DEPARTMENT OF BIOLOGICAL AND

ENVIRONMENTAL SCIENCES

Degree project for Bachelor of Science with a major in biology

BIO603, Examensarbete i biologi

First cycle

Semester/year: Spring 2019

Supervisor: Henrik Pavia, Department of Marine Sciences

Examiner: Johan Höjesjö, Department of Biological & Environmental Sciences

THE EFFECT OF LIGHT, NITROGEN AND

IRON ON THE PIGMENT CONTENT OF S.

LATISSIMA

Otto Minas

1

Contents

Sammanfattning.......................................................................................................................... 2

Abstract ...................................................................................................................................... 2

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

The nutritional value of pigments .............................................................................................. 1

Fucoxanthin ............................................................................................................................ 1

Antioxidant activity ........................................................................................................... 1

Cancer preventive activity .................................................................................................. 1

β-carotene ............................................................................................................................... 2

Provitamin A activity .......................................................................................................... 2

Cancer preventive activity .................................................................................................. 2

Violaxanthin ........................................................................................................................... 2

Zeaxanthin, Chlorophyll A & Chlorophyll C ......................................................................... 3

Environmental factors ................................................................................................................ 3

Light ........................................................................................................................................ 3

Nitrogen .................................................................................................................................. 3

Iron .......................................................................................................................................... 4

Aim ............................................................................................................................................. 4

Method........................................................................................................................................ 4

Light/Nitrogen treatment ........................................................................................................ 4

Light/Iron treatment ................................................................................................................ 5

Sample preparation ................................................................................................................. 5

Spectrophotometric analysis ................................................................................................... 6

UPLC-UV-MS analysis .......................................................................................................... 6

Statistical analysis ................................................................................................................... 6

Results ........................................................................................................................................ 6

Spectrophotometric results ..................................................................................................... 6

Light/Nitrogen treatment..................................................................................................... 7

Light/Iron treatment ............................................................................................................ 8

UPLC-UV-MS results ............................................................................................................ 8

Light/Nitrogen treatment..................................................................................................... 9

Light/Iron treatment .......................................................................................................... 10

Discussion ................................................................................................................................ 10

2

Light/nitrogen treatment ....................................................................................................... 10

Light/iron treatment .............................................................................................................. 11

Continued research ............................................................................................................... 11

Conclusion ................................................................................................................................ 11

Citations.................................................................................................................................... 12

Sammanfattning

Detta kandidatarbete undersökte om ljus, kväve och järn har någon effekt på pigmenthalterna i

Saccharina Latissima efter 48 timmars behandlingar. Detta gjordes för att undersöka möjligheten

att höja näringshalten av S. Latissima som skördas på den svenska västkusten. Några av

pigmenten som S. Latissima innehåller är karotenoiderna β-carotene, ett männskligt provitamin

A; Fucoxanthin en för brunalger originell karotenoid med antioxidanta och cancerhämmande

egenskaper och Violaxanthin, ett anti-inflammatoriskt pigment. Pigmentanalyserna gjordes med

en spectrofotometer samt med en UPLC-UV-MS. Dom spectrophotometriska resultaten visade att

klorofyll A, klorofyll C och att den totala karotenoidhalten ökade till följd av låg-ljus

behandlingen, däremot fanns ingen signifikant effekt av kvävet eller järnet. UPLC-UV-MS

analysen visade att fucoxanthin var den enda karotenoiden som påverkades signifikant av ljus

medan β-carotene visade en icke-signifikant trend att öka i lågt ljus. Detta tros vara då en

behandlingstid av 48 timmar ej är tillräckligt lång för att en reaktion på näringsämnena kväve och

järn ska ses. Då ljus är en miljöfaktor som förändras mycket snabbt i naturen medan

näringsämnen som kväve huvudsakligen förändras över säsongerna verkar denna adaptation

rimlig. För att se en effekt av kväve och järn rekommenderas längre behandlingstider än 48

timmar. Ljus visade sig vara en möjlig metod för att påverka pigmenthalterna i S. Latissima,

Abstract

This thesis studied the effect of light, nitrogen and iron on the pigment content of Saccharina

Latissima during 48h treatments. This was done to explore the possibility of increasing the

nutritional value of S. Latissima harvested from the Swedish west coast. Among other pigments

S. Latissima contains the carotenoids β-carotene, a human provitamin A; Fucoxanthin, a brown

algae specific carotenoid with antioxidant and cancer inhibiting properties and Violaxanthin, a

proven anti-inflammatory. Pigment analysis was done through spectrophotometry and UPLC-UV-

MS. The spectrophotometric results showed an increase in chlorophyll A, chlorophyll C and in

total carotenoid content in algae treated to low light conditions but no significant effect by

nitrogen. The UPLC-UV-MS results of the same treatment showed FX was the only carotenoid

significantly affected by light while BC seemed to decrease in low light.

