Important mineral for plants

Phosphorus extraction at treatment plants

Anaerobic and aerobic process

Figure 5: Pathways of PAO processes in aerobic environments

It is possible to produce PHA by either a chemical approached or biosynthesis. However,

enoyl-CoA hydratase I (figure 6) (Chen, 2010). Step 4 in the beta-oxidation 3-Ketoacyl-CoA is

Biodiesel fuels from Sludge lipids

Experimental Methods Theory

High Pressure ion chromatography (HPIC)

Thin Layer Chromatography (TLC)

𝑑𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑠𝑎𝑚𝑝𝑙𝑒

Nuclear Magnetic Resonance spectroscopy (NMR)

Gas chromatography mass spectroscopy (GC-MS)

Collective results for GC and MS

Figure 8: Flowchart of applied methods in the sludge experiment.

Sludge incubation and sample collection

Determination of concentration of P by Colorimeter

Identification of concentration of acetate by HPIC

Substrate degradation – PHA

NMR-Lipids and acetate

The TSP-d4 final concentration was calculated to be 0.54 mM.

Lipid - Dimyristoyl Phosphatidylcholine (DMPC):

Lipid - Dimyristol Phosphatidylglycerol (DMPG):

Lipid - phosphatidyl-ethanolamine (PE)

Thin Layer Chromatography analysis (TLC)

Label Samples 1st 2nd amount

Table 2: Concentration over time for each reactor

Reactor B Reactor A Reactor N

It is impossible to tell which measurements could be accurate because reactor B have no

Myristic acid, methyl ester (C14:0) 4%

Retention time (Rt) Name of the ubstance

Retention time (Rt) Name of the substance

Name of the substance Mass

Thin Layer Chromatography - TLC

entanglement, ingestion, smothering, hangers-on, hitch-hiking and alien invasions.

eutrophication. Journal of Zhejiang University SCIENCE B, 9(3), 197–209.

Appendix B: Lot and machines numbers

Standard 2 mg P/l water Ascorbic acid Acid Molybdate Final

Appendix D: NMR results

Figurxx: NMR results for acetate concentration at time 30.

Figurxx: NMR results for acetate concentration at time 60.

Figurxx: NMR results for acetate concentration at time 90.

Figurxx: NMR results for acetate concentration at time 120.

Figurxx: NMR results for acetate concentration at time 150.

Figurxx: NMR results for acetate concentration at time 180.

Figurxx: NMR results for acetate concentration at time 240.

Appendix E: NMR acetate results