Removal of Nutrients by Algae from Municipal Bachelor thesis

Removal of Nutrients by Algae from Municipal Bachelor thesis
Removal of Nutrients by Algae from Municipal
Wastewater Contaminated with Heavy Metals.
BIGYAN ARYAL
Bachelor thesis
May 1st, 2015
Environmental Engineering
2
ABSTRACT
Tampereen ammattikorkeakoulu
Tampere University of Applied Sciences
Environmental Engineering
AUTHOR : Bigyan Aryal
Removal of Nutrients by Algae from Wastewater Contaminated with Heavy Metals.
Bachelor's thesis 47 pages, appendices 11 pages.
May 1st ,2015
Selected species of algae (green algae and blue green algae) were cultivated in municipal
wastewater using PBR(photo-bioreactor)bottles. Uptake of nutrients by these algae species was measured on different dates. From the results of the experiments, it was observed
that a combination of certain blue green algae species (cyanobacteria) was able to remove
most of the nutrients from the wastewater. The presence of heavy metal ions in the
wastewater also affected the nutrient-absorbing capacity of different algae species. Further research on several blue green algae species would be helpful in utilizing these algae
species in treatment of municipal wastewater.
Key words: algae; wastewater; pH; temperature; nutrients; nitrogen; phosphorous
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CONTENTS
1 Introduction ....................................................................................................... 5
1.1 Uses of Algae in wastewater treatment ...................................................... 6
1.2 Factors that affect Algae growth in water. ................................................. 7
1.2.1 pH and temperature. ........................................................................ 7
1.2.2 Sunlight and Carbondioxide............................................................ 8
1.2.3 Nutrients .......................................................................................... 9
2 Aim of this work.............................................................................................. 12
3 Methods and materials ..................................................................................... 13
3.1 Transferring mixture of algae in PBR bottles and labelling them. .......... 13
3.2 Total Nitrogen analysis ............................................................................ 17
3.3 Total Nitrate Nitrogen (NO3- -N) analysis ............................................... 17
3.4 Total Orthophosphate (PO4 3- ) analysis ................................................. 17
3.5 pH, temperature and conductivity measurement ..................................... 18
3.6 Light measurement .................................................................................. 18
3.7 Harvesting of algae .................................................................................. 19
4 Results and discussions ................................................................................... 21
4.1 Orthophosphate(PO43-) results. ................................................................ 21
4.2 Nitrate nitrogen(NO3-N) results. .............................................................. 23
4.3 Total Nitrogen(TN) results. ..................................................................... 25
4.4 TN/TP ratio .............................................................................................. 27
4.5 pH, Temperature and Conductivity ......................................................... 29
5 Conclusions ..................................................................................................... 34
6 References ....................................................................................................... 35
7 Appendices ...................................................................................................... 37
4
ABBREVIATIONS OF TERMS
PBR - Photo-bioreactor bottles
SP- Algae species
PAS (Photo Iluminescent Algae System)
Ni – Nickel
Cu – Copper
PO3- - Orthophosphate
NO3- -N - Nitrate
TN- Total Nitrogen
TP-Total Phosphorus
ml- Millilitre
Syke- Suomen ympäristökeskus
Min- Minimum
Max- Maximum
Ave- Average
TAMK- Tampere University of Applied Sciences.
ENVE- Environmental Engineering students group
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1
Introduction
The word ‘algae’ refers to a simple but very diverse form of organism which are found in
almost every parts of the planet. From single cell autotroph floating in the freshwater to
large seaweed such as giant kelp in oceans which can be hundred feet in length, algae is
the simplest phototroph which show the greatest diversity of any major division of the
plant kingdom (Hemsley, 2000) These simples form of autotrophs can be both prokaryotes (lack nucleus or membrane bound organelles and eukaryotes (contain membrane
bound nucleus and organelles).
Within fresh water or oceans, algae can be seen as microscopic single cell floating on the
surface known as planktonic algae or they may be seen at the bottom attached with sediments, rocks known as benthic algae. Algae contain chlorophyll and other pigments that
can trap the light from the sun and use light energy to make their own food by process
called photosynthesis. These diverse form of organism serve as a primary producers in
both marine and fresh water by providing oxygen as a byproduct of photosynthesis, by
being a food source for zooplanktons, small insects, snails and in case of large filamentous
macro -algae serving as a habitat for small animals and fish and many other aquatic life.
Algae are also good bio indicators which means that their presence can provide useful
information on physical and chemical characteristics of the waterbody at a particular site
(Sigee, 2010).Many algae species are available all the year and they quickly response to
the change in the environment due to pollution. They are a good indicator of water quality
and many lakes are characterized based on their dominant phytoplankton groups
(Chowdhury, 2013) .Algae are also known to clean waterbody by their presence. Recent
studies have revealed that algae are a good source for biological purification of
wastewater and they are able to clean wastewater by accumulating nutrients, heavy metals, organic and inorganic toxins, pesticides and radioactive matters in their cells. Biological treatment system by micro algae is now thought to be as effective as conventional
wastewater treatment system and low cost alternative in treatment of municipal
wastewater (Chowdhury, 2013)
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1.1
Uses of Algae in wastewater treatment
Uses of algae is diverse. New technologies and discoveries have found several uses of
algae in so many fields that it is easy to count where algae cannot be used rather than
where it can be used. Some of the major fields were algae use is a booming market are
feed manufactures, pharmaceutical companies, cosmetic companies, chemical industries,
bio fuel producing companies, pollution control in many industries where CO2 is produced as pollutant gas, in wastewater treatment either along with conventional water treatment plant or separately to treat wastewater that is rich in nutrients that come from poultry
farming, pig and cow farming before the effluent reaches agricultural crops. (Oilgae,
2015)
In most of the wastewater treatment plant whether it is ‘domestic wastewater, municipal
wastewater or industrial wastewater’ chemical methods (use of chemicals) and biological
method(use of anaerobic and aerobic bacteria in digestion of organic matter) are used but
now interests have grown that algae could be used in different stages along with
wastewater treatment plant or separately by itself depending upon the effluent requirement .The problem with conventional wastewater treatment includes high cost of chemicals, maintenance and high energy input . Use of algae in wastewater treatment plant is
cost effective, requires low energy input and the process is sustainable. Many researches
on algae have shown that algae are able to absorb not only nutrients but also heavy metals
particles in wastewater. This property of algae is very beneficial in treatment of
wastewater especially in domestic and municipal wastewater which is rich in nutrients
like nitrogen and phosphorous and many traces of heavy metal ions (Oilgae, 2015).
Treatment of wastewater by using algae cultivation can be used in several stages with
conventional wastewater treatment plant. If the wastewater is domestic like grey water
from houses and the effluent is to be discharged directly into lakes or ponds, algae treatment facility could work without any further use of chemicals. If the wastewater is municipal wastewater coming from septic tanks or industrial wastewater that contains many
toxic chemicals and metal ions, then additional treatment process like chemical process
and future biological process may be required along with algae treatment. Algae based
treatment processes are mainly used for the removal of nitrogen and phosphorous in municipal and industrial wastewater (Oilgae, 2015).