This is believed to be because the treatment time of 48h is not long enough for a macroalgae like

S. Latissima to respond to a change in nitrogen availability whereas their response to light

availability is much faster. As light intensity can change within minutes while nitrogen

availability mainly oscillates seasonally this adaptation seems natural. Both the

spectrophotometer and UPLC-UV-MS showed the iron treated samples lacked significant results

or trends but due to the small sample size few conclusions can be drawn from them. For iron or

nitrogen to influence pigment content a treatment time longer than 48h should be tested. Light

was proven a possible method of increasing pigment content in S. Latissima.

1

Introduction

Seaweed aquaculture has the potential to be

an alternative to terrestrial biomass

production. As (terrestrial) areas subjected to

high environmental variability are expected

to expand and impact food security (Josef &

Francesco, 2007) agriculture in the stable

environment of the sea is drawing interest. In

comparison to biomass produced terrestrially

marine seaweed production does not require

any freshwater, arable land and in most cases

no fertilizer. As some of its pigments have a

positive effect on human health algae are

also of nutritional interest.

Due to an increase in interest, the global

production of seaweed has doubled in ten

years and was in 2016 30 million tons. Most

production is done in China and Indonesia,

farming 14 and 11 million tons respectively

("The State of World Fisheries and

Aquaculture,"). The seaweed is mainly used

for human consumption (directly or

processed), cosmetics, fertilizers or

pharmaceuticals. A(Stevant, Rebours, &

Chapman, 2017) rather new use for seaweed

aquaculture is as absorbents of excess

nutrients from fish farms. By utilizing excess

nutrients the seaweed decrease

environmental impact as well as increase the

total biomass yield (Stevant et al., 2017).

Swedish seaweed production is still in its

early stages, and the production is limited to

a few small scale test sites (Risinger &

Sverige. Havs- och, 2015). One of the algae

commonly grown across the north Atlantic is

the brown macroalgae Saccharina Latissima

(Boderskov et al., 2016).

The focus of this thesis is on the potential to

increase the pigment content of S. Latissima,

as some of these have positive effects on

human health. The six pigments investigated

are fucoxanthin (FX), β-carotene (BC),

violaxanthin (VI), zeaxanthin (ZE),

chlorophyll A (Chl A) and chlorophyll C

(Chl C). However FX, BC and VI are of

greatest interest due to their significant

health benefits.

The nutritional value of

pigments

Fucoxanthin

Antioxidant activity The most important function of FX in the

human body is that of an antioxidant.

Antioxidants inhibit a multitude of diseases

like the neurodegenerative diseases

Parkinson’s, Alzheimer’s and Amyotrophic

lateral sclerosis (ALS) (Chen, Guo, & Kong,

2012; Kong & Lin, 2010). As an antioxidant

it can quench the free radicals and singlet

oxygen that is produced during normal

aerobic metabolism. Free radicals and singlet

oxygens are reactive molecules that can

cause damage to DNA, proteins and lipids

by oxidizing them. FX has been shown to be

an effective scavenger of DPPH (1,1-

diphenyl-2-picrylhydrazyl), a free radical

commonly used to evaluate the activity of an

antioxidant (Sachindra et al., 2007; Yan,

Chuda, Suzuki, & Nagata, 1999). FX also

has the ability to quench singlet oxygen

(Sachindra et al., 2007). Notable is that FX

shows antioxidative activity in anoxic

conditions, unlike many other antioxidants

(Nomura, Kikuchi, Kubodera, & Kawakami,

1997) and that many human tissues under

physiological conditions have low oxygen

presence (Sachindra et al., 2007).

Cancer preventive activity Cancer is a continued global health issue and

the failure of conventional chemotherapy to

reduce mortality rates in breast-, colon-,

prostate- and lung cancer highlight the need

for new methods of preventing cancer

(Sporn & Suh, 2000). Potentially, one of

these methods is chemoprevention.

