Cleaning water with diatoms
A study of Gomphonema parvulum as a part of a
cleaning system for waste water from a land-based
fish farm
Olof Bengtsson
Degree project for Bachelor of Science in
Biology
BIO602 Biology: Degree project 15 hec
Spring 2016
Department of Biological and Environmental Sciences
University of Gothenburg
Examiner: Thomas Appelqvist
Department of Biological and Environmental Sciences
University of Gothenburg
Supervisor: Angela Wulff
Department of Biological and Environmental Sciences
University of Gothenburg
Front page image by Adil Y. Al-Handal
Cleaning water with diatoms
-a studie of Gomphonema parvulum as a part of a cleaning system for waste
water from a land-based fish farm
Olof Bengtsson
Bio 602 Degree project for Bachelor of Science in Biology, 15 hec
Supervisor: Angela Wulff1
1
Department of Biological and Environmental Sciences, University of Gothenburg, Box 461, SE 405 30 Göteborg
Sammanfattning: Landbaserade fiskodlingar är idag ett alternativ till traditionella odlingar i hav och
sjöar, med färre negativa konsekvenser som t.ex. eutrofiering. För att förhindra övergödning av anslutande
vattendrag måste näringsämnen från fiskodlingsvattnet avlägsnas innan det släpps ut. Ett steg i en sådan
process kan vara att odla mikroalger på utgående vatten. I ett experiment utfört mellan 25 april och 10 maj
2016 vid Göteborgs universitet odlades den bentiska sötvattenskiselalgen Gomphonema parvulum med tre
olika behandlingar. De bestod av tre olika medium, avfallsvatten från en gösodling med en salthalt på 3,6,
WC-medium (ett optimalt odlingsmedium för sötvattenskiselalger), samt på avfallsvatten med tillsatt
näring i samma mängd som WC-mediet. Faktorer som undersöktes var tillväxt, fotosyntetisk aktivitet,
fotosyntetiska pigment och bakteriebiomassa. Vattenprover skickades på extern analys för oorganiska
näringsämnen. Tester vid experimentets början, efter 8 dagar samt vid experimentets slut - efter 15 dagar,
med 4 äkta replikat per behandling och tillfälle. En knapp tillväxt hade skett i fiskodlingsvattnet efter 8
dagar och efter 15 dagar hade cellkoncentrationen sjunkit till omkring en femtedel av den ursprungliga.
Även i fiskodlingsvattnet med tillsatta näringsämnen observerades liknande resultat. Algerna som hade
odlats på WC-medium visade däremot en tydlig tillväxt både efter 8 och 15 dagar. Detta antyder att
fiskodlingsvattnet är ogynnsamt för odling av den undersökta kiselalgen och att det troligen inte beror på
näringsbrist. Skillnaden i salthalt kan möjligtvis förklara att tillväxten var sämre i fiskodlingsvattnet, men
är så pass liten att den inte kan förklara en minskad cellkoncentration. Resultaten pekar på att något i
fiskodlingsvattnet får algerna att dö. För att utröna vad detta är krävs en framtida studie av avfallsvattnet.
En möjlig ingång kan vara att analysera närvaro av parasiter och virus. Även skadliga halter av metaller
kan tänkas spela en roll.
Abstract: Today land based fish farms are an alternative to traditional farms in oceans and lakes, with
fewer negative consequences, i.e. eutrophication. To prevent overfertilization of connecting bodies of
waters, nutrients in the water from the fish farm has to be removed before it is released. A step in such a
process could be to grow microalgae on the outgoing water. In an experiment performed at the University
of Gothenburg between the 25th of April and the 10th of May 2016, the benthic freshwater diatom
Gomphonema parvulum was grown with three different treatments. They consisted of three different
media, waste water from a Zander farm with a salinity of 3.6, WC-medium (a medium for optimal growth
of freshwater diatoms) and waste water with nutrients added to it in the same amounts as for the WCmedium. Investigated factors were growth, photosynthetic activity, photosynthetic pigments and bacterial
biomass. Water samples were sent externally for analysis of inorganic nutrients. Tests were taken at the
beginning of the experiment, 8 days into the experiment and at the end – 15 days into the experiment, with
4 true replicates per treatment and occasion. Minor growth was observed in the waste water after 8 days
and after 15 days the cell concentration had decreased to a fifth of the original. Similar results were
observed in the waste water with added nutrients. On the other hand, algae grown on WC-medium had
grown well after 8 days as well as 15 days. This implies that the water from the fish farm is unfavorable
for the investigated diatom and that it is probably not due to nutrient limitation. The difference in salinity
could explain an impeded growth rate in the water from the fish farm but is too small to explain the
decrease in cell concentration. The results imply that something in the water from the fish farm causes
algae to die. To find out what this could be, a study of the waste water has to be performed in the future. A
possible start could be to analyze presence of virus and parasites. Harmful levels of metals could also be a
potential factor.
1. Introduction
Aquacultures with the purpose of growing fish is on a steady rise due to the ever increasing demand of
food sources in a worldwide growing demographic (Hall, 2011). By the year 2014 the amount of farmed
fish intended for human consumption matched the amount of fish caught by fisheries (F.A.O., 2016).
According to Hall (2011), aquaculture is considered less harmful to the environment than agriculture, but
that is not to say that it is completely without any negative consequences. Traditional fish farming in
natural bodies of waters, such as oceans and lakes, has sparked a lot of controversy in recent years. This is
due to the negative impacts that they can have on the surrounding environment, i.e. by sedimentation of
fecal matter and nutrients on the sea bottom affecting the biota (Wu, 1995; Iwama, 1991; Pérez, et al.,
2008). Another critique of fish farms is the often heavy use of antibiotics to prevent infections within
them, which could lead to multi resistant bacteria (Cabello, 2006; Lalumera, et al., 2004). Therefore, land
based fish farms are now being considered by some companies as a more environmental friendly option
(Tal, et al., 2009; Avnimelech, et al., 2008). To prevent eutrophication in connected water bodies due to
an excess of nutrients in the outflowing water from the fish farm, this water has to be cleaned somehow.