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Many wastewater effluent coming from dairy industries, animal feed industries, pig industries, agriculture run off water contain high amount of nutrients and fertilizers which
can pollute the fresh water body like lakes, ponds and rivers if this water is not treated
and directly discharged. Algae treatment plant can play a very vital role in capturing all
those excess nutrients and fertilizers and make water safe to be discharged at fresh water
body. The algae cultivated can be harvested in any waste water treatment plant and sent
into the bioreactor to produce biogas like methane that can be used to produce electricity
to fuel the whole treatment plant again. Apart from biogas, harvested algae is also rich in
nutrient that can be used as fertilizer in agriculture fields, also used to feed aquatic plants
and fish. The example is set up in Australia where farmers in diary industries are now
actually able to use diary effluent (wastewater) as a valuable resource. Algae cultivation
is being done on PAS (Photo-Iluminescent Algae System) which is a system of thin film
plastic which contains fluorescent dyes that alter and change the incoming sunlight that
helps algae grow. The diary effluent is sent through the algae under PAS and with the
help of light and nutrients in effluent algae grow produce methane gas as renewable energy source. The digester also concentrated nutrient stream that can be used as fertilizers
for growing crops and animal feed (Algae Enterprises, 2015)
1.2
1.2.1
Factors that affect Algae growth in water.
pH and temperature.
pH is the measurement of hydrogen ion concentration in water. pH plays a very important
role in aquatic life living in freshwater or marine water. It determines the solubility and
biological availability of many nutrients (nitrogen, phosphorous , carbon etc. )and metal
ions(zinc, copper, lead etc.) to aquatic plants and animals (Department of Ecology
,Washington, n.d.). Many metals tend to dissolve in low pH and be readily available in
water. They have toxic effects on aquatic plants and animals. Likewise, availability of
different nutrients like phosphorous and nitrogen in different forms is also determined by
the pH value .Slight change in pH can increase the nutrient availability level in lakes or
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ponds and cause rapid growth of many plants and algae giving rise to eutrophication problem (Kemer, 2013). pH is also an indication of what kind of algae is dominating the water
body. Lower pH due to increased CO2 dissolved in water gives’ green algae an competitive advantage over blue green algae’ (Weiner, 2008).
pH in fresh or marine water body may change due to several reasons that can be manmade
or natural. Manmade causes include pollution through mining, combustion in vehicles
that release CO2, sulfur oxides, nitrogen oxides and when water reacts with these compounds there is an acid rain which has low pH less than 5,0. Natural causes include
photosynthesis in surface by many phytoplankton and other aquatic organisms during day
time used more CO2 which can cause pH to rise. Likewise, at night photosynthesis stops
and CO2 from atmosphere dissolves in the water and lowers the pH (Kemer, 2013)
Environmental Protection agency (EPA) sets pH range (6,5-9,0) suitable for the growth
and survival of aquatic plants and animals (Weiner, 2008) .The optimum pH for growth
of algae is believed to be 8,2-8,7 but most species of algae grow well in pH range between
7 and 9 (FAO,Fisheries and aquaculture department)
Temperature is one another parameter that is very important in growth of algae and many
other aquatic life. Change in temperature also influences other parameters like pH ,conductivity, salinity, total dissolve oxygen, photosynthesis, compound toxicity etc. that can
change the physical and chemical properties of water. (Christine K. , 2014).The optimum
temperature for growth of cultured algae is believed to be from 20-25 ºC. Many cultured
species of micro algae would do fine and grow well in temperature range between 16-27
ºC. Temperature below 16 ºC and above 35ºC may not be suitable for algae growth.
(FAO,Fisheries and aquaculture department).
1.2.2
Sunlight and Carbondioxide
Sunlight and carbon-dioxide together are another important factor for growth of algae.
These two are the core elements needed for photosynthesis in algae. Algae including cyanobacteria (blue green algae) have chlorophyll present in their cells .These chlorophyll
capture light energy from the sun which convert the inorganic carbon to organic carbohydrate (glucose), lipids and proteins. Photosynthesis process in plants and algae releases
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oxygen so it is also called oxygenic photosynthesis (Hemsley, 2000) .Water temperature
and turbidity in lakes both affect the photosynthesis in algae. With increase in water temperature photosynthesis in most algae speeds up although there are slightly different optimum temperature requirement for different algae species. Likewise, high turbidity in
lakes slow down the photosynthetic rate in algae as amount of sunlight entering into several depth of lakes decreases( (Fitch, 2014).
Carbon dioxide in fresh or marine water is available to many phytoplankton like algae in
the dissolved gas form. Most of the carbon dioxide in water enters through the atmosphere
.Sunlight on the other hand is available only during the day so photosynthesis process
peaks during day time and declines during night (Fitch, 2014).
1.2.3
Nutrients
Nutrients play a very important role in growth of cells and enzymatic process in algal
cells. Some inorganic nutrients like nitrogen, phosphorous, carbon are primary nutrient
requirement in algae cells. There are also many other micronutrients and trace metals
which are needed in lesser amount which are silica(Si), magnesium(Mg), sodium(Na),
potassium(K), calcium(ca), sulfur(s), copper (Cu), manganese(Mn), cobalt(Co). In this
section we will focus mainly on primary nutrients like nitrogen and phosphorous and how
are they important to algal growth.
Nitrogen
Nitrogen is one of the essential nutrient required for all living cells including algae for
growth and reproduction. It is very crucial element which is found in amino acids that
forms the proteins, in nucleic acids that makes up DNA. Plants and other living organisms
cannot directly absorb atmospheric N2 as nutrient because of the strong bond in N2 molecule which is hard to break. So, atmospheric nitrogen must be broken down to several
other chemical forms .This process is called nitrogen fixation and many bacteria which
are present in water, soil pores, roots fix the atmospheric nitrogen to other chemical forms
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so that it can be absorb by plants either in soil or in water. Some fixation of nitrogen also
occurs during lighting process (Weiner, 2008).
In nitrogen cycle, nitrogen is converted to various chemical forms like ammonia, ammonium ion, nitrite, nitrate and free nitrogen. The fixed nitrogen in soil pores in the form of
ammonia and nitrogen oxides is absorbed by the roots of plants and legume plants to
make proteins, DNA and other organic nitrogen compounds. When these plants are eaten
by the animals and when they excrete or die, organic nitrogen enters into the soil in the
form of ammonia. This process is called ammonification. Ammonia is first converted to
nitrate by bacteria and nitrates are gain converted into nitrite further by oxidation and the
process is called nitrification. Nitrates in the soil are again converted to molecular nitrogen through nitric oxide by denitrifying bacteria and the process is called denitrification
through which nitrogen again returns to the atmosphere (7activestudio, 2014)
Most algae and aquatic plants receive nitrogen in the form of nitrate and ammonia. Blue
green algae (cyanobacteria) are actually able to fix atmospheric nitrogen into ammonia.
Phosphorous
Like nitrogen, phosphorous is also one of the essential nutrient for growth of plants and
animals. Plants and algae need phosphorous to make ATP, it is also needed to make DNA
and phospholipids in the cell membrane. Orthophosphate(PO43-) is the only form used by
most plants (including algae ) and other organisms to obtain phosphorous in water
(Weiner, 2008).
The conversion of phosphorous into different chemical forms is called phosphorous cycle
but it does not involve atmosphere like in nitrogen cycle. Most of the phosphorous present
in the environment is in the form of inorganic orthophosphate present in rocks and minerals in soil or in water body .The weathering of rocks and minerals helps plants and algae
to absorb inorganic phosphorous and convert them to organic form. When these plants
are eaten by animals, phosphorous again enters the soil or water when animals urinate,
excrete or when they die. The organic phosphorous in soil or water is again converted to
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inorganic phosphorous by decomposition with the help of bacteria (Beverly Biology,
2014)
Nitrogen and phosphorous are considered to be limiting nutrient in growth of algae. When
one of these nutrients is in excess amount, it triggers phytoplankton growth number. In
summer and spring times, when there is excess of nitrogen and phosphorous available in
the water body algae bloom is a common problem called eutrophication. Excess of nutrients naturally or artificially(run off of fertilizers from agricultural land that contain phosphate) in water body helps algae to grow rapidly and decrease the light penetration and
dissolve oxygen amount .This has direct effects on many aquatic animals like fish, crabs,
snails and many more. There might also be a shift of algae species from green algae to
more harmful blue green algae (Andrew R Dzialowski, 2005).These blue green algae
produce toxic that can lead the death of almost all aquatic living organisms, animals that
drink the toxic water and even humans by drinking toxic water or using it. Some toxins
like Hepatotoxins affect the liver is produced by some strains of cyanobacteria like Anabaena, Nodularia, Microcystis, Oscilatoria etc. There are also some toxins that affect
the nervous system, gastrointestinal system, kidney (WHO, 2015).
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2
Aim of this work
The aim of this project was to cultivate different mixture of algae species in photo-bioreactor bottles and compare their ability to remove nutrients and heavy metals from municipal wastewater.
This project is a continuation of previous research done in TAMK by different students
on various species of algae to purify wastewater. We tested with the selected mixture of
algae species which were already known to work better in treatment of wastewater.
This thesis mainly focuses on nutrients absorption by algae from municipal wastewater.