Chemoprevention is a pharmacological

approach that reverses/prevents the progress

of carcinogenesis (i.e. the beginning of

cancer). It is based in a change in how

researchers view cancer, not primarily as a

disease fundamentally characterized by

excessive cell proliferation but rather as “the

end stage of a chronic disease process

[carcinogenesis]” (p. 525, Sporn & Suh,

2000). In vivo studies have found FX to

have chemopreventive properties, inhibiting

2

carcinogenesis in the colon, duodenal, liver,

sarcoma and melanoma (J. Kim, Araki, Kim,

& Park, 1998; Nishino, Murakoshi, Tokuda,

& Satomi, 2009; Okuzumi et al., 1993;

Shimoda, Tanaka, Shan, & Maoka, 2010;

Wang et al., 2012). Although target

molecules of FX have been identified in

vitro the mechanisms responsible for FXs

chemopreventive activity have yet to be fully

understood in vivo.

In addition, further potential functions for

FX such as its anti-photoaging activity and

beneficial activity in the cardiovascular

system are well covered in Fucoxanthin: A

Treasure from the Sea (D'Orazio et al.,

2012) and Biosynthetic Pathway and Health

Benefits of Fucoxanthin, an Algae-Specific

Xanthophyll in Brown Seaweeds (Mikami &

Hosokawa, 2013).

β-carotene

Like FX BC is an active antioxidant and

functions as a lipid scavenger as well as a

singlet oxygen quencher (Grune et al., 2010).

Unlike FX, BC is present in many common

fruits and vegetables. For instance carrots,

tomatoes and spinach are all common

sources of BC.

Provitamin A activity

The beneficial effects of BC largely come

from its provitamin A properties. Meaning

that as humans ingest BC we metabolize it to

retinol, the alcohol called vitamin A. Being a

vitamin retinol is essential for normal

physiological functions, especially under

nutritionally demanding periods of life such

as infancy, childhood and pregnancy. These

are also the groups most commonly suffering

of vitamin A deficiency (VAD). In their 2005

report, Global prevalence of vitamin A

deficiency in populations at risk 1995–2005

WHO concluded that 33% of preschool aged

children and 15% of pregnant women

globally are at risk of VAD, a total of 210

million people. VAD is mostly prevalent in

poor communities in lower income

countries, mainly in Africa and South-East

Asia. VAD impairs many bodily functions

and therefore can have many symptoms.

Most specific to VAD is xerophthalmia, an

eye disease that causes the eye and tear duct

to dry out and which is the main cause of

preventable blindness in children (Croasdale,

1998). Anemia can also be caused by VAD

due to the roles vitamin A plays in iron

mobilization, transport and the formation of

blood cells. Furthermore VAD seems to

worsen infections (Scrimshaw, 1968) and

can cause night blindness during pregnancy

(Christian et al., 1998). Vitamin A

supplementation has shown to reduce

mortality in young children by 23-30%

(Fawzi, Chalmers, Herrera, & Mosteller,

1993; G. H. Beaton, 1993; Glasziou &

Mackerras, 1993).

Cancer preventive activity There has long been a consensus on that a

high intake of fruits and vegetables decrease

the risk of cancer, as shown by (Block,

Patterson, & Subar, 1992). Many studies

show BC to have cancer preventive

properties (Garewal, 1995; Goralczyk, 2009;

Naves & Moreno, 1998; van Poppel &

Goldbohm, 1995). For instance BC was

recently reported to inhibit neuroblastoma,

the most common extracranial tumor in

children (Y.-S. Kim et al., 2014).

Another form of cancer linked to BC is lung

cancer, yet here BC supplementation can

have adverse effects when supplemented to

smokers. In contrast a preventive effect/no

effect occurs when BC is supplemented in

antioxidant protected forms or is combined

with an antioxidant vitamin (Y.-S. Kim et

al., 2014)

Violaxanthin

Like BC, VI can be found in many plants in

addition to algae. For instance dark green

plants like spinach are good sources of VI.

VI is a proven anti-inflammatory substance.

It can inhibit the way our body responds to

viruses, bacteria and other pathogens. A part

of our inflammatory reaction consists of

producing the inflammatory agents nitric

oxide and prostaglandin E2. These agents

can cause damage due to inflammatory

disease or sepsis. VI inhibits the functions of

the complexes that produce nitric oxide and

3

prostaglandin E2 and acts as a natural and

effective anti-inflammatory

(Soontornchaiboon, Joo, & Kim, 2012).

Zeaxanthin, Chlorophyll A & Chlorophyll C

Zeaxanthin has been associated with many

health benefits (Johnson, 2012; Lam & But,

1999; Mares, 2016; Mares-Perlman, Millen,

Ficek, & Hankinson, 2002), however the

pigment was not the main focus of this thesis

as that was FX and BC.

Chl A and Chl C have not been found have

any direct association to improved health.

Environmental factors

The three environmental factors studied were

light, nitrogen and iron. As the pigments of

interest are active in both photosynthesis and

photoprotection light has been shown to

impact pigment content (Boderskov et al.,

2016).