This is often done with different filter systems (Tal, et al., 2009; Zhang, et al., 2011). A potential stage in
such a system could be to grow microalgae on waste water from the fish farm, using the nutrient rich
water as a growth medium for the algae (Lefebvre, et al., 1996).
Diatoms are unicellular microalgae belonging to the class Bacillariophyceae. They range in size between 1
µm to 2 mm (Hasle, et al., 1996; Parkinson & Gordon, 1999). There are over 250 genera of diatoms
(Round, et al., 1990) and anywhere between 20,000 to 200,000 species (Guiry, 2012). They are found all
around the globe in almost any body of water, marine and fresh (Round, et al., 1990) – even in soil
(Patrick, 1977) - and in most niches, planktonic (Guillard & Kilham, 1977) as well as benthic and
epiphytic (Moore, 1977; Patrick, 1977). The most characteristic feature of diatoms is the rigid siliceous
cell wall, the frustule (Hasle, et al., 1996). The frustule consists of two valves, one larger (epivalve) and
one smaller (hypovalve), fitting in to each other very much like a petri dish (Parkinson & Gordon, 1999).
Depending on the symmetry of the frustule, diatoms are divided in to two groups, centric (disc shaped)
and pennate (rod shaped) (Hasle, et al., 1996). The vast majority of the diatom species are autotrophic and
plays a fundamental role in the ecosystem. It is estimated that they are contributing with 20-25% of the net
world primary production (Mann, 1999; Werner, 1977). As most autotrophic organisms, diatoms contain
chlorophyll a, but also more specific pigments such as fucoxanthin, diatoxanthin and diadinoxanthin
(Hasle, et al., 1996). Diatoms are often used as indicators to monitor water quality as different species are
tolerant or sensitive to different factors (Van Dam, 1994). There are also fields where diatoms can be used
for industrial purposes, such as fertilizers or as a potential source for biofuel (Chaffin, et al., 2012). There
are even some research being done on how the silica-shell of diatoms can be utilized in nanotechnology
(Gordon, et al., 2009; Leonardo, et al., 2016; Parkinson & Gordon, 1999). Growing diatoms on waste
water could therefore create a win-win situation where nutrients are removed from the water by the
diatoms which later can be harvested and exploited.
This experiment was conducted with the primary object to find out whether a specific benthic freshwater
diatom species could be successfully grown on waste water with low salinity from a land based fish farm.
This would hopefully give insight in what factors are affecting the growth and health of the cells and
ultimately open up entries for further studies. The diatom Gomphonema parvulum was selected for the
experiment where it was grown on the waste water. For comparison it was also grown on a medium well
suited for freshwater diatom growth, a so called WC-medium (Guillard & Lorenzen, 1972) and waste
water with the same amount of nutrients as the WC-medium added to it. The experiment was performed
during two weeks inside a thermoconstant room with a controlled light cycle. To compare growth between
the media, cell concentration was estimated through manual counting. Concentration of photosynthetic
pigments were also used as an indirect measurement of biomass. To monitor the state of the algae,
photosynthetic activity was measured using fluorometry. Nutrient concentration and bacterial biomass
were also measured to get further insight in what factors might affect the growth of the diatom.
2. Material and methods
2.1 Experimental design
To see if the diatom could be successfully grown on waste water from a fish farm, three different
treatments were chosen. The treatments consisted of three different media on which the algae were grown,
namely waste water from the fish farm (Fish Water), WC-medium prepared form Milli-Q water (WC) and
WC-medium prepared from waste water instead of Milli-Q water (WC-Fish) (Figure 1). Waste water was
provided by Svensk Fiskodling, a fish farming company based in Järfälla, Sweden. They have a facility on
Ljuströ, an island in the Stockholm archipelago, where they are farming zander. Water going into the farm
is collected from their own private well in the vicinity and outgoing water goes through a sand filter called
Dynasand. The volume of water in exchange to and from the farm is 10 m3 per day, which constitutes 10%
of the total volume of the water in the farm. The tank in which the fish are farmed are pressure washed, no
biocides are used. It had a salinity of 3.6 when measured with a GMH 3431 (GHM Greisinger). The WCmedium was chosen because fresh water diatoms are known to grow well on it (Guillard & Lorenzen,
1972). This was also the medium on which the diatom had been cultured prior to the experiment. The last
treatment with WC-fish was chosen to investigate if nutrient deficiency could be a factor in potential
differences in growth. To avoid contamination, the media were filtered through a 0.7 µm filter
(Whatman™ GF/F-glass microfiber filter with a diameter of 47 mm) before algae were added.
Each treatment was incubated during 0 (initial tests), 8 and 15 days. The incubation was done with four
true replicates per treatment and incubation time (Figure 1). Each replicate consisted of 60 ml medium
with a starting concentration of approximately 2.0*106 cells/l. They were kept in 160 ml culture flasks
(Sarstedt TC flask T-75, suspension with a vented cap) which were placed lying down to maximize
growth surface area (Figure 2).
Since this diatom is a benthic species, the replicate cultures had to be suspended before tests were done to
get an even distribution of the algae throughout the culture. This was done by shaking the culture flasks
for at least 10 seconds immediately before samples were taken.
Figure 1. Experiment set-up where colored rectangles are representing culture flasks. Yellow represents fish water, red
WC-medium and blue WC-fish.
Figure 2. Picture of culture flasks on a surface in a thermo-constant room by the beginning of the experiment.
2.2 Experimental conditions
The experiment was conducted in a thermo-constant room holding a temperature of 14°C which only
occasionally fluctuated down to 13°C. This temperature was chosen because it is the average temperature
in the fish farm.
Cultures were kept on a surface that was exposed to light with an intensity ranging between 130-138 µmol
photons m-2 s-1, measured with a Biospherical Instruments Inc. QSL-2101 Scalar PAR Irradiance Sensor.
The light:dark cycle in the room was 16:8 h. Logging of temperature and light cycle was done with a
HOBO® temp/light pendant.