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3
Methods and materials
In the beginning wastewater was brought from the wastewater treatment plant Vinikanlahti, Tampere and then it was then poured in PBR(photo bioreactor bottles) to approximately 3000 ml .Three holes were made on the cap of each PBR bottle. One hole for
injection of nutrients for the growth of algae, second hole for the filtration so that the air
could come out of the bottle but nothing would enter inside and the third hole for the
aeration of wastewater.
3.1
Transferring mixture of algae in PBR bottles and labelling them.
The second stage was to transfer the algae and heavy metal (in our case Ni or Cu) inside
the new designed bottles. We named PBR bottles as belonging to set A, B, C and D. In
each set there are six bottles. Each set has 2 columns ‘first and second’ and each column
contains 3 bottles. The three bottles on first column of each set has (wastewater + algae
mixture) in it while the other three bottles on second column of each set has (wastewater+
algae mixture +heavy metal) in it. To make sure that we won’t be confused while taking
samples from each bottles to test for nutrients and heavy metals uptake, we labelled them
as below:
TABLE 1: Labelling of bottles.
Set A
Colm 1
Set B
Colm 2
Colm 1
A1a
A2a
B1a
A1b
A2b
A2c
A1c
Set C
Colm 2
Set D
Colm 1
Colm 2
Colm 1
Colm 2
B2a
C1a
C2a
D1a
D2a
B1b
B2b
C1b
C2b
D1b
D2b
B1c
B2c
C1c
C2c
D1c
D2c
In the table above A1a, A1b……..
D2c represent the labelling of the bottles.
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The project was carried out at TAMK’s green house building .There were already algae
species cultivated in separate 500 ml of bottles. These algae species were provided by
Finnish Environment Institute (SYKE) to TAMK. These 12 species of algae were:
TABLE 2: Algae species cultivated in 500 ml bottles provided by SYKE
Bottle (500 ml)
Algae Species with their phylum name
SP1
Selenastrum capricomutum ( Chlorophyta)
SP2
Pediastrum simplex (Chlorophyta)
SP4
Anabaena cylindrical (Cyanophyta)
SP6
Scenedesmus sp.(Chlorophyta)
SP7
Chlorphyta sp (Cyanophyta)
SP8
Purpuraemus sp.(No information)
SP10
Haematococcus (Chlorophyta)
SP11
Planktothrix rubescence (Cyanophyta)
SP12
Chlorella pyrenoidosa –(Chlorophyta)
SP13
Desmodesmus subspicatus (Chlorophyta)
SP14
Golekinia brevispicula (Chlorophyta)
SP15
Crucigenia tetrapedia – (Chlorophyta).
We can see from the above table that most of the algae species provided are green algae
(chlorophyta) and some blue green algae(cyanobacteria) with phylum name cyanophyta.
From earlier experiments carried out on wastewater treatment at TAMK, it had already
been known that mixture of algae species work better than the single species in
wastewater treatment so based on the same principle we transferred mixture of different
algae species in each set of PBR bottles. The figure below gives a clear picture about
which algae species was transferred to which set of PBR bottles.
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SP1.Selenastrumcapricomutum
SP2. Pediastrum simplex
SP4. Anabaena cylindrical
SP6. Scenedesmus sp.
SP7.Chlorphyta
sp
(Pekari
Set A
Strain)
SP8 .Purpuraemus sp.
SP10. Haematococcus
SP11. Planktothrix rubescence
SP12. Chlorella pyrenoidosa –
Green algae
SP13.Desmodesmus
subspi-
catus
SP14. Golekinia brevispicula
SP15. Crucigenia tetrapedia
SP1.Selenastrum
caprico-
mutum
SP2.Pediastrum simplex
Set B
SP6.Scenedesmus sp.
SP10.Haematococcus
SP4.Anabaena cylindrical
SP6.Scenedesmus sp.
Set C
SP4.Anabaena cylindrical
SP11.Planktothrix rubescence
Set D
FIGURE 1: Representation of mixture of algae used in different set of bottles.
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IMAGE 1: The setup of the experiment at greenhouse TAMK.
The image above shows the experiment set up at greenhouse TAMK. The first six bottles
of set A starting from left contain all mixture of alga species(most green +some blue
green algae) ,second six bottles of set B contain mixture of algae species SP1,SP2,SP6
and SP10(all green algae). The third six bottles of set C contain mixture of algae species
SP4 and SP 6(green + blue green algae) and the last six bottles of set D at far right corner
contain algae species SP4 and SP11(blue green algae).
It was quite challenging to transfer mixture of different algae species in different set of
bottles. For first six bottles in set A to obtain mixture of algae species, 50 ml of each algae
species from 12 cultivated bottles was collected in a separate 600 ml bottle and out of that
100ml (600/6) was transferred to each six bottles make sure mixture of algae was transferred equally in all six bottles. Similarly for second six bottles of set B, 50ml of 4 algae
species was first taken and diluted with 400 ml of water to make the total volume 600ml
and again 100 ml was transferred to each six bottles. Likewise to obtain the algae mixture
for third set of six bottles in set C, 50 ml of 2 algae species was taken and diluted with
500 ml of water to make the total volume 600 ml and 100 ml was then transferred to six
bottles. For the last six bottles in set D, the procedure was exactly same as for the six
bottles in set C; only the difference was the mixture of algae used.
The whole project was divided into two test run or experiments. Both the experiments
done were exactly the same ; we tested nutrients and heavy metal uptake by algae from
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wastewater .The only difference was in first test run Ni was used as testing metal and in
the second test run Cu was used as testing metal.
The Ni or Cu concentration to be used in wastewater was 30 mg/L based on the previous
experiment done at TAMK (Benchraka, 2010) .Since each bottle were filled with 3 litres
of wastewater we had to add 90 mg of Ni or Cu to make the concentration 30mg/L. Clean
100 ml plastic pipe lets were used to add the metal concentration.
3.2
Total Nitrogen analysis
Total Nitrogen analysis was done by using total nitrogen kit by HAACh LANGE. The kit
is named as total nitrogen 138 kit. The range for the total nitrogen analysis is (0-16) mg/L.
This Haach method of analysing total nitrogen is quite simple and the procedure is very
clearly given in the pamphlet. There are two main stages digestion which takes one hour
and then reading the sample in Haach reader.
3.3
Total Nitrate Nitrogen (NO3- -N) analysis
Total nitrate nitrogen test was done by Cadmium Reduction Method 8039. Later Haach
reader was used to get the readings .The range for measurement according to the standard
was 0.3-30.0 mg/L NO3- -N. Every time we tested for Total Nitrate Nitrogen we did two
test for each sample to make sure that the readings would match close to each other. For
somehow if the readings were not close then we had to test third time and make sure we
get the correct readings.
3.4
Total Orthophosphate (PO4 3- ) analysis
In the similar way, we also tested the reactive Orthophosphate (PO43-).The test was done
according to the PhosVer 3(Ascorbic Acid) Method1 and the reading was done in Haach
reader. The measuring range for orthophosphate is 0, 2 to 2, 50 mg/L PO4 3- .
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3.5
pH, temperature and conductivity measurement
PH, temperature and conductivity was measured using compact meter named as ‘Five
Easy PH meter’ and ‘Five Easy Conductivity meter’ made by company ‘Mettler Toledo’.
Both pH and conductivity meters are an electrical devices which consist of a probe also
known as glass electrode which is connected to the compact electric meter that measures
and displays pH and conductivity readings. The probe is a cylindrical rod like structure
made of glass and at the bottom of the probe there is a bulb. The bulb is very sensitive
and consists of the sensor that detects the pH or conductivity of the solution. The probe
has to be dipped into the solution to measure pH or conductivity. The temperature of the
solution is also displayed when measuring pH or conductivity.
Both pH and conductivity meters were first calibrated before use and the method to calibrate them could be found in the pamphlet that come with the meters. PH and conductivity
was measured every time we did the nutrients analysis.
IMAGE 2: Five Easy PH and Conductivity meter (Aa6opa, 2015)
3.6
Light measurement
The light or illuminance was measured using lux meter which measured the incoming
light in all the algae bottles and it could measure the light up to 1 square meter of area.