Nitrogen is a limiting nutrient and has been

shown to limit growth in brown macroalgae

(Chapman & Craigie, 1977; Pedersen &

Borum, 1996).

Iron was of interest as it has previously been

used to induce algal blooms to mitigate

climate change. As this was a hot topic the

effect of iron on microalgae pigmentation

was studied intensively. However, that iron

increased the content of fucoxanthin in

brown macroalgae has been shown

(Matsunaga, Suzuki, Kuma, & Kudo, 1994).

Light

Increased irradiance decrease all pigment

content in most alga and higher plants

(Larkum & Barrett, 1983). This is seen in S.

Latissima as well. In arctic populations, a

decrease in chlorophyll A and total

carotenoid content has been observed when

the sea ice breaks in the spring and light

availability increases (Aguilera, Bischof,

Karsten, Hanelt, & Wiencke, 2002).

All algal carotenoids are present in the light

harvesting complexes. However, their

functions are not only photosynthetic. BC,

FX and VI are also active during

photoprotection and their response to

changes in light is difficult to predict. The

primary function of BC in the light

harvesting complex is that of quenching

singlet oxygen (Telfer, 2002). FX can

quench the energized state of chlorophyll

associated with light stress (excited triplet

state) (Di Valentin, Büchel, Giacometti, &

Carbonera, 2012; Kosumi, Nishiguchi,

Amao, Cogdell, & Hashimoto, 2018) and VI

is an essential part of the xanthophyll cycle

(García‐Mendoza & Colombo‐Pallotta,

2007; Goss & Jakob, 2010). A method of

dissipating excess energy in the form of heat.

The photoprotective functions of these

carotenoids would suggest an increase in

their activity in high light.

The dual functions of BC, FX and VI makes

it difficult to predict their reactions in

regards to changes in light.

However, for a phototroph like S. Latissima

all light is not the same. Responses are

partially dependent on light quality. Changes

in red and blue light have greatest effect on

all pigment content as these are the

wavelengths mostly used for photosynthesis.

Carotenoids like FX also harvest green light

for photosynthesis although no responses in

FX content have been seen in response to

changes in green light irradiance (Dring,

1986). The photosynthetically active

radiation (PAR) spectrum used in this study

includes the red, blue and green light

mentioned above.

Light also works as an indicator of seasonal

change. As the day length (photoperiod)

changes with the seasons so does algal

productivity. By being able to sense seasonal

changes allows S. Latissima to “grow and

reproduce in a strategic annual rhythm

suitable for the species” (p. 209, Kain, 1989)

Nitrogen

Nutrients are an important factor of algal

growth and production, and as nutrients vary

both spatially and temporally algae have to

be able to adapt to these variations (Beardall,

Young, & Roberts, 2001). A decrease in

nutrient availability has been shown to

negatively affect pigmentation in S

Latissima (Boderskov et al., 2016).

4

The main changes in macronutrient (nitrogen

and phosphorus) availability occur

seasonally. The biomass yield of S.

Latissima peaks in late summer (Boderskov

et al., 2016), however after a five-month

period of fast growth they have depleted

most of their nitrogen reserves. And as

macronutrient availability decreases in the

late spring/summer so does the nitrogen and

phosphorus content in the blades (Conolly &

Drew, 1985). For some species of brown

algae the lack of ambient inorganic nitrogen

is what stops the period of fast growth in the

summer (Kain, 1989). The winter months

when nutrient availability is high are spent

accumulating nitrogen again.

Important to take into consideration when

treating algae with light and nitrogen

simultaneously is that nitrogen uptake can be

limited by low irradiance. The green algae

Dunaliella tertiolecta was shown to have

23x higher nitrogen assimilation under high

light than under low light (Grant & Turner,

1969). This could lessen the potential

increase in pigmentation caused by nitrogen

fertilization in low irradiance. This limitation

was not seen in a 2016 study (Boderskov et

al., 2016) on S. Latissima but should be

tested before Nitrogen fertilization is ever

attempted for other species of algae.

Iron

Iron limited strains of an Antarctic microalga

have shown to have a lower content of light

harvesting pigments than their iron

supplemented counterparts (Van Leeuwe &

Stefels, 1998).

The response has been proven to be rather

complex as the content of pigments like FX

don’t have to change for a response to have

occurred. The change occurs within a group

of molecules closely resembling FX but with

slightly different functions. In algae FX

occurs in a few different forms, namely

Fucoxanthin (FX), 19′‐Hexanoyloxyfucoxanthin (Hex‐FX), 19′‐Hexanoyloxy‐4‐ketofucoxanthin (Hex‐kFX)

and 19′‐butanoyloxyfucoxanthin (But‐FX).