2.3 Growth
2.3.1 Gomphonema parvulum
To calculate the growth rate (µ) the following formula was used: µ = (ln Cx - ln C0)/(tx - t0), where Cx
represents cell concentration at time x (tx) and C0 represents the initial cell concentration by the start of the
experiment (t0). Determining the cell concentration was done by manual counting, using a Sedgwick
counting chamber (Figure 3) and an inverted light microscope. A 3 ml sample was taken from each
replicate and was fixated with two drops of acidified Lugol’s solution. Samples were then stored in
darkness at room temperature before being counted. To estimate cell concentration, 1 ml of a fixated
sample was pipetted onto the counting chamber, which is divided into 1000 1 µl squares. Squares where
then chosen at random and the number of cells within them was counted until at least 300 cells was
counted in total. In samples where cell density was as low as 2-3 cells per square 100 squares or more
were counted instead. This could then be used to get an estimate of cells per liter. In cases when the cell
density was too high to distinguish individual cells, the sample was diluted with deionized water until a
cell density of around 10 cells per square was reached. In cases were contamination was present in the
sample, the contaminating cells were counted in the same way as the diatoms.
Figure 3. A Sedgwick counting chamber, ca 8 cm long.
2.3.2 Bacteria
To estimate concentration of bacteria, counting through flow cytometry was used. 1 ml samples were
fixated with 0.2 ml glutaraldehyde (final concentration of glutaraldehyde – 1:6) and then stored in -80 °C
before being analyzed. To limit noise during data acquisition, the glutaraldehyde was filtered through a
0.2 µm filter before being added to the sample. Analysis was done with a FACSCalibur (BD Biosciences)
flow cytometer and CellQuest (BD Biosciences) software. Samples were mixed with 5 µl of SYBRGreen,
a DNA-binding dye which makes bacteria detectable in the flow cytometer, and dark adapted for 10
minutes. To determine flow rate 10 µl of CountBright absolute counting (Life technologies) was added to
the samples immediately before being analyzed. This is a solution containing 0.54*105 microscopic
fluorescent beads per 50 µl, which makes it able to use as an internal standard.
The flow cytometer has sensors within it that detects light scattered from particles that are passing through
a laser beam. Particles of different size and shape scatter light differently and can therefore be
distinguished. Particles of similar size and shape will be forming clusters of dots when plotted in diagram
by the software and can thus be counted (Figure 4).
Figure 4. Example of a diagram in CellQuest, where particles detected in a flow cytometer are plotted as red dots. Light
colored areas represent a high density of particles.
2.4 Photosynthetic activity
Photosynthetic activity was measured to get an estimate of the state of the diatoms. This was done on the
assumption that cells will be able focus their activity on photosynthesis and growth more in favorable
conditions than when exposed to stress.
Measurements of photosynthetic activity were done by adding a 1 ml sample from each replicate into a
WALZ CUVETTE version Water-PAM fluorometer. Light that is not used for photosynthetic processes
within the chloroplasts is emitted as fluorescence or heat. Therefore, by measuring fluorescence in a
sample before and after it has been treated with a short saturation pulse, a value known as photochemical
quantum yield of photosystem II (PSII) can be acquired. If the sample has been dark-acclimated before the
measurement, maximum photochemical quantum yield of PS II (Ym) can be calculated through FV/FM,
where FV is calculated by FM – F0. FM is maximum fluorescence and is induced by a saturating light that
closes all reaction centers of PSII. F0 is the minimum fluorescence and is acquired by a low intensity
measuring light that keeps the reaction centers of PSII open. Effective photochemical quantum yield (Ye)
is acquired if the sample has been kept illuminated before the measurement and calculated by (FMʹ – F) /
FMʹ. FMʹ is relatively lower than FM due to non-photochemical quenching of PSII by the surrounding light.
F is the fluorescent level of the sample under current light conditions. Ye was calculated for replicates
from day 0 and Ym was calculated for replicates from day 8 and 15. When Ym was measured, samples
were dark-acclimated for at least 30 min before being tested.
2.5 Photosynthetic pigments
Concentration of chlorophyll a (chl. a) was used as an indirect measurement of diatom biomass. To make
sure that it was indeed chl. a from diatoms and not from other, contaminating algae, concentration of
fucoxanthin was measured since this is a pigment not found in green algae or cyanobacteria (Wright &
Jeffrey, 1987). The ratio of fucoxanthin/chlorophyll was calculated to see if cultures suspected of being
contaminated differed significantly from pure ones.
To analyze photosynthetic pigments, a certain volume of each replicate, ranging between 15 - 128 ml, was
run through a Whatman™ GF/F-glass microfiber filter with a diameter of 25 mm until a desirable
coloration of the filter was reached (Figure 5). Replicates from the same treatment were pooled if the
coloration was not satisfactory for one replicate alone. The filter was then stored in -80°C before analysis.
Pigments were extracted from the filters with 1.5 ml methanol and acetone (80:20). The extracted samples
were then ultra-sonicated for 45 seconds (Vibra Cell VC130). The high performance liquid
chromatographic (HPLC) analyses took place in a Shimadzu SPD-M20A absorbance diode-array detector
and was performed according to Wright & Jeffrey, (1997). Pigments are expressed as concentrations (ng
cell-1).
Figure 5. GF/F filter with a desirable coloration.
2.6 Inorganic nutrients
In order to determine whether different results between treatments could be due to differences in nutrient
levels, a sample of water from each replicate was taken and sent for analysis at Sven Lovén Centre for
Marine Sciences in Kristineberg. 2*10 ml from each replicate was extracted with a syringe and filtered
through a Sarstedt Filtropur S 0.2 µm filter. Samples were kept in -20 °C before being sent for analysis.
Due to an unforeseen delay of the deliverance to Kristineberg, the samples were thawed before arrival.
Upon arrival the samples were again stored in -20 °C before being analyzed. Levels of the following
inorganic nutrients were analyzed: Ammonium (NH4), nitrite (NO2) + nitrate (NO3), phosphorous (PO4)
and Silica (SiO2). This was done using colorimetric methods in accordance with Grasshoff et al. (1999).