The experiment was carried out in winter so in some days there was no sunlight. Sodium
lights were used in the green house to provide light for the growing algae and these lights
were automatic and they would shut down automatically in the evening until next morning.
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3.7
Harvesting of algae
The harvesting of the algae was done at the end of each experiments. From the beginning
of the experiment until the start of harvesting, growth of the algae and their ability to
remove excess nutrients from wastewater was observed by testing the nutrients content
in wastewater twice in every week.
Harvesting the algae at the end was necessary to determine how much nutrients and metal
algae have absorbed at the end of the experiment. The first step was to take all the
wastewater from PBR algae bottle in 45 ml of centrifuge tubes and centrifuge to remove
all the water from it for drying. We emptied one algae bottle at a time. The remaining
solid mass in centrifuge tube was then taken out with the help of a spoon into a porcelain
cup. There was always a small amount of algae left in the centrifuge bottle so ethanol
was used to remove that.as ethanol evaporates much faster than water when drying.
Before transferring the algae from centrifuge tubes to the porcelain cups, the porcelain
cups were first weighted. This was done by placing the cups in an oven for 4 hours at 100
degree Celsius. After that, they were cooled into a desiccators for half an hour and dry
weight of those cups were taken in a weighting machine. Immediately after the weight of
dry porcelain cups were taken, algae was transferred to the cup from centrifuge tube and
weight of wet algae and cup was recorded.
The cups with wet algae biomass were then again placed into oven at 55 degree Celsius
for 24 hours for drying. After 24 hours of drying the porcelain cups with dry algae biomass were again weighted.
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IMAGE 3: Porcelain cups with wet algae biomass in oven for drying.
As mentioned earlier the main aim of harvesting algae was to determine nutrients and
heavy metals left in algae biomass at the end of experiment but unfortunately the exact
method to test nutrients absorption in biomass was not found. Regarding metal absorption in biomass and the process of its extraction is mentioned in Adedayo Bello thesis
work, 2015.
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4
Results and discussions
4.1
Orthophosphate(PO43-) results.
TABLE 3: PO43- concentration(mg/l) in different PBR algae bottles with its removal
percent in the first test run.
SET A
Blank(mg/l)
Metal(mg/l)
Initial
readig
19,35
19,35
After 4th
day
29,05
38,07
After 6th After 13th After 17th %
day
day
day Removal
28,47
25,03
28,43
33,73
31,13
34,47
SET B
Blank(mg/l)
Metal(mg/l)
10,50
10,50
5,77
8,43
3,87
8,27
3,90
7,33
3,90
7,43
62,85
29,23
SET C
Blank(mg/l)
Meta(mg/l)
3,65
3,65
1,28
1,58
1,73
0,97
2,27
1,70
1,97
1,50
46,02
58,90
SET D
Blank(mg/l)
Metal(mg/l)
8,75
8,75
0,90
1,53
0,83
1,23
1,77
1,47
1,28
1,15
85,37
86,85
The above table 3 shows the orthophosphate concentration in wastewater measured in
different days and its removal percent by algae for both the blank and metal samples in
different sets. Here in this table and the following tables in other sections, ‘blank’ word
denotes the PBR algae bottle with (wastewater+algae+nutrients) in it and ‘metal’ word
denotes the PBR algae bottle with (wastewater+algae+nutrients+heavymetal) in it.
In the above table and the following tables below that represent the first test run , initial
reading of PO43- , NO3-N and TN in wastewater is taken from initial reading of second
test run. This is done because in second test run’ initial reading’ was taken after several
mixture of algae species were added in the wastewater while in first test run we took the
initial reading of wastewater before any mixture of algae species were added which can
be seen in appendix 1. Since same mixture of algae species were added in wastewater in
both test run ,we suppose it is worth taking initial reading of wastewater after different
mixture of algae species were added.
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It can be noticed that there was no PO4
3-
removal in set A while in set B, C and D
removal of PO4 3- was on average(including blank +metal) above 45% , 50% and 85%
respectively. Cyanobacteria species in set D were able to absorb more phosphate than
other algae species. Likewise, removal percent of PO4 3- is seen more in ‘blank’ bottle
than in the’ metal’ bottle.
TABLE 4: PO43- concentration(mg/l) in different PBR algae bottles with its removal
percent in second test run.
SET A
Blank(mg/l)
Metal(mg/l)
Initial
reading
19,35
19,35
After 2nd
day
4,38
6,95
After 6th
day
5,05
6,48
After 9th
day
3,77
4,17
%Removal
80,53
78,47
Set B
Blank(mg/l)
Metal(mg/l)
10,50
10,50
1,48
3,00
1,80
1,48
1,28
1,80
87,78
82,86
SET C
Blank(mg/l)
Metal(mg/l)
3,65
3,65
1,75
1,83
1,27
1,05
0,68
1,30
81,28
64,38
SET D
Blank(mg/l)
Metal(mg/l)
8,75
8,75
1,63
1,87
0,87
0,85
0,58
0,98
93,33
88,76
The table 4 above is also an illustration of orthophosphate concentration in wastewater
contained in different algae bottle sets. It can be seen that there was an average 81 %,
85%,73% and 91% phosphate removal from wastewater in both blank and metal samples
of set A,B C and D respectively.
In second test run, the absorption rate of phosphate from wastewater by different algae
species is much higher compared to the first test run and also blue green algae species in
set D have shown the highest phosphate removal from wastewater. Metal concentration
in wastewater also have affected nutrient absorbing capacity of different algae in different bottle sets. PO43- is more removed in ‘blank’ bottles than in bottles with’ metal’ in
them.
23
4.2
Nitrate nitrogen(NO3-N) results.
TABLE 5: NO3-N concentration(mg/l) in different PBR algae bottle with its removal percent in first test run.
SET A
Blank(mg/l)
Metal(mg/l)
Initial
reading
36,50
36,50
After 4th After
day 6th day
74,83
53,83
67,83
60,83
After
13th day
48,17
73,33
After %
17th day Removal
21,33
41,56
37,00
SETB
Blank(mg/l)
Metal(mg/l)
22,0
22,0
21,00
21,83
31,67
27,50
27,33
27,83
7,02
15,17
68,09
31,04
SET C
Blank(mg/l)
Metal(mg/l)
18,50
18,50
20,67
13,67
21,83
21,50
24,83
23,83
4,17
5,03
77,45
72,81
SETD
Blank(mg/l)
Metal(mg/l)
17,00
17,00
18
13,17
22,5
10,83
15,5
8,83
5,6
12,37
67,05
27,23
The above table 5 shows the NO3-N measured in wastewater in different days and its
removal percent. We can clearly see that much of the NO3-N from wastewater from set
B, C and D was removed. On an average (including both blank and metal)49%,75% and
47% nitrate nitrogen was removed from wastewater contained in set B,C and D respectively. ‘Blank’ sample in set A was able to perform better than ‘metal’ sample with above
40 % removal of NO3-N. In similar way, ‘blank’ sample bottles in other sets also have
performed better than metal sample bottles.
24
TABLE 6: NO3-N concentration(mg/l) in different PBR algae bottle with its removal percent in second test run.
SET A
Blank(mg/l)
Meta(mg/l)l
SET B
Blank(mg/l)
Metal(mg/l)
Initial
reading
36,50
36,50
After 2nd
day
42,33
48,17
After 6th
day
63,50
57,83
After 9th
day
37,50
50,83
% Removal
% Removal
22,00
22,00
28,67
39,17
39,67
42,33
42,50
35,83
SET C
Blank(mg/l)
Metal(mg/l)
% Removal
18,50
18,50
29,83
42,00
37,33
30,67
35,33
28,17
SET D
Blank(mg/l)
Metal(mg/l)
17,00
17,00
16,67
47,50
35,00
32,17
20,83
39,83
% Removal
The table 7 above illustrates the measurement dates and nitrate concentration in
wastewater algae bottles. Nitrate amount actually increased in the second test run at the
end. There have been several fluctuations like increase and decrease in readings in different days of reading. It is very hard to conclude exactly why this reading was observed.
25
4.3
Total Nitrogen(TN) results.
TABLE 7: TN concentration(mg/l) in different PBR algae bottle with its removal percent
in first test run.