A response to changing environmental

factors like iron and light can occur in the

ratio between these forms of FX. These

changes will not be visible to the analysis

used in this thesis.

An increase in FX has been observed in both

the microalgae previously mentioned

(Leeuwe, Visser, & Stefels, 2014) and in the

brown macroalgae Laminaria japonica (a

species closely related to S. Latissima) in

response to increased iron exposure

(Matsunaga et al., 1994).

Aim

As the pigments BC, FX and VI present in S.

Latissima have high nutritional value they

are of interest to the Swedish aquaculture

industry. Finding ways to increase their

content in sustainable and efficient ways

would be beneficial to both producers and

consumers.

The aim of this thesis is to understand if

raised levels of light, nitrogen and iron can

increase the pigment content in Saccharina

Latissima during a 48h treatment.

Method

Light/Nitrogen treatment

S. Latissima was collected in May from

cultivation fields used by the company

Kosteralg (www.kosteralg.se) in the Koster

archipelago on the Swedish west coast.

Individuals between 15-80cm long were

placed in 15L tanks in a climate controlled

room (14°C). A constant flow of filtered

oceanic surface water was pumped through

the aquariums. All aquariums were

additionally oxygenated as this created a

circular flow in the tank which kept the algae

from settling and dispersed the added

nitrogen/iron rapidly.

The room was kept in a 12h light/12h dark

light schedule and half of the tanks were

exposed with to high light (500-600 μmol·m-

2·s-1 of photosynthetically active radiation

(PAR)) and the other half to low light (50-60

μmol·m-2·s-1 of PAR). These intensities are

within the natural variation of light that

changes with time of day, weather and water

depth. Half of the aquariums were treated

5

with raised levels of nitrogen (Ammonium

nitrate (www.sigmaaldrich.com) whereas the

other half received the nitrogen present in

the water pumped through them. Water

samples for analysis of ambient nitrogen

exist but have yet to be analyzed. However

ambient nitrogen is estimated to vary from

0,1<1 μmol /l (SHARKweb, (SMHI)) during

the summer in shallow (0-20m) coastal

waters on the Swedish west coast. The high

treatment had a total exposure of ambient

nitrogen + 5 μmol/l Ammonium nitrate. This

is comparable to the seasonal changes in

nitrogen availability. A winter treatment

(ambient nitrogen + 5 μmol/l) and a summer

treatment (abinent). Each treatment had 8-11

replicates.

Light/Iron treatment

An identical setup was used for the

Light/Iron treatment as for the

Light/Nitrogen treatment. Half were treated

with ambient iron and the other half were

treated with a raised iron concentration (Iron

chloride hexahydrate

(www.sigmaaldrich.com)).The ambient iron

is roughly estimated to be around 0,1<1

μmol /l (McGaraghan & Kudela, 2012) but

due to the uncertainty in this estimate the

high treatment was exposed to ambient iron

+ 10 μmol /l. This is not based on the

seasonal variation of iron as this is not

known. This is based on annual averages of

the coast of California and a 10x increase.

Each treatment had 5-6 replicates.

All treatments were done over 48h and

sampling was done by cutting roughly a

10x10cm piece of tissue from the blade just

above the stipe. Samples were kept in -80 °C

until pigment analysis.

Sample preparation

The pigments were quantified using two

methods, spectrophotometry and UPLC-UV-

MS (ultra performance light chromatography

- ultra violet - mass spectrometry). The two

methods differ from each other, the

spectrophotometer is a fast and easy way of

quantifying pigments, however it lacs the

ability to differentiate FX, BC and VI from

each other and groups them together as “total

carotenoids”. The UPLC-UV-MS can detect

the six pigments of interest separately,

however it is slower and more complex. It

also and had to be slightly adjusted (see

below) for this experiment. Therefore both

spectrophotometry and UPLC-UV-MS were

used.

The spectrophotometer sends light of

specific wavelengths through the sample and

senses how much of the light the sample

absorbs. As the absorbance of pigments at

different wavelengths is known, the

absorbance of my samples can be used to

quantify the pigments in my samples.

However, as the spectrophotometric analysis

only quantifies chlorophyll a, chlorophyll c

and total carotenoids and not each carotenoid

individually this method was paired with a

UPLC-UV-MS.