2.7 Statistical analysis
Cochran’s test for homogeneity of variance was done on data sets in Microsoft Excel before being
analyzed with a univariate analysis of variance. Level of significance was set to α = 0.05. If the variance
was heterogenetic, the original values were log transformed. Univariate analysis of variance was done in
SPSS to see if there were significant differences between any of the treatments. Null hypothesis of there
being no significant differences between treatments was rejected when p < 0.05. Tukey HSD Post Hoc test
was used to determine which treatments that differed from each other, again differences were considered
significant when p < 0.05.
3. Results
3.1 Growth
3.1.1 Gomphonema parvulum
Cell concentrations are presented as means and are given in cells per liter followed by standard deviations.
Initial cell concentration for all treatments were 2.0*106 cells/l. This number was estimated by adding
culture with a known cell concentration to the media, not by manual counting of the initial samples.
Therefore it was not calculated as a mean and is not presented with standard deviations.
After 8 days there was a significant difference in cell concentration between the treatments (F(2, 9) =
119.047, p = 0.000). Data was log transformed for homogenate variances but were not normally
distributed. In a post hoc test, differences between WC and WC-Fish (p = 0.000), and WC and Fish Water
(p = 0.000) showed to be significant. There was no significant difference between WC-Fish and Fish
Water (p = 0.292). Fish Water had the lowest mean concentration of diatoms - 2.6*106 ± 1.6*105 cells/l.
The mean concentrations of WC and WC-Fish were 3.0*107 ± 8.6*106 cells/l and 3.5*106 ± 1.1*106 cells/l
respectively (Figure 6). After 15 days there was a significant difference in cell concentration between the
treatments (F(2, 6) = 534.426, p = 0.000). Data was log transformed for homogenate variances but were not
normally distributed. In a post hoc test, differences between all treatments were shown to be significant,
Fish Water and WC (p = 0.000), WC and WC-Fish (p = 0.000), and Fish Water and WC-Fish (p = 0.003).
Both Fish Water and WC-Fish had a lower cell concentration than the initial one. The Fish Water had a
mean concentration of 0.7*106 ± 0.6*105 cells/l – the lowest of the three media. WC and WC-Fish had
mean concentrations of 4.0*107 ± 7.2*106 cells/l and 1.4*106 ± 2.6*105 cells/l respectively (Figure 6).
Growth rates are presented as means and are given in divisions per day followed by standard deviations. A
significant difference in growth rate was observed between the treatments after 8 days (F(2, 9) = 119.066, p
= 0.000). Data was not normally distributed. In a post hoc test differences between WC and Fish Water (p
= 0.000), and WC and WC-Fish (p = 0.000) was shown to be significant, but not between Fish Water and
WC-Fish (p = 0.291). Algae grown on Fish Water had the lowest growth rate with a mean growth rate of
0.028 ± 0.0077 divisions/day. In WC and WC-Fish growth rates were 0.33 ± 0.032 division/day and 0.063
± 0.042 divisions/day respectively (Figure 7).
All treatments had slower growth rates from day 0 to day 15 than from day 0 to day 8. There was a
significant difference in growth rate between the treatments at day 15 (F(2, 6) = 534.426, p = 0.000). Data
was not normally distributed. In a post hoc test there showed to be significant differences between all
treatments, Fish Water and WC (p = 0.000), WC and WC-Fish (p = 0.000), and Fish Water and WC-Fish
(p = 0.003). Algae grown on Fish Water and WC-Fish had negative growth rates after 15 days. The mean
growth rates in these two media were -0.075 ± 0.0061 divisions/day and -0.024 ± 0.013 divisions/ day
respectively. The algae grown on WC had a growth rate of 0.20 ± 0.012 divisions/day (Figure 7).
cells/l *107 Mean concentra1on of G. parvulum 5 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0 D8 Fish Water D8 WC D8 WC Fish D15 Fish D15 WC D15 WC Water Fish Figure 6. Mean concentration of algae (cells/l) in different media at day 8 (D8) and at day 15 (D15). Error bars show
standard deviations. The colors of the bars represent different media. Yellow – Fish Water, red – WC and blue – WCFish.
divisions/day Mean growth rate of G. parvulum 0,4 0,35 0,3 0,25 0,2 0,15 0,1 0,05 0 -­‐0,05 -­‐0,1 -­‐0,15 D8 Fish Water D8 WC D8 WC Fish D15 Fish D15 WC D15 WC Water Fish Figure 7. Mean growth rates (divisions/day) of algae grown in different media after 8 (D8) and 15 (D15) days. Error bars
show standard deviations. The colors of the bars represent different media. Yellow – Fish Water, red – WC and blue –
WC-Fish.
3.1.2 Bacteria
When plotted in CellQuest, bacteria formed clusters in different areas of the diagram depending on
medium (Figure 8). This was interpreted as an indication of it being different species of bacteria in the
different treatments. Therefore cell concentrations are only compared within – not between - the
treatments.
Mean concentrations of bacteria are presented as cells/l followed by standard deviations. Mean
concentration of bacteria in Fish Water was 3.9*106 ± 1.6*106 cells/l on day 0, 3.9*107 ± 1.4*107 cells/l on
day 8 and 8.8*107 ± 7.2*107 cells/l on day 15 (Figure 9). In WC mean concentration of bacteria was
6.3*105 ± 3.9*104 cells/l on day 0, 6.1*106 ± 1.9*105 cells/l on day 8 and 9.2*106 ± 6.6*105 cells/l on day
15 (Figure 10).
In WC-Fish the mean concentration of bacteria was 1.5*106 ± 1.3*105 cells/l on day 0 and 4.9*107 ±
1.8*107 cells/l on day 8. On day 15 it was 3.9*107 ± 1.8*107 cells/l (Figure 11).
Figure 8. Diagrams over three different media, acquired from CellQuest where bacteria detected in a flow cytometer are
plotted as red dots. Light colored areas represent a high concentration of bacteria. From left to right: Fish Water, WC and
WC-Fish.
Mean concentra1on of bacteria -­‐ Fish Water 18 16 cells/l * 107 14 12 10 8 6 4 2 0 Init D8 D15 Figure 9. Mean concentrations of bacteria (cells/l) in Fish Water at day 0 (Init), day 8 (D8) and day 15 (D15). Error bars
show standard deviations.