Set A
Blank(mg/l)
Metal(mg/l)
Initial
reading
90,00
90,00
After
4th day
58,23
64,20
After
After
6th day 13th day
89,27
70,27
125,00
85,03
After
%
17th day Removal
38,13
57,63
47,63
47,07
Set B
Blank(mg/l)
Metal(mg/l)
54,80
54,80
46,13
65,47
40,77
59,53
35,60
44,50
30,83
36,63
43,74
33,15
Set C
Blank(mg/l)
Metal(mg/l)
48,70
48,70
31,56
31,41
26,4
43,9
20,36
25,76
20,7
20,8
57,49
57,28
Set D
Blank(mg/l)
Metal(mg/l)
45,50
45,50
25,30
33,37
24,30
47,03
14,80
39,60
11,07
29,27
75,67
35,67
The table 7 above shows the TN concentration in wastewater measured in different dates
in first test run. There was an average(including both blank and metal) 52 %, 38%, 57%,
and 55% of TN removal from wastewater in algae bottle set A, B, C and D respectively.
We can see that blank samples without metals have done pretty well in removing TN from
wastewater .Blue green species in set D seem to be affected more with presence of metal
in wastewater as there is a significant difference in removal percent of TN in blank and
metal sample in set D. The same ‘blue green algae species’ in set D have also the highest
TN removal if we compare the blank sample bottle with other sets.
26
TABLE 8: TN Concentration(mg/l) in different PBR algae bottle with its removal percent
in second test run.
SET A
Initial
reading
After 2nd
day
After 6th
day
After 9th
day
% Removal
Blank(mg/l)
90,00
75,53
70,33
65,83
26,85
Metal(mg/l)
90,00
77,80
79,03
63,60
29,33
Blank(mg/l)
54,80
34,53
35,90
36,77
32,91
Metal(mg/l)
54,80
43,77
42,63
40,97
25,24
Blank(mg/l)
48,70
35,97
39,27
36,50
25,05
Metal(mg/l)
48,70
36,97
24,83
35,23
27,65
Blank(mg/l)
45,50
35,83
37,83
34,87
23,37
Metal(mg/l)
45,50
40,73
30,40
44,27
2,71
SET B
SET C
SET D
The table 8 above shows the TN concentration in wastewater measured in different days
in second test run. There was an average(including both blank and metal) 28 %, 29%,
26%, and 13% of TN removal from wastewater in algae bottle set A, B, C and D respectively.
By having a close look at the table above we can see again that TN removal was better in
‘blank’ sample than in the ‘metal’ sample. TN removal is not that much higher than it
was seen in the first test run. But overall, absorption of nutrient nitrogen was seen in all
algae bottles.
27
4.4
TN/TP ratio
TABLE 9: Nitrogen to phosphorus mass ratio in First test run.
SET A
Blank
Metal
SET B
Blank
Metal
SET C
Blank
Metal
SET D
Blank
Metal
Initial N/P
ratio
14,25
14,25
After 4th
day
6,14
5,17
After 6th
day
9,61
11,36
After 13th After 17th
day
day
8,60
4,11
8,37
4,23
15,99
15,99
24,50
23,80
32,28
22,06
27,97
18,60
24,23
15,11
40,89
40,89
75,56
60,92
46,76
138,69
27,49
46,44
32,20
42,49
15,94
15,94
86,15
66,84
89,72
117,17
25,62
82,55
26,50
78,00
The table 9 above is the representation of nitrogen to phosphorous mass ratio in each set
of wastewater in different days in first test run.
TN/TP ratio is helpful in determining which of the nutrient(nitrogen or phosphorous) is
the limiting factor for algae growth in water. Study suggest that when TN/TP ratio is less
than 10, a lake is nitrogen limited, when TN/TP ratio is between 10-17 each of the nutrient
either nitrogen or phosphorous might be limited and if TN/TP ratio is greater than 17
phosphorous is the limiting factor (Florida, 2015). In many cases phosphorous threshold
values also help to determine which nutrient is limited. In lakes where TP concentrations
is above 0,1mg/L there is a chance that nitrogen might be the limiting factor rather than
phosphorous and their TN/TP ratio is generally less than 17. In lakes where TP concentration is less than 0,05mg/ , phosphorous is the limiting factor (Florida, 2015)
As we can see from the table that ,TN/TP ratio for algae bottle sets B,C and D is usually
greater than 17 which shows that phosphorous was the limiting nutrient in algae growth
while in set A TN/TP ratio is below 10 (excluding initial ratio TN/TP>10) , nitrogen was
the limiting nutrient.
TN/TP ratio is not only helpful to know which of this nutrient is the limiting factor in
growth of algae or any phytoplankton in waterbody but also helpful in predicting if blue
green algae are growing more and dominating the green algae or vice versa. At lower
28
TN/TP ratio usually below 22 or 30, blue green algae also known as cyanobacteria start
to increase in number by absorbing most of the nutrients .Although in this situations nitrogen can be limited but some cyanobacteria are able to utilize dissolve nitrogen gas
.They have competitive advantage when nitrogen is limited and phosphorous is not limiting. Nitrogen fixing cyanobacteria such as Anabeca spp and Aphanzomenon flos-aquae
are found to be more radially present in lakes with TN/TP ratio less than 30:1 or 22:1.
(Schaedel, 2011).
In set ‘A’ which is a mixture of green algae and blue green algae TN/TP ratio is far below
22 which shows that blue green algae might have increased in number and grown well
than green algae species. It could be assumed that the competition between these two
algae species might be the reason for no PO43- removal in set A. Likewise, TN/TP ratio
for blank sample in set D on the 13th and 17th day of measurement is below 30 so this
might have caused the increase in blue green algae population. In the same set D when
we see the metal sample ,TN/TP ratio is greater than 30 which might have affected the
blue green algae to grow well. This might be the reason why we saw more nutrients removal in blank sample than in metal sample of set D.
TABLE 10 : Nitrogen to Phosphorus ratio in Second test run.
SET A
Blank
Metal
Initial N/P
ratio After 2nd day After 6th day After 9th day
14,25
52,85
42,68
53,51
14,25
34,30
37,37
46,74
15,99
15,99
71,50
44,71
61,12
88,27
88,03
69,75
Blank
40,89
62,99
94,76
164,49
Metal
SET D
40,89
61,91
72,47
83,05
Blank
15,94
67,36
133,25
184,24
Metal
15,94
66,75
109,60
138,43
SET B
Blank
Metal
SET C
The table above is the representation of nitrogen to phosphorous ratio in each set of
wastewater in second test run in different days of measurement. We can see that TN/TP
ratio is above 17 in all sets of wastewater which shows that phosphorous was the limiting
factor for algae growth. It is hard to relate TN/TP ratio with nutrient removal results in
29
second test run. TN/TP ratio suggest that phosphorous was the limiting nutrient and nitrogen was readily available but there has been no nitrate nitrogen (NO3- N )removal and
not much TN removal but much of the orthophosphate was removed from wastewater.
4.5
pH, Temperature and Conductivity
FIGURE 2: Variation of pH during the Nickel test run.
30
FIGURE 3: Variation of pH during the Copper test run.
We can see from the above figure ‘2’ and ‘3’ that pH remained on the range of 6-8 which
has favoured the growth of algae.
Temperature on the other hand was within the range of (18-22)ºC in first test run and in
the second test run within the range of (15-22)ºC.
pH and temperature play a very important role in the growth of phytoplankton in any
water body. The information provided by FAO (Food and Agriculture Organization of
the United Nations) says that the optimum pH for the growth of phytoplankton is believed
to be from 8,2 - 8,7 when cultured while most of the cultured algae species would do fine
or grow in pH between 7-9 ( (FAO,Fisheries and aquaculture department). Some studies
have revealed that at pH 7 ,algae growth is increased and maximum amount of heavy
metal and nutrients( phosphorous and nitrogen) are removed from wastewater. In studies
done with marine macro-algae Caulerpa taxifolia and red algae Kappaphycus alvarezii
to test the heavy metal and nutrient absorption in wastewater , it has been found that at
pH 7 maximum amount of heavy metal zinc and nutrients phosphorous and nitrogen were
removed from wastewater (R.Mithra, 2012).