This method has two steps. The UPLC

separates the pigments from each other

depending on polarity and the UV-MS

identifies and quantifies the pigments as they

come out of the UPLC. The UPLC used a

hydrophilic liquid phase that transports the

sample through a hydrophobic channel. The

pigments with hydrophobic properties have

greater affinity to the channel while

pigments with hydrophilic properties have

low affinity with the channel and will pass

through it faster. Therefor the UPLC

separates the pigments through elution time.

As pigments exit the channel their

absorbance is recorded by the UV detector.

By measuring the pigments absorbance at

different wavelengths it can be identified.

Lastly the MS (mass spectrometer) measures

the mass-to-charge ratio of the particles. The

elution time, absorbance’s and masses of the

particles were used to quantify the pigments.

All samples were freeze dried and 50mg of

each was prepared for HPLC-UV-MS by the

method described in (Fu, Magnúsdóttir,

Brynjólfson, Palsson, & Paglia, 2012) with

some modifications. α-Tocopherol acetate

was used as an internal standard and

butylated hydroxytoluene was exchanged to

6

2,6-di-tert-butyl-4 methylphenol

(www.sigmaaldrich.com). The

ethanol/hexane 2:1 (v/v) containing 0,1%

(w/v) 2,6-di-tert-butyl-4methylphenol was

evaporated before addition of water and

hexane, this because fucoxanthin would

otherwise separate from the other pigments

and could not be analyzed together with

them.

Spectrophotometric analysis

Half of the sample prepared as above was

evaporated once more and prepared for

spectrophotometry by dissolving the

pigments in 2ml of 90% acetone. The

absorbance was measured at 480, 510, 630

and 665 nm and were blanked against 90%

acetone. The following equations were used

for chlorophyll a, chlorophyll c and total

carotenoid quantification (Veide Vilg et al.,

2015).

Chl a= (11,43·A664) - (0,40·A630)

Chl c= (3,80·A664) + (24,88·A630)

Total Carotenoids= (7·A480) - (1,49·A510)

UPLC-UV-MS analysis

The UPLC was set up according to the

following parameters.

LC system: Agilent 1100 Series

Column: Agilent Poroshell 120 EC-C18,

2.1 x 100 mm, 2.7µ

Flow: 400.00 µL/min

Injection voume:4 µl

Solvent A: Water + 0.1% ammonium

acetate

Solvent B: Acetonitrile + methanol + tert-

butyl-methyl ether (7:2:1)

Gradient timetable:

Time Solvent A

%

Solvent B

%

1.00 40 60

4.00 25 75

14.00 0 100

28.00 0 100

29.00 2 98

Post tune time: 10.00 min

MS system: Agilent 6540 UHD accurate

mass TOF

Ionization: electro spray ionization

Acquisition: MS mode, 100-3000 m/z, 1

spectra/s

Statistical analysis

The data was analyzed for outliers, normal

distribution and homogeneity of variance

before being tested with a 95% confidence

limit in a two way ANOVA in SPSS. Due to

most samples having undetectable amounts

of ZE in UPLC-UV-MS the results were

tested through a Chi-square test to see if the

frequency of detectable/undetectable

amounts of ZE was dependent on treatment.

Results

Spectrophotometric results

The content of chlorophyll A, chlorophyll C

and of total carotenoids per mg dry weight is

presented in Charts 1 and 2.

7

Light/Nitrogen treatment In the light/nitrogen treated samples light

had a significant effect on chlorophyll A

chlorophyll C and total carotenoid content.

The content of all pigments measured

increased in low light treatments compared

to high light treatments. Nitrogen did not

show a significant effect on chlorophyll A,

chlorophyll C or on total carotenoids.

Although not significant, an indication of

interaction between light and nitrogen is

visible as the effect of light seems to be

larger in high nitrogen than in low nitrogen

(see Chart 1).

All significance levels are presented in Table

1. All significant results are marked with an

asterix.

0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

0.0007

0.0008

0.0009

High lightHigh nitrogen

High lightLow nitrogen

Low lightHigh nitrogen

Low lightLow nitrogen

μg

pig

men

t/m

g d

wSpectrophotometry

light/nitrogen

Total Cartoenoids Chlorophyl A Chlorophyl C

Chart 3. The average content of chlorophyll A, chlorophyll C and total carotenoids in μg /mg dry weight. Bars show means

±standard error.

0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

0.0007

0.0008

0.0009

High lightHigh iron

High lightLow iron

Low lightHigh iron

Low lightLow iron

μg

pig

men

t/m

g d

w

Spectrophotometry light/iron

Total Cartoenoids Chlorophyl A Chlorophyl C

Chart 1. The average content of chlorophyll A, chlorophyll C and total carotenoids in μg /mg dry weight. Bars show means

±standard error

Chart 2. The average content of chlorophyll A, chlorophyll C and total carotenoids in μg /mg dry weight. Bars show means

±standard error

8

Light/Iron treatment The light/iron treated samples showed no

significant effect of light or iron on pigment

content. No significant interaction between

the factors was found. The significance

levels are visible in Table 2.