Mean concentra1on of bacteria -­‐ WC 1,2 cells/l * 107 1 0,8 0,6 0,4 0,2 0 Init D8 D15 Figure 10. Mean concentrations of bacteria (cells/l) in WC at day 0 (Init), day 8 (D8) and day 15 (D15). Error bars show
standard deviations.
Mean concentra1on of bacteria -­‐ WC-­‐Fish 8 cells/l * 107 7 6 5 4 3 2 1 0 Init D8 D15 Figure 11. Mean concentrations of bacteria (cells/l) in WC-Fish at day 0 (Init), day 8 (D8) and day 15 (D15). Error bars
show standard deviations.
3.1.3 Bacteria/Gomphonema parvulum
Concentration of G. parvulum and concentration of bacteria were compared by determining the ratio of
bacteria/diatom at the three different days for each medium. Again comparison was only done within and
not between the different media.
In Fish Water the mean bacteria/diatom ratio was 1.9 ± 0.77 on day 0, 13 ± 2.0 on day 8 and 150 ± 110 on
day 15 (Figure 12). The mean bacteria/diatom ratio in WC was 0.31 ± 0.019 on day 0, 0.21 ± 0.049 on day
8 and 0.23 ± 0.054 on day 15 (Figure 13). The mean bacteria/diatom ratio in WC-Fish was 0.72 ± 0.061
on day 0, 14 ± 6.5 on day 8 and 27 ± 12 on day 15 (Figure 14).
Mean bacteria/diatom ra1o -­‐ Fish Water 300 bacteria/diatom 250 200 150 100 50 0 Init D8 D15 Figure 12. Mean bacteria/diatom ratio in Fish Water at three different occasions, on day 0 (Init), day 8 (D8) and day 15
(D15). Error bars show standard deviations.
Mean bacteria/diatom ra1o -­‐ WC 0,35 bacteria/diatom 0,3 0,25 0,2 0,15 0,1 0,05 0 Init D8 D15 Figure 13. Mean bacteria/diatom ratio in WC at three different occasions, on day 0 (Init), day 8 (D8) and day 15 (D15).
Error bars show standard deviations.
Mean bacteria/diatom ra1o -­‐ WC-­‐Fish 45 bacteria/diatom 40 35 30 25 20 15 10 5 0 Init D8 D15 Figure 14. Mean bacteria/diatom ratio in WC-Fish at three different occasions, on day 0 (Init), day 8 (D8) and day 15
(D15). Error bars show standard deviations.
3.1.4 Contamination
When manually counted there were some presence of contamination in WC-Fish on day 8 and day 15.
What species the contaminating cells belonged to could not be determined, but they were thought to be
some kind of unicellular chlorophyte. The state of these cells was not well and looked partly lysed (Figure
15). On day 8 the mean concentration of contaminating cells was 2.5*106 ± 1.1*106 cells/l, and on day 15
the mean concentration was 1.4*106 ± 3.6*105 cells/l (Figure 16). To compare number of contaminating
cells with number of G. parvulum a ratio (contaminating cells/diatom) was calculated. On day 8 there was
0.77 ± 0.33 contaminating cells/diatom and on day 15 there was 0.96 ± 0.16 contaminating cells/diatom
(Figure 17). An unknown amount of cyanobacteria was also observed in these cultures, but were hard to
distinguish from cracks and dust on the counting chamber and were too small to be counted manually.
Figure 15. Image of a contaminating cell (right) next to a diatom (left) fixated in Lugol’s Solution under a light
microscope.
Mean concentra1on of contamina1ng cells 4 3,5 cells/l * 106 3 2,5 2 1,5 1 0,5 0 D8 D15 Figure 16. Mean concentration of contaminating cells (cells/l) in WC-Fish at day 8 (D8) and day 15 (D15). Error bars show
standard deviations.
Mean contamina1ng cell/diatom ra1o WC-­‐Fish contamina1ng cell/diatom 1,2 1 0,8 0,6 0,4 0,2 0 D8 D15 Figure 17. Mean contaminating cell/diatom ratio in WC-Fish at day 8 (D8) and day 15 (D15). Error bars show standard
deviations.
3.2 Photosynthetic activity
Photosynthetic activity is presented as mean photochemical quantum yield – either effective (Ye) or
maximum (Ym) - followed by standard deviation.
On day 0 there were no significant differences in effective photochemical quantum yield between the
media (F(2, 9) = 0.477, p = 0.636). The data was not normally distributed. Mean Ye was 0.0065 ± 0.0041 for
algae in Fish Water. For algae in WC and WC-Fish Ye was 0.012 ± 0.012 and 0.0078 ± 0.0076
respectively (Figure 18). On day 8 there was a significant difference in maximum photochemical quantum
yield between the media (F(2, 9) = 58.980, p = 0.000). The data was not normally distributed. In a post hoc
test the differences between WC and the two other media were shown to be significant (p = 0.000), but not
between Fish Water and WC-Fish (p = 0.949). Algae grown on Fish Water and WC-Fish had mean values
of Ym that were less than half of that from the algae grown on WC. For algae grown in Fish Water Ym was
0.14 ± 0.034. For algae grown in WC and WC-Fish Ym was 0.32 ± 0.011 and 0.14 ± 0.032 respectively
(Figure 19). After 15 days there was a significant difference in maximum photochemical quantum yield
between the media (F(2, 9) = 9.266, p = 0.007). The data did not pass Cochran’s test for homogeneity of
variance, even when it was log transformed and square root transformed. In the end untransformed data
was used. In a post hoc test, there was found to be a significant difference only between WC and Fish
Water (p = 0.005), not between Fish Water and WC-Fish (p = 0.126) and WC and WC-Fish (p = 0.142).
Algae grown on Fish water had again the lowest value of Ym of the three media - 0.23 ± 0.095. For algae
grown in WC and WC-Fish Ym was 0.41 ± 0.016 and 0.32 ± 0.017 respectively (Figure 19).