31
Regarding temperature, the optimum temperature for many cultured species of algae
ranges from 20 to 24 ºC. It is also believed that many cultured species of micro algae will
tolerate the temperature from (16 -27)ºC. Below 16 ºC the growth rate slows down and
above 35 ºC many algae species may start to die (FAO,Fisheries and aquaculture
department) .
In another case study done on blue green algae in Lake Mendota, Wisconsin it was found
that the optimum temperature for photosynthesis by blue green algae was usually between
20 and 30 degree Celsius (Allan Konopka, 1978). Recent news published in ‘Biotechniques ,the international Journal of life Science methods’ , suggest that some red algae
species like Galidieria sulphuraria could be able to survive in extreme environment like
hot sulphur springs. They are able to tolerate extreme pH and temperature. These algae
species are able to survive this extreme conditions by borrowing genes from bacteria and
archaea that protect them from unbearable heat and toxic conditions (Chi, 2013).
No extreme temperature or pH was observed that could have harmed the growth of algae
in both the test run. Somewhere in second test run the temperature of wastewater in different algae bottles have also dropped below 16 ºC when outside temperature was also
very low but it has remained usually above 18 ºC in many measurements.
32
FIGURE 4: Variation of conductivity during the Nickel test run.
FIGURE 5: Variation of conductivity during the Copper test run.
33
The above figure ‘4’ and ‘5’ represent the variation of conductivity in wastewater during
nickel and copper test run respectively. We can see that wastewater which has metal have
higher conductivity than the wastewater in blank sample in most of the sets. A decrease
in conductivity can also be seen in many algae bottle sets in both the test run which also
indicates that with absorption of nutrients by algae from water, conductivity of water has
also decreased. In set ‘A’ conductivity values were higher because it had more algae species than other sets so more nutrients were available in the wastewater from the beginning
that gave up higher conductivity values.
Conductivity is one another important parameters that measures water quality. Conductivity is the early indicator that show the change in water system . (Christine, 2014). It
measures the dissolved ions in the water through which water can conduct electricity.
Dissolved ions in water come from various sources like dissolve salts, inorganic ions like
chlorides, sulphides, alkalis, carbonate compounds, metal ions like zinc, copper, nickel,
lead ,mercury, silica, inorganic nitrate ,phosphate and many more . A typical freshwater
body might have conductivity from 100-2000(µs/cm), industrial wastewater (10000
µs/cm), sea water (55000 µs/cm), distilled water (0,5-3 µs/cm) (Christine, 2014) .Conductivity and temperature are directly related. With the increase in temperature conductivity of water also increases as mobility of ions increases as well as dissolving capacity
of many salts and minerals increases. Conductivity of any waterbody can go up in the day
time when temperature is warmer due to sunlight and go low at night when temperature
is cool (Christine, 2014).
34
5
Conclusions
From the results of both test run, it can be seen that ‘Set D’ which is a mixture of blue
green algae have proved to work better in removing most of the nutrients from
wastewater. This also tells us that if blue green algae are grown under controlled conditions (monitoring their growth and toxicity) to purify wastewater they can be very useful.
Similarly, presence of metal in wastewater have also affected the nutrient absorbing capacity of different algae. Unfortunately, due to limited time we were not able to do more
research on how metal binding on algae cells affect their nutrient absorption property.
Further research on how metal binding on algae affect their ability to absorb nutrients is
necessary.
Parameters like pH(6-8) and temperature(16-22)ºC in this project were in favour of algae
growth. TN/TP ratio results on the other hand was helpful in predicting which nutrient
was limiting factor for algae but did not provide sufficient information on how it affected
growth of several mixtures of algae. More research is needed to exactly know how TN/TP
ratio affects the growth of several algae species .
Nutrient absorption in algae biomass was not analysed. This could have provided more
detailed information on how much nutrients was absorbed by different algae species in
different sets of wastewater bottles.
Overall, this project has revealed that algae are definitely able to absorb nutrients from
wastewater and blue green algae were able to perform better than green algae .It can be
concluded that further research would be useful to test several blue green algae species
in treatment of wastewater.
35
6
References
7activestudio.
(2014,
March
4).
Nitrogen
cycle.
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https://www.youtube.com/watch?v=LbBgPekjiyc
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Algae Enterprises. (2015). Advanced Algae bioremediation System. Retrieved from
Waste Water Treatment: http://www.algaeenterprises.com/waste-water-treatment
Allan Konopka, T. D. (1978, August 15). Effect of Temperature on Blue green
Algae(Cyanobacteria)
in
Lake
Mendota.
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from
http://aem.asm.org/content/36/4/572.full.pdf
Andrew R Dzialowski, S. H.-C. (2005). Nutrient limitation of phytoplankton growth in
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https://publications.theseus.fi/search?query=chouaib&submit=Go
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https://www.youtube.com/watch?v=c5KqwhX1dvk
Chi, K. R. (2013, 8 3). Algae hijack genes to survive. (Biotechniques) Retrieved 4 22,
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Survive/biotechniques-340978.html#.VTesQ1Wqqko
Chowdhury, W. E. (2013). Water Treatment. Intech.
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Fundamentals
of
Environmental
Mesaurements:
http://www.fondriest.com/environmental-measurements/parameters/waterquality/conductivity-salinity-tds/
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Understanding
and
monitoring
Lakes
and
Streams:
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Algae,
Phytoplankton
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http://www.fondriest.com/environmental-measurements/parameters/waterquality/algae-phytoplankton-chlorophyll/
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37
7
Appendices
Appendix 1: Initial Wastewater Nutrients and Heavy Metal (Ni) Readings (first test run)
Date
6.2.2015
PH
7,2
conductivity
Temperature
NO3- -N
PO43-
TN
Nickel
(µs/cm)
ºC
(mg/L)
(mg/L)
(mg/L)
(mg/L)
1073
22,1
21
60,76
0
3,2
Appendix 2: Mesurement on 10.2.2015(first test run)
ConducPO43- Av. PO43- NO3Av. NO3 –
tivity
Sample (mg/l) (mg/l)
N(mg/l) N(mg/l)
TN(mg/l) pH (µs/cm) Temp°C
31,1
60
A1a
32,2
31,65
59
59,5
28,4 7,86
5580
22
19,6
88
A1b
20,8
20,2
86
87
53,5 6,76
5581
22,3
38,4
78
A1c
32,2
35,3
73
75,5
92,8 7,19
5582
23,3
42,7
85
A2a
42,7
42,7
83
84
86 7,59
2360
21,9
50
89
A2b
50
50
67
78
49,3 7,87
1030
21,4
21,5
42
A2C
21,5
21,5
41
41,5
57,3 7,4
940
22,1
4,5
19
B1a
4,5
4,5
18
18,5
37,9 7,51
641
21,6
5,2
13
B1b
5
5,1
12
12,5
53,4 7,59
706
21,3
7,6
33
B1c
7,8
7,7
31
32
47,1 7,29
609
23,6
10
24
B2a
10,9
10,45
22
23
66,9 7,55
853
21,7
9,8
24
B2b
10,1
9,95
21
22,5
82,3 8,09
705
21,1
4,9
20
B2c
4,9
4,9
20
20
47,2 8,04
647
21,2
0,7
16
C1a
0,6
0,65
17
16,5
35,6 7,97
624
22
1,9
18
C1b
2,1
2
16
17
31,3 7,93
620
21,7
1,3
29
C1c
1,1
1,2
28
28,5
27,8
8
560
21,4
2,7
28
C2a
2,6
2,65
27
27,5
47,1 7,56
736
22,2
0,7
10
C2b
0,5
0,6
10
10
38,8 7,7
712
21,4
C2c
1,4
1,45
13
12,5
8,34 7,82
779
22,2
38
D1a
D1b
D1c
D2a
D2b
D2c
1,5
1
1,1
1
0,9
0,7
0,7
1,5
1,4
1,4
1,4
1,7
1,7
1,05
0,95
0,7
1,45
1,4
1,7
12
19
18
19
17
18
17
12
11
14
13
15
14
18,5
24,6 7,83
634
21,9
18
33,3 7,76
611
21,2
17,5
18 7,79
614
21,4
11,5
35,8 7,81
801
22,3
13,5
32,3
788
21
14,5
32
779
20,5
39
Appendix 3: Measurement on 12.02.2015 (first test run)
PO43Av.