Note the substantial increase in Chl C

content compared to the spectrophotometric

analysis of the light/nitrogen treatments

(Chart 1). One outlier was removed.

UPLC-UV-MS results

The UPLC-UV-MS results are presented in

charts 3 and 4.

As can be seen on the y-axis, all results in

Chart 3 are approximately half of what is in

the other three charts. This is presumably

due to an analytical error and not the effect

of the treatment. Chl A in chart 1 is from the

same treatments and the same samples and

does not show the same reduction in pigment

content.

Table 1. Significance levels for the spectrophotometric

analysis of the light/ nitrogen treatments.

Table 2. Significance levels for the spectrophotometric

analysis of the light/ iron treatments.

0

0.00005

0.0001

0.00015

0.0002

0.00025

0.0003

0.00035

0.0004

High lightHigh nitrogen

High lightLow nitrogen

Low lightHigh nitrogen

Low lightLow nitrogen

μg

pig

men

t/m

g d

w

UPLC-UV-MS light/nitrogen

Zeaxanthin β-carotene Fucoxanthin Chlorophyl AChart 3, The average content of ZE, BC, FX and Chl A in one mg of sample. Bars show means ±standard error.

9

In Charts 3 and 4, Chl A content is slightly

lower and has a SE larger than the same

pigment analyzed with the

spectrophotometer. The SE is also

substantially larger for Chl A than for the

other three pigments analyzed in Charts 3

and 4. This is presumed to be because of Chl

A degrading to pheaophytin a before

analysis. If the degradation varied between

samples the variance of Chl A content

increased, explaining the large SE.

Light/Nitrogen treatment As seen in Table 3, FX was significantly

affected by light in the light/nitrogen

treatments. The samples exposed to high

light had lower contents of FX than samples

exposed to low light. No other pigments

were significantly affected by light or

nitrogen, and the two factors did not affect

each other.

Although not significant, Β Carotene seems

to be negatively affected by increasing light.

0

0.0001

0.0002

0.0003

0.0004

0.0005

0.0006

0.0007

0.0008

0.0009

High lightHigh iron

High lightLow iron

Low lightHigh iron

Low lightLow iron

μg

pig

men

t/m

g d

wUPLC-UV-MS

light/iron

Zeaxanthin β-carotene Fucoxanthin Chlorophyl A

Chart 4, The average content of ZE, BC, FX and Chl A in one mg of sample. Bars show means ±standard error. Note the change

in scale on the y axis compared to table 3.

Table 3, Significance levels for the UPLC-UV-MS analysis

of the light/ nitrogen treatments.

Table 4, Significance levels for the UPLC-UV-MS analysis

of the light/ iron treatments.

10

The frequency of detectable and

undetectable amounts of ZE was not

associated to treatment.

The absence of VI is due to the content being

too low in all samples for the mass

spectrometer to detect it.

Light/Iron treatment None of the light/iron treatments were

significantly affected by iron or light. Chl A

seems to have degraded to pheaophytin a in

these samples as well. See Chart 4.

The frequency of detectable/undetectable

concentrations of ZE was not associated to

treatment.

All light/iron treated samples had VI

concentrations too low for the mass

spectrometer to detect and it is therefore

absent from these tables.

Discussion

Light/nitrogen treatment

Chl A, Chl C and total carotenoids in the

light/nitrogen treated samples showed a

significant effect of light on pigment

content. This confirms that the overall

pigment content increases in lower light

intensities after 48h, as seen in Chart 1. As

the function of Chl A and Chl C are

photosynthetic their increase was expected.

For accurate conclusions about carotenoids,

it is more informative to discuss the UPLC-

UV-MS analysis as this shows their

individual reactions. However, before the

results are discussed some sources of errors

must be mentioned.

Firstly, all data in Chart 3 (UPLC-UV-MS

analysis of the light/nitrogen treated

samples) is roughly halved compared to

Charts 1, 2 and 4. As Charts 1 and 3 show

the same treatment groups and show that the

results in Chart 3 are not due to the

treatment. It is also not a miss calibration of

the UPLC-UV-MS as the results from Chart

3 were analyzed simultaneously as the

results shown in Chart 4. The halving of all

pigment content in Chart 3 is probably due

to an error in data handling, although this

error was not found.