Mean effec1ve yield -­‐ day 0 0,03 0,025 Yield 0,02 0,015 0,01 0,005 0 Fish Water WC WC-­‐Fish Figure 18. Mean effective photochemical quantum yield of algae grown in three different media at day 0. Error bars show
positive standard deviations. . The colors of the bars represent different media. Yellow – Fish Water, red – WC and blue –
WC-Fish.
Mean maximum Yield 0,45 0,4 0,35 Yield 0,3 0,25 0,2 0,15 0,1 0,05 0 D8 Fish Water D8 WC D8 WC Fish D15 Fish D15 WC D15 WC Water Fish Figure 19. Mean maximum photochemical quantum yield of algae grown in three different media at day 8 (D8) and day 15
(D15). Error bars show standard deviations. . The colors of the bars represent different media. Yellow – Fish Water, red –
WC and blue – WC-Fish.
3.3 Photosynthetic pigments
On day 0 all replicates from Fish Water and WC had to be pooled to get a good enough coloration
(enough biomass). The mean concentration of photosynthetic pigments for these two treatments are
therefore only based on one value, hence they have no standard deviations. Replicates from WC-Fish were
pooled two by two on day 0 to get a satisfactory coloration of the filters.
3.3.1 Chlorophyll a
Mean concentrations of chl. a are given in µg/l followed by standard deviations. The mean concentration
of chl. a on day 0 was 0.98 µg/l in Fish Water. In WC and WC-Fish the mean concentration of chl. a was
1.63 µg/l and 1.39 ± 0.0056 µg/l respectively (Figure 20). On day 8 there was a significant difference in
concentration of chl. a between the media (F(2, 9) = 23.743, p = 0.000). The data was not normally
distributed. In a post hoc test differences between WC and Fish Water (p = 0.000) and WC and WC-Fish
(p = 0.001) were significant but not between Fish Water and WC-Fish (p = 0.516). Mean concentration of
chl. a was– 4.1 ± 0.43 µg/l in Fish Water. In WC and WC-Fish the mean concentration of chl. a was 21 ±
5.5 µg/l and 7.0 ± 3.3 µg/l respectively (Figure 20).
On day 15 there was a significant difference in concentration of chl. a between the media (F(2, 9) = 278.306,
p = 0.000). Data was log transformed for homogeneity, but was not normally distributed. In a post hoc test
differences between WC and Fish Water (p = 0.000), and WC and WC-Fish (p = 0.000) were shown to be
significant but not between Fish Water and WC-Fish (p = 0.572). Fish Water had a mean concentration of
1.26 µg/l. In WC and WC-Fish the mean concentration of chl. a was 44 ± 14 µg/l and 1.5 ± 0.36 µg/l
respectively (Figure 20).
Mean concentra1on of chl. a 70 60 µg/l 50 40 30 20 10 0 Init Init Init Fish WC WC Water Fish D8 D8 D8 Fish WC WC Water Fish D15 D15 D15 Fish WC WC Water Fish Figure 20. Mean concentrations (µg/l) of chlorophyll a in three different media at day 0 (Init), day 8 (D8) and day 15
(D15). Error bars show standard deviations. Values for Init Fish and Init WC are based solely on one value each and thus
have no error bars. The colors of the bars represent different media. Yellow – Fish Water, red – WC and blue – WC-Fish.
3.3.2 Fucoxanthin
Mean concentrations of fucoxanthin are given in µg/l followed by standard deviations. On day 0 mean
concentration of fucoxanthin was 0.65 µg/l in Fish Water, 0.75 µg/l in WC and 0.74 ± 0.01 µg/l in WCFish (Figure 21). On day 8 there was a significant difference in concentration of fucoxanthin between the
treatments (F(2, 9) = 62.601, p = 0.000). Data was not normally distributed. In a post hoc test, differences
between WC and the two other media were shown to be significant (p = 0.000), but not between Fish
Water and WC-Fish (p = 0.099). Mean concentrations were 2.02 ± 0.23 µg/l in Fish Water, 12.20 ± 1.38
in WC and 4.26 ± 1.88 µg/l in WC-Fish (Figure 21). On day 15 there was a significant difference in
concentration between the treatments (F(2, 9) = 2.982, p = 0.000). Data was log transformed for
homogeneity, but was not normally distributed. In a post hoc test differences between WC and the two
other media were shown to be significant (p = 0.000), but not between Fish Water and WC-Fish (p =
0.572). Mean concentrations were 0.72 ± 0.09 µg/l in Fish Water, 16.79 ± 3.08 µg/l in WC and 0.85 ±
0.13 µg/l in WC-Fish (Figure 21).
Mean concentra1on of fucoxanthin 25 µg/l 20 15 10 5 0 Figure 21. Mean concentrations (µg/l) of fucoxanthin in three different media at day 0 (Init), day 8 (D8) and day 15 (D15).
Error bars show standard deviations. Values for Init Fish and Init WC are based solely on one value each and thus have
no error bars. The colors of the bars represent different media. Yellow – Fish Water, red – WC and blue – WC-Fish.
3.3.3 Fucoxanthin/chlorophyll a ratio
On day 0 the mean fucoxanthin/chlorophyll a ratio was 0.68 in Fish Water, 0.88 in WC and 0.54 ± 0.0097
in WC-Fish (Figure 22). On day 8 there was no significant difference between the treatments (F(2, 9) =
4.004, p = 0.057). The data did not pass Cochran’s test for homogeneity of variance, even when it was log
transformed and square root transformed. In the end untransformed data was used. The mean ratio was
0.50 ± 0.0074 in Fish Water, 0.60 ± 0.11 in WC and 0.61 ± 0.023 in WC-Fish (Figure 22). On day 15
there was a significant difference between the treatments (F(2, 9) = 6.893, p = 0.015). In a post hoc test,
differences between WC and WC-Fish (p = 0.010) and WC and Fish Water (p = 0.011) were shown to be
significant, but not between Fish Water and WC-Fish (p = 0. 965). The mean ratio was 0.57 ± 0.030 in
Fish Water, 0.39 ± 0.074 in WC and 0.57 ± 0.11 in WC-Fish (Figure 22).