Conduc(mg/l Av. PO43- (NO3 (mg/l)(NO TN(mg/l
tivity(µs/cm Temp°
Samples )
(mg/l)
N)(mg/l)
N)
)
pH
)
C
3
34,2
51
A1a
33,1
33,7
53
52
97,2 6,47
982
20,6
16,6
48
A1b
16,4
16,5
53
50,5
74,4 6,39
966
20,8
35,6
50
A1c
34,8
35,2
68
59
96,2 6,33
1032
21
40,3
61
A2a
39,9
40,1
72
66,5
141 8,02
1310
20,3
24
59
A2b
24,1
24,1
69
64
117 7,94
1140
20
37
59
A2C
37
37
45
52
117 8,01
1030
19,9
2,5
27
B1a
2,2
2,4
32
29,5
37,5 7,74
693
20,4
1,4
34
B1b
1,4
1,4
27
30,5
38,5 7,69
689
20,4
7,6
31
B1c
8
7,8
39
35
46,3 6,92
724
20,6
9,6
27
B2a
9,7
9,7
29
28
72,6 8,17
914
20,3
10,9
26
B2b
10,8
10,9
28
27
68,3 8,23
903
20
4,2
27
B2c
4,1
4,2
28
27,5
37,7 7,99
694
20,4
3
19
C1a
5
4
20
19,5
29,3 7,57
642
20,8
0,5
24
C1b
0,7
0,6
22
23
29,7 7,59
635
20,4
0,6
23
C1c
0,5
0,6
23
23
20,2 7,44
677
21
1,2
28
C2a
1,4
1,3
30
29
50,2 7,83
769
20,6
0,3
15
C2b
0,5
0,4
15
15
42,1 8,02
771
20,3
1
21
C2c
1,3
1,2
20
20,5
39,4 7,98
844
20,8
0,9
21
D1a
0,8
0,9
19
20
24,5 7,76
631
20,7
0,8
20
D1b
0,7
0,8
26
23
24,9 7,78
618
21
0,9
28
D1c
0,6
0,8
21
24,5
23,5 7,65
613
21
40
1
D2a
1,5
9
1,3
10
9,5
46,9 8,22
2190
19,5
D2b
1,1
1,3
1,6
1,2
10
9
13
9,5
45,7 8,22
821
19,8
D2c
0,8
1,2
14
13,5
48,5 8,12
1118
19,6
Appendix 4: Measurement on 16.02.2015(first test run)
Samples pH
Conductivity(µs/cm) Temp°C
A1a
6,28
10004
18,5
A1b
A1c
A2a
A2b
A2C
6,81
5,92
7,25
6,99
7,73
964
1294
1295
1131
983
18,3
18,5
18,5
18,4
18,5
B1a
7,47
717
18,5
B1b
B1c
7,61
7,57
656
752
17,9
18,4
B2a
B2b
B2c
C1a
C1b
8,12
7,96
7,53
7,67
7,7
905
864
690
665
634
18,7
17,9
18,4
18,7
18,1
C1c
C2a
C2b
7,56
7,9
8,12
699
753
737
19,3
18,7
18,1
C2c
D1a
7,99
7,87
793
638
18,9
18,4
D1b
D1c
7,98
7,83
613
623
17,3
18,6
D2a
8,1
786
18,6
D2b
D2c
8,13
8,06
775
774
17,8
18,4
41
Appendix 5: Measurement on 19.2.2015(first test run)
Sample
A1a
A1b
A1c
A2a
A2b
A2C
B1a
B1b
B1c
B2a
B2b
B2c
C1a
C1b
C1c
C2a
C2b
C2c
D1a
D1b
D1c
D2a
D2b
Av.
PO43- PO4 3NO3Av. NO3ConducTemp
(mg/l) (mg/l)
N(mg/l) N(mg/l)
TN(mg/l) pH tivity(µs/cm) °C
30,4
50
30,5
30,5
45
47,5
63 5,95
1022 20,9
11,5
40
12,1
11,8
50
45
64,3 6,91
972 20,9
32,5
51
33,1
32,8
53
52
83,5 6,01
1060 21,4
40,8
74
41,1
41
65
69,5
80,8 7,74
1302 21,2
26,2
106
26,2
26,2
70
88
89,3 6,83
1209 20,6
26,4
61
26
26,2
64
62,5
85 6,94
985
21
3,6
33
3,3
3,5
28
30,5
40,3 7,93
901 20,8
2,6
26
2,8
2,7
27
26,5
34 7,97
864 20,4
5,4
25
5,5
5,5
25
25
32,5 7,86
748 20,8
10,2
23
10,2
10,2
22
22,5
51,6 8,04
900 20,9
9,9
32
10,1
10
40
36
48,3
8
868 20,5
1,8
24
1,7
1,8
26
25
33,6 8,01
693 20,9
1,5
26
1,7
1,6
26
26
25,7 8,04
671 21,1
1,8
20
1,6
1,7
21
20,5
13,6 8,1
642 20,8
3,4
26
3,6
3,5
30
28
21,8 7,9
734 21,3
0,9
49
0,9
0,9
52
50,5
28,1 8,27
732
21
2,3
11
2,2
2,3
9
10
26,3 8,1
764 20,9
1,8
11
1,9
1,9
11
11
22,9 7,83
775 21,2
1,3
17
1,1
1,2
18
17,5
12,5 8,08
643 21,2
1,9
16
2,1
2
16
16
16,6 8,06
626 20,9
2
14
2,1
2,1
12
13
15,3 8,07
629 21,3
1,8
9
1,8
1,8
13
10,5
39,7 7,97
789
21
1,2
1,2
9
8
46,3 7,93
787
21
42
D2c
1,2
1,4
1,3
1,4
7
8
8
8
32,8 7,99
750
Appendix 6: Measurement on 23.2.2015(first test run)
Sample
A1a
A1b
A1c
A2a
A2b
A2C
B1a
B1b
B1c
B2a
B2b
B2c
C1a
C1b
C1c
C2a
C2b
C2c
D1a
D1b
(mg/l)
(mg/l) Av.
(mg/l) Av.(mg/l) (mg/l)
ConducTemp
PO43- PO43(NO3- N NO3-N
TN
pH
tivity(µs7cm) °C
32,8
24
34,6
33,7
14
19
43 6,14
1133
20,9
15,2
20
15,2
15,2
14
17 28,4 7,16
989
21,3
37,2
28
35,6
36,4
28
28
43 6,25
1087
22,1
46,2
36
54,2
50,2
30
33
52 7,55
1320
20,8
30
50
29,2
29,6
36
43 51,2 6,34
1270
20,9
23,6
36
23,6
23,6
34
35 39,7
6,5
1019
21,6
4
9
3,7
3,85
9
9 28,9 7,68
731
20,8
4
1,1
3,8
3,9
7
4,05 30,1 7,64
729
20,9
3,9
9
4
3,95
7
8 33,5 8,16
799
21,9
10,5
7
10,4
10,45
7
7 38,2 8,18
862
20,9
8
34
8,1
8,05
30
32 54,4 8,26
820
20,8
3,9
7
3,7
3,8
6
6,5 17,3
8,4
698
21,7
2,4
3
2,2
2,3
6
4,5
19 8,08
678
20,9
2
4
1,9
1,95
3
3,5
21 8,19
634
20,8
1,6
5
1,7
1,65
4
4,5 22,1 8,05
767
21,6
1,3
3,4
1,3
1,3
3,7
3,55
18
8,1
735
21,1
2,3
2,6
2,3
2,3
2,6
2,6 22,3 8,09
751
21
1
9
0,8
0,9
8,9
8,95 22,1 8,29
671
21,5
1
3,9
1
1
3,7
3,8 10,9
8,4
629
20,9
1,5
7,2
1,5
1,5
7,4
7,3 10,4 8,38
610
20,8
21,2
43
D1c
D2a
D2b
D2c
1,4
1,3
1,1
1,3
1
1
1,2
1,3
5,7
5,7
8,1
8
4,8
4,5
24,4
24,4
1,35
1,2
1
1,25
5,7
11,9
8,42
625
21,7
8,05
32,5
8,27
722
20,6
4,65
30,7
8,24
727
21
24,4
24,6
8,64
651
21,7
Appendix 7: Measurement on 17.3.2015 (initial readings for second test run)
3-
PO4
Samples (mg/l)
Av. PO4
(mg/l)
A1
Group
19,8
A2
Group
18,9
B1
Group
10,2
B2
Group
10,8
C1
Group
3,6
C2
Group
3,7
D1
Group
8,9
D2
Group
8,6
3-
19,35
Av.