Secondly, as the UPLC-UV-MS analysis

was done one week after the

spectrophotometric analysis the extracted

pigments were kept in a dark, -20°C freezer

for seven days. During this time some of the

Chl A presumably degraded into

pheaophytin a (a compound found in

pigment samples treated and analyzed

similarly. As this was not discovered until

after the analysis the presence of

pheaophytin a was not measured during

analysis). This degradation of Chl A seems

to have happened at different rates in the

samples, creating a large variation in Chl A

content and therefor large SE (visible in

Charts 3 & 4).

With regards to these two sources of error,

the results in Chart 3 still have value. It is

clearly visible that the carotenoid response to

light is more complex than the

spectrophotometric analysis shows.

FX is the only carotenoid significantly

affected by light. A slight increase in content

can be seen in low light compared to high

light. This indicates that the photosynthetic

function of FX is of greater value to the

algae than its photoprotective function. This

can only be said for the light intensity es

(Kain, 1989)function might become more

important in light intensities greater than

500-600 μmol·m-2·s-1. The increase in FX

content is also very slight, and since the

application of this research is to increase the

nutritional value of S. Latissima the light

levels and treatment times used just barely

achieve that purpose. A more substantial

increase might be achieved under lower light

intensities or longer treatments.

BC was not significantly affected by light

but showed a trend that suggested high light

could have a positive effect on BC content.

This would suggest that at the light

intensities tested, the photoprotective

11

function of BC is of greater importance than

its photosynthetic function. If this was the

case, increasing the content of FX and BC in

S. Latissima simultaneously could not be

done through light treatment, as one

increases under low light and the other

potentially does so under high light. To

determine if the trend seen in Chart 3 is

actually a result of light treatment further

research is needed.

ZE was undetectable in most samples and

the frequency of detectable/undetectable

amounts was not significantly affected by

treatment (tested with a chi-square). For

further analysis on ZE using a UPLC-UV-

MS the starting dry weight sample should be

heavier than 50mg to ensure detectability.

This study finds it hard to conclude anything

about ZE as the standard error is

comparatively large, almost going to zero μg

/mg dw in all treatments.

VI was not detectable in any treatments and

its positive effect on health can be

disregarded if a product was prepared like

the light/nitrogen treatments.

Nitrogen did not have a significant effect on

any of the pigments analyzed by the

spectrophotometer or by the UPLC-UV-MS.

There is however an indication of an

interaction between light and nitrogen,

visible in both Chart 1 and in Table 1. The

interaction was not significant and further

research is needed for a definitive

conclusion. The trend visible in Chart 1

indicates that nitrogen has an effect under

low light conditions, were the production of

figments is presumed to be most active. This

interaction might become larger if the

treatment time was longer, as a response in

pigmentation du to nutrient levels has been

seen after a five day treatment (Boderskov et

al., 2016). Further research can hopefully

show if and how nitrogen affects the

pigment content under low light.

Light/iron treatment

As no significant effect of light, nitrogen or

an interaction between them was seen in the

light/iron treatments no conclusions can be

made from the treatment. In Chart 2 there is

an indication that low light increases the

pigment content, which was seen in the

light/nitrogen treatment. A larger sample

size might have resulted in a significant

effect from light. There are no apparent

trends from iron treatment. It could be that

the response to a higher iron content is not

fast enough to lead to a response in two

days, the study that fund an increase in FX

under high iron was done over 15 days

(Matsunaga et al., 1994).

Continued research

The potential effect of photoperiod on the

pigmentation of S. Latissimar could be of

future interest. S. Latissima is thought to be

an” anticipating” species of alga. It is

believed to use photoperiod as an indicator

of seasonal changes and can therefor “grow

and reproduce in a strategic annual rhythm

suitable for the species” (p. 209, Kain,

1989).

If photoperiod is a trigger of seasonal change

it could be used to manipulate the growth

and pigmentation of the species.

Conclusion

As the aim of this thesis was to understand if raised levels of light, nitrogen and iron can increase

the pigment content in Saccharina Latissima during a 48h treatment.

A two-day treatment of low light increases the content of Chlorophyll A, Chlorophyll C and

Fucoxanthin, fore greater changes different treatment times and light intensities should be tested.

Nitrogen could potentially increase the effect of a low light treatment, but further research is

needed to see if and how this interaction works.

Iron has not been shown to affect the pigment content but due to small sample size little weight

should be put in this conclusion. Further research is needed to see if there is an effect.

12

Special thanks to; Matthew Hargave and Swantje Enge who lent their time, guidance and

patience to making my thesis possible; Henrik Pavia, the supervisor of this thesis and all others at

Tjärnö who answered my questions this summer.

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