Mean fucoxanthin/chl. a ra1o 1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0,0 Init Init Fish WC Water Init WC-­‐
Fish D8 D8 WC D8 Fish WC-­‐
Water Fish D15 D15 D15 Fish WC WC-­‐
Water Fish Figure 22. Mean fucoxanthin/chl. a ratio in three different media at day 0 (Init), day 8 (D8) and day 15 (D15). Error bars
show standard deviations. Values for Init Fish and Init WC are based solely on one value each and thus have no error
bars. The colors of the bars represent different media. Yellow – Fish Water, red – WC and blue – WC-Fish.
3.3.4 Chlorophyll b and zeaxanthin
No chlorophyll b (proxy for green algae) or zeaxanthin (proxy for cyanobacteria when chl b is absent)
were detected in any of the samples.
3.4 Inorganic nutrients
Concentrations of inorganic nutrients are presented as µ moles per liter (µM). No nutrient was completely
depleted in any of the treatments, though SiO2 levels were greatly reduced in all media (Table 1). Silica
showed to be the limiting nutrient when nutrients were assumed to be taken up according to the RedfieldBrzezinski ratio Si:N:P = 15:16:1 (Brzezinski 1985).
Table 1. Mean concentration of inorganic nutrients in three different media – Fish Water, WC and WC-Fish – at three
different occasions – day 0 (Init), day 8 (D8) and day 15 (D15).
Treatment
Init Fish Water
Init WC
Init WC-Fish
NO2+NO3NH4+
HPO42SiO2
µM
SD
µM
SD2
µM
SD3
µM
SD4
1 720.1
7.8
65.6
0.3
20.2
0.4
87.8
8.4
951.5
162.5
1.6
0.8
25.4
1.9
58.3
3.1
2 794.0
61.0
42.6
2.0
38.7
3.5
144.8
15.8
D8 Fish Water
D8 WC
D8 WC-Fish
1 436.2
899.4
2 172.9
182.2
5.6
18.9
3.6
0.8
18.2
0.8
0.5
3.5
7.2
13.8
13.5
1.0
1.1
1.9
1.0
0.5
1.2
0.2
0.1
0.1
D15 Fish Water
D15 WC
D15 WC-Fish
1 185.7
843.9
2 090.3
194.6
15.2
301.5
4.1
7.6
42.4
2.6
1.4
1.7
4.8
11.7
2.6
0.2
0.7
0.4
0.9
0.2
1.1
0.1
0.0
0.1
4. Discussion
The significant differences in cell concentration and growth rate between WC and the two media
containing waste water from the fish farm (Fish Water and WC-Fish) strongly implies that something in
the waste water makes it unfavorable for diatom growth and even causes them to die. Manual counting is
of course a method susceptible to human error, but the differences were so clear in this case that such an
error is negligible. That the growth rate is lower on day 15 than on day 8 in WC means that the algae is
not in an exponential growth phase during this period, quite possibly this phase even stopped before day 8.
This can be linked to the reduced levels of SiO2 after day 0 and is discussed further below.
Concentration of bacteria was expected to have a positive correlation to chl. a, i.e. diatom biomass, as
shown by White, et al. (1991). Since results of chl. a concentration followed the same trend as diatom
concentration in this case, concentration of bacteria in relation to concentration of diatoms was looked at
instead. This was more interesting since it is a more direct measurement. Unfortunately bacteria formed
different clusters in the different media (Figure 8), which means that they were of different size and likely
different species making it hard to compare the bacteria/diatom ratio between the treatments. Another
inconvenience was the seemingly unreasonable low number of bacteria detected, much lower than would
have been expected (Cole, 1982). It could be that a large portion of the bacterial population was attached
to the diatoms (Amin, et al., 2012). Other reasons could be of methodical nature, e.g. that SYBRGreen did
not attach successfully to the bacterial DNA or that the concentration of the Count Bright bead solution
differed from the labeled one, leading to miscalculation of the flow rate. What can be said is that the
concentration bacteria in WC seemed to live up to the expectation of correlating with the concentration of
diatoms, where the bacteria in Fish Water and WC-Fish showed a different trend. The bacteria in these
two media could have been favored by dead algae as a food source. The bacteria themselves could have
had a negative impact on the diatom concentration (Amin, et al., 2012), resulting in the low concentration
of diatoms in Fish Water and WC-Fish.
It is interesting that contamination was present in WC-Fish, and only in WC-Fish. There could potentially
have been an undetected contamination in the original culture which hadn’t grown in detectable numbers
due to unfavorable conditions. Being a nutrient loaded medium, WC-fish might be favorable for
opportunistic species. Precautions were taken to prevent this. The algae where e.g. handled with
autoclaved instruments in a laminar flow (LAF) cabinet before being added to the media. However, due to
lack of space under the LAF cabinet media had to be filtered on a laboratory bench instead. Even though
all equipment and surfaces that were in contact with the media had been sterilized with 70% ethanol, there
is a risk that airborne contamination slipped through the defense. In the future a better method to prevent
contamination of media should be investigated, e.g. autoclaving. Autoclaving of the media was not done
in this case to prevent degradation of the vitamins in the WC-medium and other organic molecules that
might be present in the water from the fish farm. Another, perhaps more likely, possibility is that a small
number of unwanted cells already present in the unfiltered media slipped through the GF/F-filter when the
media were filtered. This might explain why the cultures grown in Fish Water and WC were not
contaminated. However, these cells did not seem to outgrow the diatoms, especially when looking at the
high concentration of cells that were found in WC, and was not thought to have had any negative impacts
on the diatoms. When photosynthetic pigments are discussed later in the text this assumption is further
strengthened.
Effective- and not maximum photochemical quantum yield was measured on day 0 due to human error.