NO3 – N
(mg/l)
–
NO3
N(mg/l)
35
36,5
TN(mg/l) pH
90
36
10,5
23
22
54,8
21
3,65
19
18,5
48,7
18
8,75
17
17
17
Conductivity
Temp
(µs/cm) °C
45,5
7,25
1116
22,3
7,12
1083
21,2
7,4
826
21,4
7,38
818
21
7,48
790
22,1
7
813
22,2
7,03
1014
21
7,27
791
22,5
44
Appendix 8: Measurement on 19.3.2015(second test run)
(mg/l)
Av.
(mg/l) Av.
(mg/l)
(mg/l)
Conduc33Sample PO4
PO4
(NO 3 N) NO 3 N TN(mg/l) pH tivity(µs/cm) Temp°C
5
38
A1a
3,3
4,15
44
41
65 6,68
919
15,1
5,1
46
A1b
4,9
5
47
46,5
78 6,46
929
14,6
4
39
A1c
4
4
40
39,5
83,6 6,43
857
15,2
10,9
53
A2a
11,1
11
51
52
75,1 6,46
1045
14,8
4,4
49
A2b
4,2
4,3
52
50,5
76,4 7,29
856
14,5
5,6
43
A2C
5,5
5,55
41
42
81,9 6,94
951
16,1
1,7
26
B1a
1,9
1,8
25
25,5
32 6,83
681
16,2
1,3
34
B1b
1,3
1,3
34
34
39,7 6,79
708
15,6
1,5
26
B1c
1,2
1,35
27
26,5
31,9 6,43
678
15,9
2,9
30
B2a
3,6
3,25
32
31
43,3 6,43
764
15,7
2,7
50
B2b
2,9
2,8
51
50,5
46 6,57
703
14,9
2,9
35
B2c
3
2,95
37
36
42 6,4
747
16,3
1,7
40
C1a
1,8
1,75
41
40,5
49,3 6,47
653
16,5
1,9
36
C1b
1,9
1,9
36
36
33,3 6,36
682
15,7
1,3
12
C1c
1,9
1,6
14
13
25,3 7,06
692
16,8
1,7
52
C2a
1,9
1,8
53
52,5
40,9 7,13
683
17,4
2,1
17
C2b
1,8
1,95
16
16,5
40,2 7,9
727
16,3
1,8
57
C2c
1,7
1,75
57
57
29,8 7,52
675
18,2
1,8
11
D1a
2,1
1,95
10
10,5
30,7 7,52
794
18,2
1,4
10
D1b
1,4
1,4
12
11
41,9 7,87
705
17,3
1,2
27
D1c
1,9
1,55
30
28,5
34,9 6,71
656
17,9
2,2
49
D2a
1,9
2,05
48
48,5
44,9 7,43
680
18,4
D2b
2
1,95
40
40
41,7 6,9
687
17,9
45
D2c
1,9
1,5
1,7
1,6
40
55
53
54
35,6 6,82
682
18,5
Appendix 9: Measurement on 23.3.2015(second test run)
Samples
A1a
A1b
A1c
A2a
A2b
A2C
B1a
B1b
B1c
B2a
B2b
B2c
C1a
C1b
C1c
C2a
C2b
C2c
D1a
D1b
Conduc(mg/l) (mg/l)
(mg/l)
Av.(mg/l)
tivity(µs/c Temp
PO4 3- Av. PO43- (NO 3 -N)
NO3- N
TN(mg/l) pH m)
°C
6,5
72
6,6
6,55
74
73
69,8
6
913
20,4
4,9
61
4,9
4,9
72
66,5
93,6 5,9
981
20,7
3,7
51
3,7
3,7
51
51
47,6 6,2
871
20,5
8,5
66
8,3
8,4
63
64
100 7,8
1029
20,2
3,9
48
3,7
3,8
49
48,5
43,7 8,2
846
20,2
7,2
67
7,3
7,25
55
61
93,4
6
1004
20,9
1,7
46
1,6
1,65
47
46,5
34,7 5,9
717
20,2
2,2
47
2,2
2,2
48
47,5
46,9 5,6
792
20
1,6
25
1,5
1,55
25
25
26,1 6,8
701
20,9
1,6
29
1,6
1,6
30
29,5
39 5,8
793
20
1,5
69
1,4
1,45
54
61,5
31,9 7,4
736
19,8
1,4
36
1,4
1,4
36
36
57 6,1
796
20,7
1,2
52
1,2
1,2
55
53,5
33,5 5,6
701
20,2
1,2
45
1,4
1,3
45
45
63,9 5,5
776
20,4
1,3
14
1,3
1,3
13
13,5
20,4 7,5
638
20,8
1,2
39
1,1
1,15
41
40
23,3 7,6
654
20,1
0,8
15
0,8
0,8
15
15
29 8,3
729
20
1,2
38
1,2
1,2
36
37
22,2 7,7
681
20,6
0,8
33
0,7
0,75
32
32,5
27,6 7,6
598
20,1
1,2
36
1,4
1,3
36
36
50,4 7,5
585
19,8
46
D1c
D2a
D2b
D2c
0,6
0,5
1,2
1,4
0,6
0,5
0,6
0,8
0,55
1,3
0,55
0,7
38
35
36
34
25
25
37
36
36,5
35,5
5,5
700
20,6
35
32,6
7
697
20,2
25
23
7
728
20,1
36,5
35,6
7,2
707
20,8
Appendix 10: Measurement on 26.3.2015(second test run)
Sample
A1a
A1b
A1c
A2a
A2b
A2C
B1a
B1b
B1c
B2a
B2b
B2c
C1a
C1b
C1c
C2a
C2b
C2c
(mg/l)
Av.
(mg/l) Av.
(mg/l)
(mg/l)
ConducTemp
PO43- PO43(NO3-N)
NO3 - N
TN(mg/l) pH tivity(µs/cm) °C
4,2
37
5,6
4,9
41
39
57,2
6
957
20,1
4,5
46
4,3
4,4
43
44,5
74,9
6
1057
20
2
29
2
2
29
29
65,4
7
906
19,8
4,8
45
4,5
4,65
47
46
75,5
8
1060
20,3
1,9
37
2
1,95
40
38,5
42,7
8
844
19,9
5,8
69
6
5,9
67
68
72,6
5
1073
20,2
1,2
37
1,3
1,25
38
37,5
39,4
5
800
20,1
1,5
43
1,5
1,5
39
41
43,8
6
875
19,7
1,1
46
1,1
1,1
52
49
27,1
6
743
20,5
1
25
1,2
1,1
24
24,5
41,8
6
820
20,1
4
39
3
3,5
37
38
31,6
8
753
20
0,9
45
0,7
0,8
45
45
49,5
6
824
20,3
0,7
35
0,7
0,7
38
36,5
41,4
6
741
20,5
0,8
45
0,9
0,85
49
47
47,1
6
872
20,2
0,6
23
0,4
0,5
22
22,5
21
7
615
20,6
1,1
47
1
1,05
47
47
30
8
673
19,9
1,8
23
2,1
1,95
21
22
41,3
8
766
19,8
0,9
15
0,9
0,9
16
15,5
34,4
8
675
20,5
47
D1a
D1b
D1c
D2a
D2b
D2c
0,5
0,5
0,7
0,7
0,6
0,5
1
1
1
0,8
1,1
1
0,5
0,7
0,55
1
0,9
1,05
18
18
13
15
31
30
49
47
35
35
34
39
18
25,7
8
590
19,9
14
26,3
8
572
19,6
30,5
52,6
5
762
20,4
48
38
7
737
20,2
35
56,9
7
771
19,8
36,5
37,9
7
723
20,6
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