However, this is not of any major concern since the object was to investigate how the treatments affected
the algae after a time of incubation. What is worth to note from this test is that the algae initially did not
differ significantly with respect to photochemical activity. When looking at photosynthetic activity on day
8, diatoms in WC have a significantly higher yield than the other two media, indicating that this medium
is more favorable to them which is in line with the results on growth. Nutrient deficiency is often
explained to be the cause of a lower photochemical quantum yield (Beardall & Roberts, 2001; Saeck, et
al., 2016), however, this is not probable in this case as will be discussed later. According to Kruskopf &
Fynn (2006), photochemical quantum yield might not even be a good way of monitoring nutrient stress.
Viral infection has been shown to impede photosynthetic activity of algae (Juneau, et al., 2003; Van Etten,
et al., 1983) and it cannot be excluded as a factor in this case. On day 15 algae in Fish Water again had a
significantly lower photosynthetic activity than in WC, giving support to the assumption that this medium
is unfavorable. That algae in WC-Fish did not differ from any of the other media is hard to explain. Since
the Water-PAM does not discriminate between species, it is also possible that fluorescence measured in
WC-Fish partly came from the contamination that was present. However, when looking at the
fucoxanthin/chl. a ratio, WC-Fish did not stand out as having a lower value than the other media on day
15. This indicates that whatever contamination was present did not contribute much to the total chl. a
concentration/algae biomass.
Concentrations of chl. a followed the same pattern as the concentrations of diatoms with a significantly
higher concentration in WC than in the two other media on day 8 and 15, making it clear that the media
containing waste water is unfavorable for the diatoms. As expected concentrations of fucoxanthin also
follows this pattern. As discussed above the fucoxanthin/chl. a ratio was not lower in WC-Fish as would
have been expected if there was high levels of contamination present. That this ratio is significantly lower
in WC is hard to explain but the answer might be found in the different nutrient and/or light levels (e.g. by
self-shading) (Mouget, et al., 1999; Schlüter, et al., 2000). That no chlorophyll b was present in any of the
samples further indicates that chlorophytes were not present in any significant numbers (Jeffrey, 1976).
Neither was zeaxanthin found, making the same case for cyanobacteria (Wulff & Wängberg, 2004).
Nutrient deficiency is ruled out as a factor due to the poor growth in the nutrient rich WC-Fish-medium.
Results from the analysis of inorganic nutrients further strengthens this assumption. Fish Water and WC-
Fish had higher levels of nutrients than WC – with the exception of Fish Water having a slightly lower
phosphate level than WC – at the beginning of the experiment. From a different perspective, the high
nutrient levels may actually be harmful to the diatoms. High levels of ammonium has been shown to have
toxic and/or inhibitory effects on diatoms and their growth (Admiraal, 1977). On the other hand, G.
parvulum has been shown to have a high tolerance to ammonium (Hürlimann & Schanz, 1993), making
this reason unlikely. For further studies, growth of the diatom in different concentrations of ammonium
should be investigated. SiO2 was the only nutrient that was nearly depleted, which was true for all
treatments already on day 8. Since it is a necessary nutrient for diatom growth (Bidle & Adam, 1999;
Martin-Jézéquel, et al., 2000), this can explain why growth rates were lower on day 8 than on day 15 for
algae in all treatments. This problem can potentially be avoid by constantly exchanging old medium with
new, as would be the case in the fish farm where there is a steady outflow of waste water and inflow of
water from the well. A method for accomplishing this while minimizing the risk of contamination should
be created to better mimic the conditions in the fish farm. Interestingly SiO2 was almost depleted in Fish
Water and WC-Fish, even though the growth was poor in these treatments, but could possibly be
explained by precipitation, e.g. due to the higher salinity (Bien, 1959). There is a possibility that there is
concentrations of elements in the waste water, e.g. heavy metals, which are harmful to the diatoms. In the
future a deeper analysis of the waste water should be performed to get a full picture of elements and
molecules present in it to be able to determine if some of them might have a negative impact on diatom
growth.
Salinity could play a role in why the waste water was unfavorable since the growth rate was lower in the
media containing waste water, which as previously stated had a salinity of 3.6 as opposed to 0 in WC.
This would be in line with findings of Lionard et al. (2005), where impeded growth rate was explained as
an effect of increased salinity on fresh water diatoms. However, according to their results increased
salinity would not cause diatoms to die, so this would not explain the declining concentration of cells from
day 8 to day 15 in Fish Water and WC-Fish. A fourth treatment with WC-medium with the same salinity
as the water coming from the fish farm could have been tested as well to be able to investigate salinity as
an influential factor on growth, and should be considered for further studies. Lionard et al. (2005) also
raised an interesting question if viruses or parasites present in the water could be the cause of cells dying,
which as stated earlier is a possible cause of the lower photochemical activity in Fish Water and WC-Fish.
Time could have been used as a factor in the statistical tests since true replicates were used, but was
decided against since the object was to determine if the treatments differed from each other and not how
the diatoms reacted over time. Another reason is because the exponential growth rate phase probably
ended before or around day 8. When grown as a part of a filter system, algae would be harvested during
the exponential growth phase and not a week after it had ended. The object of the experiment was to see if
there was a difference between the treatments, not to define general effects of different conditions,
therefore univariate analysis of variance was used even when data was not normally distributed. Often
differences could also been seen clearly by just looking at the data and graphs over the data.
5. Conclusion
The experiment showed that the waste water was unfavorable for diatom growth and potentially harmful.
It is not clear what the cause of this is but a few things stand out as potential factors to this. One is high
levels of ammonium, another salinity. It is also conceivable that there were viruses, parasites or harmful
elements such as heavy metals present in the water, explaining why cell concentrations were lower on day
15 than on day 0. In the future new methods should be developed to investigate these factors more
thoroughly and the waste water should be analyzed for viruses, parasites and heavy metals. To be able to
refine a successful method of cleaning waste water from the fish farm other species of diatoms and mixed
communities of diatoms should also be investigated.
Acknowledgements: Angela Wulff, supervisor. Mikael Hedblom, Gustav Knutsson and Justin Pearce from
Swedish Algae Factory for helping out with methods and data analysis. Jenny Andersson, Monica
Appelgren and Anders Torstensson for helping out with experiments. Adil Y. Al-Handal for pictures and
help with analysis. Ola Öberg from Svensk Fiskodling for providing water and information about the fish
farm.
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